ASSAY FOR PROGNOSIS OF COVID-19 DISEASE
20230038748 · 2023-02-09
Inventors
- Ernestas SIRKA (London, GB)
- Adam CRYAR (London, GB)
- Markus RALSER (Berlin, DE)
- Johannes HARTL (Berlin, DE)
- Ziyue WANG (Berlin, DE)
- Michael MÜLLEDER (Berlin, DE)
- Vadim DEMICHEV (Berlin, DE)
Cpc classification
G01N2800/60
PHYSICS
International classification
Abstract
The present invention relates to a method for predicting and monitoring the severity of COVID-19 disease following infection of a subject with the SARS-CoV-2 virus. It also relates to a method for the treatment of a subject with COVID-19 disease. It also relates to kits for use in the methods of the invention.
Claims
1. A method for predicting and/or classifying the severity of COVID-19 disease in a subject, the method comprising: (i) preparing a biological sample from the subject for assay by incubating the sample with a protease to form a proteolytic digest of proteins in the sample; and (ii) assaying the proteolytic digest of proteins of step (i) for the presence of a proteolytic peptide of at least one protein selected from the group of proteins as shown in Table 1, said group consisting of Proteoglycan 4, Inter-alpha-trypsin inhibitor heavy chain H1, Plasminogen (EC 3.4.21.7), Actin (Actin, aortic smooth muscle; Actin, cytoplasmic 1; Actin, cytoplasmic 2; Actin, gamma-enteric smooth muscle), Complement C1q subcomponent subunit C, Cystatin-C, Protein ORM2, Alpha-1-antichymotrypsin, Serotransferrin, Apolipoprotein B-100, EGF-containing fibulin-like extracellular matrix protein 1, von Willebrand factor, C-reactive protein, Lysozyme C (EC 3.2.1.17), Apolipoprotein A-I, Alpha-2-HS-glycoprotein, Histidine-rich glycoprotein, Beta-2-microglobulin, N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28), Transthyretin, Plasma kallikrein (EC 3.4.21.34), Antithrombin-III, Heparin cofactor 2, Afamin, Plasma protease C1 inhibitor, Transferrin receptor protein 1, Low affinity immunoglobulin gamma Fc region receptor III-A, Monocyte differentiation antigen CD14, Insulin-like growth factor-binding protein complex acid labile subunit, Immunoglobulin heavy variable 5-51, or Complement C3, wherein the presence of said proteolytic peptide is assayed for using mass spectrometry with reference to a corresponding labelled and/or unlabelled reference proteolytic peptide.
2. The method of claim 1 comprising: (ii) assaying the proteolytic digest of proteins of step (i) for the presence of one or more proteolytic peptides as shown in Table 9 Cohort 1, said group consisting of: TABLE-US-00015 (SEQ ID No.: 11) WEMPFDPQDTHQSR (SEQ ID No.: 12) EQLSLLDR (SEQ ID No.: 27) CNLLAEK (SEQ ID No.: 21) GYSIFSYATK (SEQ ID No.: 26) TVVQPSVGAAAGPVVPPCPGR (SEQ ID No.: 13) GDVAFVK (SEQ ID No.: 14) WCALSHHER (SEQ ID No.: 25) ATEHLSTLSEK (SEQ ID No.: 24) AHVDALR (SEQ ID No.: 43) LVLLNAIYLSAK (SEQ ID No.: 8) ALDFAVGEYNK (SEQ ID No.: 10) SDVMYTDWK (SEQ ID No.: 42) LLDSLPSDTR (SEQ ID No.: 23) YWCNDGK (SEQ ID No.: 52) VHQYFNVELIQPGAVK (SEQ ID No.: 34) GSPAINVAVHVFR (SEQ ID No.: 1) GLPNWTSAISLPNIR (SEQ ID No.: 6) FNAVLTNPQGDYDTSTGK (SEQ ID No.: 33) TFTLLDPK (SEQ ID No.: 18) ILTSDVFQDCNK (SEQ ID No.: 49) LAELPADALGPLQR (SEQ ID No.: 16) EQHLFLPFSYK (SEQ ID No.: 17) ADQVCINLR (SEQ ID No.: 7) TNQVNSGGVLLR (SEQ ID No.: 35) AADDTWEPFASGK (SEQ ID No.: 32) GCPDVQASLPDAK (SEQ ID No.: 38) ANRPFLVFIR (SEQ ID No.: 2) GHMLENHVER (SEQ ID No.: 41) TINPAVDHCCK (SEQ ID No.: 40) HFQNLGK (SEQ ID No.: 37) IAYGTQGSSGYSLR (SEQ ID No.: 20) ESDTSYVSLK (SEQ ID No.: 15) IAELSATAQEIIK (SEQ ID No.: 22) STDYGIFQINSR (SEQ ID No.: 39) TSCLLFMGR (SEQ ID No.: 44) ILNIFGVIK (SEQ ID No.: 48) DFALQNPSAVPR (SEQ ID No.: 3) EAQLPVIENK (SEQ ID No.: 28) GGEGTGYFVDFSVR and (SEQ ID No.: 4) CQSWSSMTPHR.
3. The method of claim 1 comprising: (ii) assaying the proteolytic digest of proteins of step (i) for the presence of one or more proteolytic peptides as shown in Table 9 Cohort 2, said group consisting of: TABLE-US-00016 (SEQ ID No.: 33) TFTLLDPK (SEQ ID No.: 32) GCPDVQASLPDAK (SEQ ID No.: 20) ESDTSYVSLK (SEQ ID No.: 21) GYSIFSYATK (SEQ ID No.: 27) CNLLAEK (SEQ ID No.: 26) TVVQPSVGAAAGPVVPPCPGR (SEQ ID No.: 13) GDVAFVK (SEQ ID No.: 36) DSVTGTLPK (SEQ ID No.: 24) AHVDALR (SEQ ID No.: 25) ATEHLSTLSEK (SEQ ID No.: 14) WCALSHHER (SEQ ID No.: 49) LAELPADALGPLQR (SEQ ID No.: 48) DFALQNPSAVPR (SEQ ID No.: 41) TINPAVDHCCK (SEQ ID No.: 12) EQLSLLDR (SEQ ID No.: 40) HFQNLGK (SEQ ID No.: 2) GHMLENHVER (SEQ ID No.: 11) WEMPFDPQDTHQSR (SEQ ID No.: 5) EITALAPSTMK (SEQ ID No.: 8) ALDFAVGEYNK (SEQ ID No.: 47) VLDLSCNR (SEQ ID No.: 38) ANRPFLVFIR (SEQ ID No.: 29) IADAHLDR (SEQ ID No.: 37) IAYGTQGSSGYSLR (SEQ ID No.: 51) VEGTAFVIFGIQDGEQR (SEQ ID No.: 19) YAGSQVASTSEVLK (SEQ ID No.: 39) TSCLLFMGR (SEQ ID No.: 3) EAQLPVIENK (SEQ ID No.: 28) GGEGTGYFVDFSVR (SEQ ID No.: 15) IAELSATAQEIIK (SEQ ID No.: 17) ADQVCINLR (SEQ ID No.: 35) AADDTWEPFASGK (SEQ ID No.: 18) ILTSDVFQDCNK (SEQ ID No.: 34) GSPAINVAVHVFR (SEQ ID No.: 16) EQHLFLPFSYK (SEQ ID No.: 4) CQSWSSMTPHR (SEQ ID No.: 42) LLDSLPSDTR (SEQ ID No.: 23) YWCNDGK (SEQ ID No.: 43) LVLLNAIYLSAK (SEQ ID No.: 6) FNAVLTNPQGDYDTSTGK (SEQ ID No.: 22) STDYGIFQINSR (SEQ ID No.: 52) VHQYFNVELIQPGAVK (SEQ ID No.: 45) VSASPLLYTLIEK (SEQ ID No.: 44) ILNIFGVIK (SEQ ID No.: 10) SDVMYTDWK (SEQ ID No.: 1) GLPNWTSAISLPNIR (SEQ ID No.: 7) TNQVNSGGVLLR and (SEQ ID No.: 46) DSGSYFCR.
5. The method of claim 1 comprising: (ii) assaying the proteolytic digest of proteins of step (i) for the presence of one or more proteolytic peptides as shown in the top-right panel of Supplementary
6. A method for the treatment of a subject with COVID-19 disease, the method comprising: (i) preparing a biological sample from the subject for assay by incubating the sample with a protease to form a proteolytic digest of proteins in the sample; (ii) assaying the proteolytic digest of proteins of step (i) for the presence of a proteolytic peptide of at least one protein selected from the group of proteins as shown in Table 1, said group consisting of Proteoglycan 4, Inter-alpha-trypsin inhibitor heavy chain H1, Plasminogen (EC 3.4.21.7), Actin (Actin, aortic smooth muscle; Actin, cytoplasmic 1; Actin, cytoplasmic 2; Actin, gamma-enteric smooth muscle), Complement C1q subcomponent subunit C, Cystatin-C, Protein ORM2, Alpha-1-antichymotrypsin, Serotransferrin, Apolipoprotein B-100, EGF-containing fibulin-like extracellular matrix protein 1, von Willebrand factor, C-reactive protein, Lysozyme C (EC 3.2.1.17), Apolipoprotein A-I, Alpha-2-HS-glycoprotein, Histidine-rich glycoprotein, Beta-2-microglobulin, N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28), Transthyretin, Plasma kallikrein (EC 3.4.21.34), Antithrombin-III, Heparin cofactor 2, Afamin, Plasma protease C1 inhibitor, Transferrin receptor protein 1, Low affinity immunoglobulin gamma Fc region receptor III-A, Monocyte differentiation antigen CD14, Insulin-like growth factor-binding protein complex acid labile subunit, Immunoglobulin heavy variable 5-51, or Complement C3, wherein the presence of said proteolytic peptide is assayed for using mass spectrometry with reference to a corresponding labelled and/or unlabelled reference proteolytic peptide; and (iii) treating the subject with a therapeutic agent or treatment according to the severity of the COVID-19 disease detected in the subject.
7. A kit for predicting and/or classifying the severity of COVID-19 disease in a subject according to a method as claimed in claim 1, comprising: a plurality of sample preparation media for analysis of a sample by mass spectrometry for the presence of a proteolytic peptide of at least one protein selected from the group of proteins as shown in Table 1, said group consisting of Proteoglycan 4, Inter-alpha-trypsin inhibitor heavy chain H1, Plasminogen (EC 3.4.21.7), Actin (Actin, aortic smooth muscle; Actin, cytoplasmic 1; Actin, cytoplasmic 2; Actin, gamma-enteric smooth muscle), Complement C1q subcomponent subunit C, Cystatin-C, Protein ORM2, Alpha-1-antichymotrypsin, Serotransferrin, Apolipoprotein B-100, EGF-containing fibulin-like extracellular matrix protein 1, von Willebrand factor, C-reactive protein, Lysozyme C (EC 3.2.1.17), Apolipoprotein A-I, Alpha-2-HS-glycoprotein, Histidine-rich glycoprotein, Beta-2-microglobulin, N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28), Transthyretin, Plasma kallikrein (EC 3.4.21.34), Antithrombin-III, Heparin cofactor 2, Afamin, Plasma protease C1 inhibitor, Transferrin receptor protein 1, Low affinity immunoglobulin gamma Fc region receptor III-A, Monocyte differentiation antigen CD14, Insulin-like growth factor-binding protein complex acid labile subunit, Immunoglobulin heavy variable 5-51, or Complement C3.
8. A pharmaceutical composition comprising a therapeutic agent for use in a method of treatment of a subject with COVID-19 disease, wherein the COVID-19 disease of the subject has been classified according to a method as claimed in claim 1.
9. A kit for use in the treatment of a subject with COVID-19 disease according to a method as claimed in claim 6, comprising: (i) a plurality of sample preparation media for analysis of a sample by mass spectrometry for the presence of a proteolytic peptide of at least one protein selected from the group of proteins as shown in Table 1, said group consisting of Proteoglycan 4, Inter-alpha-trypsin inhibitor heavy chain H1, Plasminogen (EC 3.4.21.7), Actin (Actin, aortic smooth muscle; Actin, cytoplasmic 1; Actin, cytoplasmic 2; Actin, gamma-enteric smooth muscle), Complement C1q subcomponent subunit C, Cystatin-C, Protein ORM2, Alpha-1-antichymotrypsin, Serotransferrin, Apolipoprotein B-100, EGF-containing fibulin-like extracellular matrix protein 1, von Willebrand factor, C-reactive protein, Lysozyme C (EC 3.2.1.17), Apolipoprotein A-I, Alpha-2-HS-glycoprotein, Histidine-rich glycoprotein, Beta-2-microglobulin, N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28), Transthyretin, Plasma kallikrein (EC 3.4.21.34), Antithrombin-III, Heparin cofactor 2, Afamin, Plasma protease C1 inhibitor, Transferrin receptor protein 1, Low affinity immunoglobulin gamma Fc region receptor III-A, Monocyte differentiation antigen CD14, Insulin-like growth factor-binding protein complex acid labile subunit, Immunoglobulin heavy variable 5-51, or Complement C3; and (ii) a therapeutic agent for treatment of COVID-19 disease.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0121] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
[0122] Reference is made to the following drawings in which:
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[0137] Global healthcare systems continue to be challenged by the COVID-19 pandemic, and there is a need for clinical assays that can help to optimise resource allocation, support treatment decisions and accelerate the development and evaluation of new therapies. We developed a multiplexed proteomics assay and performed a stratification and prediction study on 2 cohorts of patients with COVID-19. The assay quantifies 50 peptides, derived from 30 known and newly introduced COVID-19 related protein markers, in a single measurement using analytical flow rate liquid chromatography and multiple reaction monitoring (LC-MRM), on equipment that is broadly available in routine laboratories. Technical analytical validation of the targeted, mass spectrometry-based peptide panel showed that it enables reproducible (i.e. inter-batch CV of 10.9%) absolute quantification of 47 peptides with high sensitivity (i.e. median LLOQ of 1.6 ng/ml) and accuracy (median 97.3%). Applied to two COVID-19 inpatient cohorts treated before and after dexamethasone became standard of care, composed of 30 and 164 patients, respectively, the assay reproducibly captured hallmarks of COVID-19 infection and severity, as it distinguished healthy subjects, mild, moderate and severe COVID-19. In the post dexamethasone cohort, the assay predicted survival with an accuracy of 0.83 (108/130), and death with an accuracy of 0.76 (26/34) in the median 2.5 weeks before the outcome, thereby outperforming compound clinical risk assessments such as SOFA, APACHE II, and ABCS scores. Disease severity and clinical outcomes of COVID-19 patients can be well stratified and predicted by this scalable and standardised protein panel assay that combines known and novel COVID-19 biomarkers. The prognostic value of the peptide panel assay should be prospectively assessed in larger patient cohorts for future support of clinical decisions, including evaluation of sample-flow in routine setting. The possibility to objectively classify COVID-19 severity can be helpful for monitoring of novel therapies, especially in early clinical trials.
[0138] The clinical presentation of COVID-19 is extremely diverse, ranging from asymptomatic infection to fatal disease, and can change rapidly. Timely assignment of the appropriate level of care substantially improves outcome in COVID-19. Objective and easy-to-apply tools to anticipate the patient's risk of deterioration, maximum disease severity and outcome based on validated biomarkers are fundamentally required, particularly in situations with overstrained healthcare resources. Risk assessment, as informed by established ICU outcome predictors, such as APACHE II or SOFA, as well as prognostic markers that depend on established clinical chemistry, have so far proven to be of limited predictive value in COVID-19. Moreover, the pandemic situation has prompted an unseen amount of repurposing attempts of existing antiviral and immunomodulatory drugs. However, small and rapidly conducted clinical trials can fail to yield reliable results with regard to clinical benefit and patient safety. Plasma and serum proteome studies have recently shifted to the centre stage to provide added value in COVID-19 patient classification and outcome prediction. Proteomic assays could overcome limitations surrounding the accuracy of early clinical testing. However, proteome analyses are thus far restricted to research settings, as the technology employed in discovery proteomics does not meet the requirements of technical stability and ease of implementation as required in the routine laboratory. In order to bridge the gap between research based discovery proteomics and the clinical application of a proteomic marker panel, we used proteomic datasets recorded in a deeply phenotyped COVID-19 patient cohort. We select a panel of 50 peptides derived from 30 proteins whose functions are associated with the COVID-19 host response and which can classify disease severity in COVID-19. Assembled on the basis of observational criteria, our panel contains a set of established clinical markers, but is also based on new protein markers that are not in use in the routine so far. We then develop and analytically validate a scalable and standardised proteomic panel assay that may be performed on instrumentation common in certified laboratories. Applying the assay to two independent cohorts, we demonstrate accurate disease classification, and show that the marker panel is prognostic about outcome. There is value in using the human plasma proteome in severity classification, risk assessment and outcome prediction in COVID-19. Missing so far is a translation of this research evidence into a routinely applicable assay. We show that a proteomic marker panel, which predicts survival in COVID-19 with high accuracy, can be used in routine laboratory testing. The described proteomic marker panel has the potential to substantially improve clinical risk assessment for patients with COVID-19 by translating discovery proteomics findings to patient care.
[0139] The invention will now be further described with reference to the Examples which are present for the purposes of reference only and are not to be construed as limitations on the invention.
EXAMPLE 1: CHARACTERIZATION OF PLASMA PROTEOME AND PATIENT RESPONSE TO COVID-19 DISEASE
[0140] 1. Venous blood was collected from patients suspected or diagnosed to have COVID-19
[0141] 2. EDTA plasma was prepared from the collected blood samples using standard hospital pathology laboratory protocols.
[0142] 3. Plasma samples were prepared for mass spectrometry analysis by spiking with heavy isotope-labelled peptides shown in Table 1 and digestion with trypsin to release tryptic peptides from plasma proteins for analysis on the targeted LC-MS/MS platform.
[0143] 4. LC-MS/MS analysis was performed on the prepared plasma samples by measuring tryptic peptides. This occurred on a pre-configured targeted LC-MS/MS system which was “programmed” according to the specifications in Table 2 to detect and quantify a specific set of peptides in Table 1.
[0144] 5. Tryptic peptide concentration in μg/ml or ng/ml was determined for all 52 analytes using heavy isotope-labelled internal standards and synthetic reference peptides at known concentration as shown in Table 1.
[0145] 6. Tryptic peptide concentration with a patient's age was used as input in a linear regression statistical model to generate an overall risk score for each individual patient.
[0146] 7. The test results were sent to a treating clinician in order to make a treatment decision, taking other clinical readouts and their judgment into account.
EXAMPLE 2: COVID-19 ANTIGEN CALIBRATION CURVE PREPARATION
[0147] (i) solution preparation: [0148] Peptide Storage Solvent: 50% acetonitrile, 50% ddH.sub.2O. [0149] Denaturation Buffer: 8M Urea, 100 mM ammonium bicarbonate, 50 mM DTT. [0150] Alkylation Agent: 100 mM IAA. [0151] 100 mM ammonium bicarbonate. [0152] Surrogate Matrix: 40 mg/ml bovine serum albumin in ddH.sub.2O.
[0153] (ii) Internal standard Spike Solution (Peptide Mix A) [0154] To an Eppendorf labelled as “Internal Standard Mix”, 20 μl each of all 52 internal standard peptide stock solutions were added.
[0155] Final volume of pooled IS mix was 1.04 ml. [0156] A 5 ml Eppendorf was labelled with “Peptide Mix A”. [0157] To “Peptide Mix A”, 200 μl of “Internal standard Mix” was added. [0158] “Peptide Mix A” was dried down to completion and stored at −20° C. until calibration sample digestion.
[0159] (iii) Calibration Curve Preparation [0160] A 5 ml Eppendorf was labelled with “Cal 0”. [0161] To “Cal 0”, 55 μl of each of the 52 native peptide stock solution was added. Total volume of the native pool was 2.86 ml. [0162] A set of ×20 1.5 ml Eppendorf tubes was labelled with “Plasma Cal 1”-“Plasma Cal 18”, “Plasma IS Only” and “Plasma Blank”. [0163] A 2.sup.nd set of ×20 1.5 m Eppendorf tubes was labelled with “BSA Cal 1”-“BSA Cal 17”, “BSA IS Only” and “BSA Blank”. [0164] To all ×40 tubes that have been labelled during steps 3.3. & 3.4, 650 μl of peptide storage solvent was added. [0165] To “Plasma Cal 1”, 650 μl of “Cal 0” was added and the sample was mixed well. [0166] To “Plasma Cal 2”. 650 μl of “Plasma Cal 1” was added and mixed well. [0167] The serial dilution process was continued until “Plasma Cal 18” was reached. [0168] 650 μl of sample was removed from “Plasma Cal 17” and discarded to achieve required concentration. [0169] Nothing was added at this point to either “Plasma IS only” or “Plasma Blank”. [0170] The serial dilution process detailed in steps 3.6-3.10 was repeated using the 2.sup.nd set of tubes that were labelled in step 3.4 (“BSA Cal 1 . . . ”). [0171] All samples were dried down to complete dryness. C0 stock was not dried down and remaining sample was kept in the fridge for repeat curves.
[0172] (iv) Calibration Sample Tryptic Digestion [0173] Denaturation buffer was thawed. [0174] To all ×19 “Plasma Cal . . . ” samples, equal volumes of pooled human plasma was added. [0175] To all ×19 “BSA Cal . . . ” samples, equal volumes of 40 mg/ml BSA was added. [0176] To the dried “Peptide Mix A”, denaturation buffer was added. [0177] “Peptide Mix A” was vortexed in order to mix well. [0178] “Peptide Mix A” was left to mix gently for 10 minutes in order to fully solubilise internal standards. [0179] To “Plasma Cal Blank” & “BSA Cal Blank”, equal volumes of denaturation buffer was added. [0180] To all other calibration samples, equal volumes of “Peptide Mix A” was added. [0181] Samples were spun briefly to ensure all solution was collected at bottom of tubes. [0182] Samples were left mixing at 30° C. for 1 hour. [0183] Equal volumes of alkylation agent was added to samples. [0184] Samples were mixed briefly and then spun briefly. [0185] Samples were incubated at room temperature in the dark for 30 minutes. [0186] Equal volumes of 100 mM ammonium bicarbonate was added to the samples. [0187] Samples were then spun briefly. [0188] Mass spectrometry grade trypsin was made up to a final concentration of 0.1 μg/μl in 100 mM ammonium bicarbonate. [0189] Equal volumes of trypsin solution was added to all samples. [0190] Samples were incubated at 37° C. overnight with gentle mixing. [0191] The following morning, equal volumes of formic acid was added to all samples to quench the digest and prepare samples for SPE clean up. [0192] Samples were mixed and proceeded to SPE processing.
EXAMPLE 3: A MULTIPLEXED, TARGETED LIQUID CHROMATOGRAPHY MASS SPECTROMETRY-BASED PROTEIN PANEL ASSAY DETERMINES COVID-19 DISEASE SEVERITY AND IS PROGNOSTIC ABOUT OUTCOME
[0193] Abstract
[0194] Global healthcare systems continue to be challenged by the COVID-19 pandemic, and there is a need for clinical assays that can both help to optimize resource allocation and be used to monitor clinical trials. Several recent studies reported a huge potential for plasma and serum proteomics in classifying COVID-19 disease severity and to provide accurate outcome prediction weeks in advance. However, these investigations are so far based on shotgun and discovery proteomic platforms using nanoflow chromatography and relative quantification, which are difficult to implement in clinical settings and routine laboratories. Here, we present an absolute quantitative proteomic panel assay for the assessment of COVID-19 disease severity and outcome. For the ease of implementation, the assay is based on analytical flow rate chromatography and multiple reaction monitoring (LC-MRM), and runs on equipment available in routine and CLIA laboratories. We demonstrate classification of severe COVID-19 patients according to treatment choices in two cohorts. Moreover, we show that the panel assay substantially outperforms established risk assessments such as the Charlson comorbidity Index, the SOFA score, and the Apache II in predicting COVID-19 outcome. Our study hence shows that the combination of discovery and targeted plasma proteomics based on analytical-flow rate chromatography has become a powerful platform that facilitates the rapid translation of proteomic findings into routinely applicable clinical assays.
[0195] Introduction
[0196] COVID-19 continues to challenge healthcare systems worldwide despite vaccination efforts and novel treatments. This is particularly apparent in areas with limited vaccine uptake or supply. Currently, the outlook remains uncertain even in countries with high vaccination rates as the immunity conferred by the vaccines appears to diminish over time (2-5) and SARS-COV-2 variants with varying capacity to evade vaccine induced immunity continue to emerge (2, 6-8).
[0197] Biomarker tests that can classify patients, predict disease severity and are prognostic could help optimise resource allocation, specifically during a pandemic (9-11). Indeed, clinical manifestation of COVID-19 is highly variable, which does create challenges in timely clinical decision making. For instance, ‘happy hypoxia’ describes situations where COVID-19 patients report minor impairments, while molecular indicators such as blood oxygen levels, indicate they are, in fact, severely ill (12). Furthermore, in situations when healthcare systems reach maximum capacity, prognostic markers could support difficult clinical decisions, for instance, to navigate through triaging situations (13). Prognostic and disease severity tests could further help to increase the likelihood of success and accelerate clinical trials, by improving the assessment of treatment efficacy of COVID-19 therapies or stratify patient populations that are to be included in the trials. Indeed, COVID-19 prompted the rapid repurposing and development of new treatments. However, during a pandemic, there is pressure to conduct trials in a timely manner. Furthermore, specifically in ICU settings, study cohorts are limited in size, and when underpowered, clinical trials can lead to false positive and false negative results (14)(15). So far however, the reliability of several risk assessment scores conventionally used in ICU settings, such as the Acute Physiology And Chronic Health Evaluation (APACHE II) and Sequential Organ Failure Assessment (SOFA) scores, appear to be limited for COVID-19 cases (16). Moreover, combinations of generic clinical readouts, e.g. blood oxygen saturation, interleukin-6 concentration, have been considered for COVID-19 outcome prediction at various disease severity stages (Vatansever and Becer 2020), however, several predictive models based on clinical parameters and routine tests were reported to be vulnerable to bias and not suitable for the clinic (17,18).
[0198] Protein biomarker signatures from plasma present a promising alternative. Proteomic datasets have repeatedly been successful to classify and predict COVID-19 severity and outcome (9, 10, 16, 19-21). For instance, we recently presented a proteomics biomarker signature that stratified COVID-19 severity grades and successfully predicted disease progression and outcome, such as survival of hospitalised COVID-19 patients (13, 22, 23). Furthermore, specifically early in the pandemic, proteomics successfully characterized the antiviral host response, which greatly improved our understanding of the COVID-19 disease (9, 10, 16, 19-21, 23).
[0199] The success of shotgun proteomics in the clinical routine is so far however limited for technical and economic reasons. Most of the research platforms require a high degree of expert knowledge to use and they are also susceptible to interference and batch effects. Moreover, with only recent exceptions (15,22), most discovery proteomics platforms make use of nano-flow chromatography, which limits the throughput of proteomics while creating high maintenance efforts. The objective of this study was to translate selected biomarkers from this discovery based approach into a routinely applicable proteomics platform that could be deployed for clinical use within existing regulatory frameworks and on broadly available analytical instruments. Triple quadrupole mass spectrometers coupled to high flow liquid chromatography were the optimal choice for rapid test development and deployment as they are used in the clinic in other areas, for instance rare diseases, newborn screening and steroid hormone analysis (24-27). Further, they are widely available in large hospital laboratories, diagnostic laboratories and contract research organizations. Biomarker tests developed on this platform can be accredited to existing regulatory standards in GCP, ISO:17025, ISO:15189 and CLIA environments, standardised and transferred across different instruments, manufacturers and laboratories, and thus deployed at scale rapidly. Triple quadrupole mass spectrometry-based tests are cost effective to run at scale as the sample preparation is amenable to automation, consumables costs are typically <£5 per test and the instrument uptime is typically >95%. The tests also integrate with existing workflows at clinical and analytical laboratories.
[0200] We have mined discovery proteomics data from COVID19 patients, and selected peptides that are informative about COVID19 disease progression and severity. The biomarkers were chosen for i) being prognostic of remaining duration of hospitalisation, disease aggravation, or being differentially concentrated in plasma depending on the treatment escalation level, used as a measure of disease severity, and ii) participating in biological processes that contribute to COVID-19 pathology, and iii) to be technically and analytically suitable for the assay. The COVID-19 severity biomarkers chosen include proteins that function in inflammation (e.g. C-reactive protein), coagulation and vascular dysfunction (e.g. von Willebrand factor), complement cascade (e.g. Complement C1q subcomponent subunit C) and diverse biological processes detected to be altered by COVID-19 (e.g. Cystatin C). We then established a targeted proteomics assay utilising multiple reaction monitoring (MRM) data acquisition mode. Employing calibration curves with synthetic reference and stable isotope labelled internal standards, the assay provides absolute quantification of 50 surrogate tryptic peptides arising from 30 plasma proteins (Table 5). These includes proteins that function in inflammation (e.g. C-reactive protein), coagulation, vascular dysfunction (e.g. von Willebrand factor), complement cascade (e.g. Complement C1q subcomponent subunit C) and other biological processes detected to be altered by COVID-19 (e.g. Cystatin C, which is a known marker of kidney dysfunction). We have validated the assay technically, and implemented it in two analytical laboratories employing two triple quadrupole LC-MS/MS platforms from different vendors. The assay was applied to two cohorts with a total of XX patients and XX samples. We demonstrate that the presented MRM assay captures host response to SARS-COV-2 and, based on it, classifies and predicts COVID-19 disease severity and is predictive about outcome in severely ill individuals. We found that the biomarker panel is predictive about survival weeks before outcome and outperforms several commonly used risk assessment scores.
[0201] Results
[0202] Peptide Selection and Optimisation of an MRM Based, Targeted COVID-19 Biomarker Assay.
[0203] To select peptides for a COVID-19 biomarker panel assay, we used shotgun plasma proteomic data recorded on a deeply phenotyped COVID-19 patient cohort treated at Charit{tilde over (e)} Universitätsmedizin Berlin (13,22). On the basis of xx proteomes recorded for xx patients, we selected 50 proteotypic peptides that corresponded to 30 plasma proteins. These peptides were chosen based on their ability to predict the future worsening of COVID-19 over time, the remaining time in hospital for respective COVID-19 patients, which in turn serves as a treatment-insensitive proxy for COVID-19 severity (
[0204] To quantify the 50 proteotypic peptides, we obtained synthetic reference standards. For each peptide 2 synthetic standards were manufactured: 1) with a natural isotope distribution (‘native’) and 2) with a C-terminal stable isotope-labelled (SIL) amino acid (either [13C6,15N2]-lysine or [13C6,15N4]-arginine) to act as an internal standard. Internal standards contained a short tryptic tag to account for the digestion efficiency (Table 5). The native peptides were employed to optimise liquid chromatography-mass spectrometry (LC-MS/MS) data acquisition method and quality of the Q1/Q3 (MRM) transitions. To select optimal MRM transitions for each peptide, we first predicted the transitions (consisting of several precursor ion charge states and respective product ions) using Skyline v21.1.0.146 (30). We infused each native peptide solution into the LC-MS/MS system and selected 1 precursor ion per peptide with the highest relative intensity and 5 most abundant product ions for collision energy optimization which was also provided by Skyline. From these 5 product ions, we ultimately selected 2-5 experimentally optimised ion transitions per native peptide based on i) highest relative signal intensity, ii) optimal chromatographic peak shape and iii) absence of interfering signals. Product ions of <300 Da were excluded where possible to ensure specificity. Precursor and product ion-matched SIL internal standard transitions were included. Lastly, all selected transitions were combined into one dynamic MRM method, which was subsequently analytically validated. In the designed method, the most abundant transition for each peptide is used for quantification and 1-4 less abundant transitions are used as qualifiers (Tables 6 & 7). In order to establish the assay on a chromatographic system that could be run in a routine setting, we chose analytical flow-rate (800 μl/min) reversed phase chromatography using an 1290 Infinity II (Agilent) binary pump. The selected 50 peptides were well distributed along a 8.6 minute linear gradient, and quantifiable with the chosen total runtime of 10 minutes (
[0205] Analytical Validation of the Peptide Panel MRM Assay.
[0206] To evaluate the general applicability of the method for a clinical assay we tested performance of the selected peptides/transitions with respect to intra- and inter-batch repeatability, linearity, limits of quantification, accuracy, and potential matrix effects.
[0207] First, we determined the intra- and inter-batch repeatability. We calculated the coefficient of variation (CV) between three independently prepared replicate calibration curves.
[0208] These were constructed from serial dilutions of native peptide standards in BSA, covering a concentration range ˜5×105, and measured in technical pentuplicates (N=15). We used BSA as a surrogate matrix to test the analytical performance achieved on the standards in the absence of the endogenous plasma peptides (31) and achieved a median inter-batch CV of 10.9% across low (LLOQ), medium ((LLOQ+ULOQ)/2) and high (ULOQ) concentration points (Table 8). CV was determined from the response ratios, calculated by dividing native peptide area by internal standard peptide area. Additionally, we determined the limits of quantification as the highest and lowest concentration points on the linear calibration curve where the CV of the inter-batch repeatability was 520%. 37/50 peptides fulfilled the ≤20% CV cutoff requirement. Since analytical validation requirements for clinical assays are purpose and context dependent, and are influenced by the magnitude of change of target analyte levels in control versus disease samples, we subsequently expanded the CV cutoff to 540%. This enabled us to determine LOQ of 10 additional peptides and prevented missing values in downstream data analysis. Thus, we eventually determined limits of quantification and generated calibration curves for 47 peptides, with a median LLOQ of 1.6 ng/ml. Obtained calibration curves also showed excellent linearity within the determined LOQs (R2>0.99) and typically spanned 3-4 orders of magnitude (Table 8).
[0209] As LC-MS measurements can suffer from matrix effects (32), we next evaluated how quantification in surrogate matrix (BSA) compares to human plasma. We compared the slopes obtained from calibration samples measured in commercial human plasma samples (manufacturer), with those measured in the surrogate matrix. 41/47 quantified peptide biomarkers that passed the above described validation showed no statistically significant matrix effects when comparing the slopes between matrices (P>0.05) as expected for an assay using SIL internal standards (33,34). 6 peptides (AADDTWEPFASGK (SEQ ID No: 35), ASDTAMYYCAR (SEQ ID No: 50), ATEHLSTLSEK (SEQ ID No: 25), EQLSLLDR (SEQ ID No: 12), GDVAFVK (SEQ ID No: 13), IADAHLDR (SEQ ID No: 29)) differed significantly and matrix factor (slope plasma/slope BSA×100%) was calculated. The matrix factor was within the acceptable +/−20% limits and these peptides were included in the final method.
[0210] To test if peptide quantities from actual patient samples would be covered within the (linear) range of the calibration curves, we performed absolute quantification in plasma samples obtained from COVID-19 patients (pooled COVID-19 patient samples of different WHO severity grades; see methods). Absolute quantification was performed by calculating the relative response ratio (peak area ratio of native peptide over its corresponding ISTD peptide) in pooled samples, followed by absolute concentration interpolation from the above described BSA calibration curve. The peptide concentrations obtained from patient samples were covered within the determined linear range of the assay and were measured with a median accuracy of 97.3% across low (LLOQ), medium ((LLOQ+ULOQ)/2) and high (ULOQ) concentration points (Methods; Table 8).
[0211] Diagnostic Performance of the Peptide Panel Assay in an Early COVID-19 Cohort.
[0212] Next, we assessed how the absolute concentration of the selected potential biomarkers changed as a function of the COVID-19 treatment escalation level, a proxy for disease severity, as expressed by the WHO ordinal severity scale. To establish this relationship, we applied the MRM assay on a COVID-19 cohort that was also analysed with a discovery proteomics platform (23). This cohort was sampled as citrate plasma (in contrast to EDTA plasma used in the cohorts employed to select the peptides), and included healthy controls, as well as samples from patients hospitalized during the first wave of the pandemic between March 2020 and XX with mild to severe forms of the disease (23).
[0213] For robust and reliable sample preparation, we employed the recently presented procedure that enables tryptic digest and solid phase extraction in a semi-automated way (22). 40/50 peptides could be reliably detected and quantified on the Agilent 6495c LC-MS/MS platform (Table 9). The concentration of 32 of these both up- and down-regulated peptides changed with the severity of the COVID-19 disease according to treatment escalation: i.e. from uninfected (WHO 0) to mild (WHO3), moderate (WHO 4, 5) and severely (WHO 6, 7) COVID-19 affected individuals (
[0214] Analytical Cross-Platform and Cross-Laboratory Validation.
[0215] To evaluate transferability of the assay, samples from the above described cohort were further employed for a cross-platform, cross-laboratory validation of the LC-MS/MS assay. Post-digested samples were analyzed on a triple-quadrupole platform from a different vendor, with independently optimised MRM transitions (Table 7), in a different laboratory. For the majority of selected peptides, we obtained an excellent correlation between the concentration measured in respective samples on both instruments (
[0216] Similarly, the absolute quantities obtained for individual peptides were highly similar, with some exceptions where there was a high correlation, but some discrepancies in the obtained absolute concentration, suggesting differences in the employed calibration curves (see for instance the peptide CNLLAEK (SEQ ID No: 27) in
[0217] Overall, as we detected and quantified the majority of peptides on both platforms with high precision, the peptides that differentiate between different stages of COVID-19 can be quantified on both platforms, demonstrating the general applicability of the assay independent of the employed analytical platform.
[0218] Severity Stratification and Prediction of Disease Progression in a Longitudinal COVID-19 Cohort.
[0219] After establishing that the assay successfully captured hallmarks of COVID-19 disease in a small patient cohort, we next measured a large, longitudinal collection of samples obtained during the second wave of the pandemic in Germany. This cohort was selected as i) it provided a large number of samples, and thus more statistical power to evaluate the potential of the MRM assay to stratify disease severity, and ii) the longitudinal nature of the study, which allowed us to assess the predictive value of the MRM panel. Reassuringly, despite the large number of samples acquired (n=655 including quality controls) split over three batches and measured over 10 days, variation was low and no significant batch-effect was observed (
[0220] To evaluate the ability of the whole MRM panel to differentiate COVID-19 severity, we first analyzed the earliest sample obtained for each patient of this cohort (n=165). We were able to reliably detect and quantify 47/50 peptides, and the majority (33 peptides, 11 up-regulated, 22 downregulated) had a significant trend between patients according to the WHO ordinal outcome scale for clinical improvement with patients capturing a WHO score from relatively mild (WHO 3) to very severe cases (WHO 7) (Supplementary
[0221] To further explore the possibilities to classify COVID-19 severity with the MRM assay, we constructed a support vector machine trained to differentiate between three different treatment groups: WHO grade 3 (mild COVID-19, hospitalised, but no oxygen therapy necessary), WHO grade 4/5 (severe COVID-19, hospitalised, non-invasive oxygen therapy) and WHO grade 6/7 (severe COVID-19, hospitalised, mechanical ventilation).
[0222] The data was split in a training and a validation set in a cross-validated manner. The SVM successfully predicted the WHO grade in the prediction validation set (
[0223] Finally, we examined the capability of the measured peptides for predictions. An outcome predictor (SVM) was trained in a cross-validated manner to differentiate patients who survived COVID-19, from patients who had a fatal disease (First sample taken for each patient, n=165, n_died=34) (
[0224] Plotting the predicted outcome with respect to the time until the death (Kaplan-Meier survival analysis,
[0225] To evaluate how well the predictor performs compared to the clinically established scores, we determined the Sepsis-related Organ Failure Assessment score (SOFA), Acute Physiology And Chronic Health Evaluation (APACHE II) and Charlson Comorbidity Index (CCI), all which are in clinical use. The proteomics predictor significantly outperformed all three clinical scores (AUC of the ROC-curves). The SOFA-score and the APACHE II-score, which are directly linked to the severity of the patient, performed the best among the three “conventional”/established scores tested. For both SOFA and APACHE II scores one can observe an early enrichment of critical cases who will die. However, due to only capturing the current state of the disease, a future prediction especially for non-critical patients can not be performed reliably.
[0226] In conclusion, we demonstrate the development of a fast, multiplexed, sensitive LC-MS peptide assay that captures hallmarks of COVID19 disease severity and progression. The assay is easily translatable, sensitive, and specific, and thus well suited for rapid translation into a clinical test. Further, the fast and multiplexed nature of the presented method gives suitable throughput for large patient cohorts or routine diagnostic applications.
[0227] Discussion
[0228] Novel infectious diseases such as COVID-19 that lack immediate treatment options can quickly challenge health systems on a global scale. Considering the enormous pressure on intensive care units, there is an urgent clinical need for assays that capture and monitor the individual response of patients. Such personalized tests can support an objective clinical decision making that otherwise depends on confounders like age, and could guide development of novel treatments. While a range of such assays and clinical scoring systems have been applied, they often rely on a limited number of biomarkers that do not necessarily capture all major features associated with complex diseases such as COVID-19.
[0229] Here, we presented how the combination of high-throughput proteomics for identification of biomarker signatures, followed by the development and validation of a targeted, clinically translatable and scalable LC-MS/MS based assay is a powerful strategy to rapidly transition from a discovery approach to a platform that can be deployed in a clinical setup.
[0230] Targeted mass spectrometry using multiple reaction monitoring (MRM) is a method of choice for quantification of multiplex or multiparametric marker panels in biofluids. This is because LC-MRM i) provides excellent sensitivity and specificity, ii) has the possibility to easily include internal standards that give the assay precision and enable control over potential matrix effects (33, 34), iii) facilitates absolute quantification, enabling cross-platform transferability (35, 36). iv) has a large dynamic range (4 orders of magnitude of linear range in the presented assay) (37), which makes it possible to compare biomarkers with large abundance differences within one run, facilitating multiplexing of many biomarkers in parallel; and further, it facilitates sample preparation as matrix depletion is not necessarily required (38). Given the simplicity, flexibility, and multiplexability of LC-MS based MRM assays, the initial panel of peptides can be large, increasing the chance of finding a reliable set of reproducible biomarkers based on hierarchical filtering and selection of the most suitable markers during establishment of the targeted assay (39,40). Finally, although LC-MRM assays require analytical expertise and are labor-intensive to set up, they are highly cost-effective to run. In our study, we also overcome a common limitation of proteomic assays for their routine use—their dependency on low flow rate-chromatography (Bian et al. 2020; Song et al. 2017; Gao et al. 2020). Exploiting the high sensitivity of contemporary triple-quadrupole mass spectrometers, we demonstrate the accurate quantification of the peptide panel using analytical flow rate chromatography, which not only is robust and fast, but also available in typical clinical laboratories, greatly simplifying the application our assay in the routine.
[0231] The developed biomarker panel includes 50 proteotypic peptides derived from 30 plasma proteins. The peptides were selected from discovery proteomic data (13, 22, 23, 28) for their ability to predict the patient stay in hospital and to indicate the likelihood of future worsening, and were found to change in abundance depending on the treatment escalation level according to the WHO ordinal scale, introduced as of April 2020 (41), used as a measure of COVID19 disease severity. These proteins were all found to belong to biological processes important for the COVID19 host response, like the coagulation system, the complement cascade or metabolism. The assay is hence monitoring processes causal to the disease progression. In this study, we established the assay on two routine-laboratory compatible LC-MRM platforms, and performed an elaborate analytical validation procedure of the technical aspects of the assay. We demonstrate excellent reproducibility across two different mass spectrometric technologies, and reveal for each peptide the optimal measurement parameters, dynamic range, as well as sensitivity to matrix effects. As such, the assay could easily be translated into a multiplexed, high-throughput clinical assay that captures hallmarks of the COVID-19 pathophysiology.
[0232] We confirm in two independent patient cohorts, that the panel assay captures the severity of the COVID19 affected individual, and is discriminatory about the treatment levels. Moreover, the assay is prognostic about the outcome for the most severely affected COVID19 patients. Thus, the panel assay parameters could be used to assess the current state of the patient, help to monitor efficacy of novel treatments, or stratify patients based on their responsiveness to novel clinical interventions for COVID-19 therapies. Furthermore, the assay can be employed to predict the progression of COVID-19 disease, as exemplified by the performed prediction of disease outcome weeks into the future. Such knowledge can help guide clinical decision making, and optimise hospital resource planning, specifically in critical situations of the pandemic that threatens the capacities of hospitals. [0233] Proteomics can be prognostic for outcome in patients with similar disease severity, e.g. among severely affected patients, that are difficult to distinguish by clinical parameters. This means that the prognosis of survival using targeted proteomics could be improved beyond what was shown in this study for ‘within-severity-group’ prognosis, if biomarker panels specifically for stratification within the respective COVID-19 severity group would be selected. Indeed, particularly within the group of severely affected individuals, some patients were predicted incorrectly, i.e. survived despite being predicted as non-survivors, or vice versa. We assessed on a patient-by-patient basis whether there are medical reasons that could explain wrong predictions. Plotting outcome with respect to the time until death denotes no clear tendency for the correct and false predictions. However, we noted that the three samples with the smallest decision function across wrongly predicted patients with fatal outcomes belonged to WHO 4 patients that had DNI (‘do not intubate’) orders in place. It is hence plausible that the assay correctly identified a milder form of COVID-19 in these three individuals; i.e. that without a strong comorbidity or a DNI order in place, these might have had a good chance to survive COVID-19. In a similar fashion, we recently reported two cases where the proteomic signatures of patients correctly distinguished an Influenza B from a COVID-19 infection, and that highlighted a patient that had to undergo chemotherapeutic cancer treatment just days before a COVID-19 infection. Thus, protein signatures could in principle distinguish different (respiratory) infections or comorbidities, while additional research will be required to establish this for the presented protein panel. COVID-19 will remain a central public health issue for the foreseeable future as new variants of concern with capacity to evade vaccine induced immunity continue to emerge. The underpinning targeted proteomics platform supports rapid iteration of the panel composition in case additional prognostic biomarkers are discovered. Taken together, this peptide panel and the underlying analytical platform, holds potential to support a broader, continuous pandemic response in addition to its utility in hospitalised patient cohorts which we demonstrate in the present study
[0234] Methods
[0235] Patient Cohorts
[0236] Patient samples were collected as described previously (13, 22, 23, 28) as part of a prospective observational cohort study Pa-COVID-19 at Charité—Universitätsmedizin Berlin. The study protocol has been described in detail before (Kurth et al., 2020). The study is registered in the German and the WHO international registry for clinical studies (DRKS00021688).
[0237] Reagents and Peptide Standards
[0238] Synthetic reference peptides were from Pepmic (Suzhou, China). Native peptides were synthesised at ≥95% purity and internal standard peptides—at ≥70% purity. Internal standards contained 4-6 amino acid tryptic tags mimicking the sequence in a corresponding human plasma protein and were labelled on C-terminal lysine (K) or arginine (R) amino acids with stable isotopes (K(U-13C6,15N2) or R(U-13C6,15N4)). Water was from Merck (LiChrosolv LC-MS grade; Cat #115333), acetonitrile was from Biosolve (LC-MS grade; Cat #012078), trypsin (Sequence grade; Cat #V511X) was from Promega, 1,4-Dithiothreitol (DTT; Cat #6908.2) from Carl-Roth, iodoacetamide (IAA; Bioultra; Cat #11149) and urea (puriss. P.a., reag. Ph. Eur.; Cat #33247) were from Sigma-Aldrich, ammonium bicarbonate (Eluent additive for LC-MS; Cat #40867) and Dimethyl sulfoxide (DMSO; Cat #41648) were from Fluka, formic acid (LC-MS Grade; Eluent additive for LC-MS; Cat #85178) was from Thermo Scientific™, bovine serum albumin (BSA) (Albumin Bovine Fraction V, Very Low Endotoxin, Fatty Acid-free; Cat #47299) was from Serva, commercial human plasma samples (Human Source Plasma, LOT #20CILP1034) was from zenbio.
[0239] All peptide stock solutions were prepared at 1 mg/ml concentration in 50:50 ddH2O: acetonitrile mix, except for STDYGIFQINSR (SEQ ID No: 22) and VEGTAFVIFGIQDGEQR (SEQ ID No: 51) where 200 μl of DMSO were added to solubilise the peptides at 5 mg/ml which were then diluted to small aliquot of 1 mg/ml with 50:50 ddH2O: acetonitrile mix before each sample preparation). Internal standard mix was prepared by pooling 20 μl of each heavy isotope-labeled peptide, evaporating 200 μl of this mix to dryness and reconstituting in a denaturation buffer to the final concentration of 1.4 μg/ml for each peptide. Cassetted calibration curves were prepared by serial dilution of pooled native reference peptide standards as described in Analytical method validation. After serial dilution, these samples were treated identically to respective clinical samples.
[0240] Peptide Selection
[0241] Peptides were selected based on discovery LC-MS/MS data from our prior studies (Messner et al., 2020; Demichev et al., 2021), where we identified a protein biomarker signature predictive of COVID-19 outcome. We selected a total of 50 proteotypic reference peptides from 30 human plasma proteins (1-2 peptides per protein) that had the highest predictive power in previous studies. The predictive performance in the following statistical tests was used for selection: (i) prediction of the remaining time in hospital for mild patients, (ii) prediction of future worsening for patients of all severity grades and (iii) stratification of patients of different severity grades. For each protein, only peptides that showed a good signal in the SWATH data as well as were predicted to be suitable for MRM by the Peptide Analyzing Tool (Thermo) were selected. Each native reference peptide was unique to a corresponding protein within the human proteome in a Uniprot BLAST search. Isotope-labelled internal standards were designed based on the selected native reference peptides.
[0242] Sample Preparation
[0243] Samples were prepared as described previously (Messner et al., 2020) with minor modifications. Briefly, clinical samples and calibration lines in Cohort 1 were prepared as follows: 5 μl of plasma or serum sample were added to 55 μl of denaturation buffer, composed of 5 μl 8M Urea, 100 mM ammonium bicarbonate, 5 μl 50 mM dithiothreitol (DTT) and peptide internal standard mix. The samples were incubated for 1 h at room temperature before addition of 5 μl of 100 mM iodoacetamide (IAA). After a 30 min incubation at room temperature the samples were diluted with 340 μl of 100 mM ammonium bicarbonate and digested overnight with 23 μl of 0.1 μg/μl trypsin at 37° C. The digestion was quenched by adding 50 μl of 10% v/v formic acid. The resulting tryptic peptides were purified on a 96-well C18-based solid phase extraction (SPE) plate (BioPureSPE Macro 96-well, 100 mg PROTO C18, The Nest Group). The purified samples were resuspended in 120 μl of 0.1% formic acid and 20 μl were injected into the LC-MS/MS system.
[0244] Samples in cohort 2 were prepared as described above, with one modification. As these samples had already been prepared for a discovery proteomics study, internal standards were digested separately and added to pre-digested clinical and calibration line samples before their injection into the LC-MS/MS system. Quality control (QC) samples consisted of pooled commercial control and COVID-19 human plasma samples (as described in a previous publication (Messner et al. 2020)), and were prepared alongside clinical and calibration curve samples in each cohort.
[0245] The COVID-19 sample pools used for the analytical validation were generated by pooling 5 μl of patient plasma from cohort 2 according to their WHO treatment severity score. Only samples of patients that had not received dexamethasone were used.
[0246] Liquid Chromatography-Tandem Mass Spectrometry
[0247] Tryptic peptides were quantified on 2 liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS) platforms—7500 (Sciex) and 6495c (Agilent).
[0248] 6495c (Agilent) LC-MS/MS Method
[0249] All clinical samples were analysed on the Agilent 6495c mass spectrometer, coupled to Agilent 1290 Infinity UHPLC system. Prior to MS analysis, samples were chromatographically separated on Agilent InfinityLab Poroshell 120 EC-C18 1.9 μm, 2.1×50 mm column heated to 45° C. and with a flow rate of 800 μl/min. Linear gradients employed were as follows (time, % of mobile phase B): 0 min, 3%; 1 min, 3%; 7.5 min, 35%; 8 min 98%; 8.5 min, 98%; 8.6 min, 3%; 10 min, 3% where mobile phase A & B are 0.1% formic acid in water and 0.1% formic acid in acetonitrile respectively.
[0250] The 6495c mass spectrometer was controlled by Agilent's MassHunter Workstation software (LC-MS/MS Data Acquisition for 6400 series Triple Quadrupole, Version 10.1) and was operated in positive electrospray ionisation mode with the following parameters: 3500 V capillary voltage (positive), 0 V nozzle voltage (positive), 12 L/min sheath gas flow at a temperature of 280° C., 17 L/min gas flow at a temperature of 170° C., 40 psi nebulizer gas flow, 166 V default fragmentor voltage, 5 V default cell accelerator potential. Samples were analysed in dynamic MRM mode with both quadrupoles operated in unit resolution. All other MRM parameters, including monitored transitions and scheduling are provided in the Table 6.
[0251] 7500 (Sciex) LC-MS/MS Method
[0252] Samples from the Kubler cohort (cohort 2) were analysed on a SCIEX 7500 mass spectrometer coupled to an ExionLC AD UHPLC system (SCIEX, UK) in addition to the analysis on the Agilent platform. Prior to MS analysis, samples were chromatographically separated on a Phenomenex Luna Omega Polar 3 μm, 100×2.1 mm column heated to 40° C. and with a flow rate of 500 μl/min. Linear gradients employed were as follows (time, % of mobile phase B): 0 min, 3%; 1 min, 3%; 7.5 min, 30%; 8 min 95%; 8.5 min, 95%; 8.6 min, 3%; 10 min, 3% where mobile phase A & B are 0.1% formic acid in water and 0.1% formic acid in acetonitrile respectively.
[0253] The 7500 triple quadrupole mass spectrometer was operated in positive electrospray ionisation mode with the following ion source parameters: 1750 V Ionspray voltage, 40 psi curtain gas, 40 psi Ion source gas 1, 70 psi ion source gas 2 and 500° C. temperature. Samples were analysed in MRM mode with both quadrupoles operated in unit resolution. All other MRM parameters, including monitored transitions and scheduling are provided in the Table 7.
[0254] Establishment of the MRM Based Assay
[0255] The assay was first set up on the 6495C (Agilent) system. Preliminary transitions for the 50 selected putative biomarkers (consisting of several precursor ion charge states and respective product ions) were predicted by Skyline v21.1.0.146. The native peptide standard solution was then infused into LC-MS/MS system and 1 precursor ion per peptide with the highest relative intensity and 5 most abundant product ions were selected for collision energy optimisation as provided by Skyline. From these 5 product ions, 2-5 experimentally optimised ion transitions per native peptide were ultimately selected in the panel based on the following criteria: i) highest relative signal intensity, ii) optimal chromatographic peak shape and iii) absence of interfering signals. Product ions of <300 m/z were excluded where possible to ensure specificity. Precursor and product ion-matched ISTD transitions were also included. Lastly, all selected transitions were combined into one scheduled MRM method, which was subsequently analytically validated. In the designed method, the most abundant transition for each peptide was used for quantification and 1-4 less abundant transitions were used as qualifiers (data not shown). For analytical cross-platform and cross-laboratory validation, the assay was set up on the 7500 (SCIEX) system in parallel following that approach (data not shown).
[0256] Mass Spectrometry Data Processing
[0257] Mass spectrometry data processing was performed with vendor-specific software: MassHunter Quantitative Analysis, v0.1, Agilent Technologies and SCIEX OS Software v2.0.1. Peak selection and integration were manually assessed before exporting the peak area values to .csv for further analysis. Peptide absolute concentration was determined from calibration curves, constructed with native and heavy isotope-labelled synthetic reference standards. Of note, SIL internal standards for 5 corresponding native peptides could not be detected on the 6495C system. To quantify these native peptides, we used other, closely eluting SIL internal standards in the assay: AADDTWEPFASGK(U-.sup.13C.sub.6,.sup.15N.sub.2 (SEQ ID No: 87) was used for ASDTAMYYCAR (SEQ ID No. 50), GYSIFSYATK(U-.sup.13C.sub.6,.sup.15N.sub.2) (SEQ ID No: 73) for GSPAINVAVHVFR (SEQ ID No: 34) and WEMPFDPQDTHQSR (SEQ ID No: 11), ANRPFLVFIR(U-.sup.13C.sub.6,.sup.15N.sub.4) (SEQ ID No: 90) for LAELPADALGPLQR (SEQ ID No: 49) and VSASPLLYTLIEK(U-.sup.13C.sub.6,.sup.15N.sub.2) (SEQ ID No: 97) for VEGTAFVIFGIQDGEQR (SEQ ID No: 51). In addition, due to low signal intensity of pre-assigned quantifier transitions (transitions with matched precursor and product ions across native and SIL peptides), we chose other transitions with higher signal intensity for 4 SIL peptides, even if they did not match the fragmentation pattern of their respective native peptides. The transitions used for quantification on both 6495C and 7500 LC-MS/MS platforms are shown in Tables 6 and 7 respectively. Linear regression analysis of each calibration curve was performed in Rstudio or Sciex OS (with 1/x weighting) and the respective peptide concentration in patient samples was expressed in ng/ml.
[0258] Analytical Method Validation
[0259] Method analytical validation was performed based on FDA Bioanalytical Method Validation criteria (42) where sensitivity, specificity, intra, inter-repeatability, accuracy and matrix effects have been assessed. A total of 5 independent calibration curves were prepared by serial dilution of native reference peptide standards in assay buffer (1), surrogate matrix (3) and pooled human plasma (1) across the final sample peptide concentration range of 0-1.63 μg/ml. Surrogate matrix (40 mg/ml BSA) calibration curves were prepared and analysed across 3 separate batches and all calibration curve samples were analysed in quintuplets. Linear 1/x weighted calibration curves were obtained for all native peptide reference standards in order to check the linearity of the response. Lower limit of quantification (LLOQ) was defined as the lowest calibration sample on the linear curve with a CV 20%. Ten peptides where low endogenous concentrations were observed in a pooled clinical subset of samples, LLOQ criteria were expanded to CV≤40% to prevent missing values where these peptides that were highly differentially expressed between COVID-19 severity groups were successfully quantified. Upper limit of quantification (ULOQ) was defined as the highest calibration sample on the linear curve with a CV≤20%. Accuracy was assessed by treating 1 of the 5 replicates in each calibration curve in the surrogate matrix as pseudo-unknown samples and quantifying with the curve generated from the remaining 4 replicates. The final accuracy was determined as the median of all calculated accuracy of each peptide. Matrix effects were measured by comparing the slopes of curves from calibration samples prepared in a BSA matrix and pooled human plasma. Here an Extra Sum of Square F test was used for statistical comparison with a p-value<0.05 indicating potential matrix effects.
[0260] Data Analysis and Visualisation, Statistics
[0261] Significance testing of the trend between absolute peptide concentrations and the ordinal classification as provided by the WHO treatment escalation scale (levels as indicated) was performed using Kendall's tau (KT) statistics as implemented in the “EnvStats v2.4.0” R package “kendallTrendTest” function. For cohort 1 the KT statistics was calculated as the trend of peptide quantities against the following WHO groups: 0, 3, 4, 5, 6, 7; for cohort 2, peptide quantities were tested against the following WHO groups: 3, 4, 5, 6, 7. A full summary of statistical test results is provided in Table 9. Multiple testing correction was performed by controlling for false discovery rate using the Benjamini-Hochberg procedure (43) as provided by the R package “stats v4.1.0”-“p.adjust” function. Principal component analysis was performed using the R function “prcomp” from the “stats 4.1.0” package and visualized using “ggplot2 v3.3.5”.
[0262] Prediction of WHO Grade and Disease Outcome
[0263] Clinical scores (CCI [39], SOFA [40], APACHE II [41], and ABCS [22]) were extracted from the clinical information system or, where missing, manually calculated. CCI and APACHE II were determined at time of admission, while SOFA was calculated for time of sampling. ABCS was calculated for admission and time of sampling. For ABCS, up to two missing laboratory values (either lymphocytes, blood urea nitrogen (BUN) or aspartate aminotransferase (ASAT)) were imputed by using the median value of patients within the same maximum WHO severity group. Note that due to imputation of the ABCS score memory leakage between training and test data for the ABCS score models can not be excluded. For the prediction of the current WHO grade and for the outcome prediction a Support Vector Machine was used as implemented in scikit-learn 0.23.2 (sklearn.svm.SVC) (44) using default parameters (rbf-kernel) and balanced class weights (class_weight=“balanced”). For one peptide (VSASPLLYTLIEK (SEQ ID No: 45)) negative values were present after external calibration. Those values were replaced by the minimal positive value of the respective peptide measured over all samples. Two peptides were removed (ASDTAMYYCAR (SEQ ID No: 50) and LVGGPMDASVEEEGVRR (SEQ ID No: 9)) as they were not reliably quantified leaving 48 peptides for the analysis. For every patient the first sample measured was selected (n=165). All patients with unknown WHO grade/outcome were neglected. All data were log 2-transformed and scaled to 0 mean and 1 variance fitted on the training data (sklearn.preprocessing.StandardScaler). The model was trained and validated using a shuffled stratified 5-fold cross-validation (sklearn.model_selection.StratifiedKFold) to assure that every split has a comparable case-to-control ratio and that every sample was used in 4 runs for training and in the remaining run for validating the trained model not including this sample. For reproducibility the seed was fixed to 42. For models trained on severity scores, only samples for which the respective score was determined were included in model construction and testing.
[0264] Decision function, ROC-Curve, accuracy, sensitivity and specificity were calculated using scikit-learn 0.23.2. For the Kaplan-Meier estimate lifelines 0.26.0 (45) was used. The data were divided in positive and negative predicted cases. For the true positive and false negative predicted cases, the days until death were included in the model. The samples for people who left the hospital alive were censored. Samples with missing time until outcome for patients who died were neglected. The confidence intervals were calculated using Greenwood's Exponential formula as implemented in lifelines 0.26.0 (alpha=0.05).
[0265] In addition, a predictor based on the extra-trees algorithm implemented in scikit-learn 0.23.2 (sklearn.ensemble.ExtraTreesClassifier) was evaluated. The same approach as described above was applied with the differences that the data weren't log 2-transformed and scaled as this isn't needed for a tree-based classifier. In addition, the maximal depth of the trees was set to 3 (max depth=3) to compensate for overfitting issues due to limited data set size.
REFERENCES
[0266] 1. Dong E, Du H, Gardner L. An interactive web-based dashboard to track COVID-19 in real time. Lancet Infect Dis. 2020; 20:533-4. [0267] 2. Pouwels K B, Pritchard E, Matthews P C, Stoesser N, Eyre D W, Vihta K-D, et al. Effect of Delta variant on viral burden and vaccine effectiveness against new SARS-CoV-2 infections in the U K. Nat Med [Internet]. 2021; Available from: http://dx.doi.org/10.1038/s41591-021-01548-7 [0268] 3. Kearns P, Siebert S, Willicombe M, Gaskell C, Kirkham A, Pirrie S, et al. Examining the Immunological Effects of COVID-19 Vaccination in Patients with Conditions Potentially Leading to Diminished Immune Response Capacity—The OCTAVE Trial [Internet]. 2021 [cited 2021 Nov. 16]. Available from: https://papers.ssrn.com/abstract=3910058 [0269] 4. Chemaitelly H, Tang P, Hasan M R, AlMukdad S, Yassine H M, Benslimane F M, et al. Waning of BNT162b2 Vaccine Protection against SARS-CoV-2 Infection in Qatar. N Engl J Med [Internet]. 2021; Available from: http://dx.doi.org/10.1056/NEJMoa2114114 [0270] 5. Levin E G, Lustig Y, Cohen C, Fluss R, Indenbaum V, Amit S, et al. Waning Immune Humoral Response to BNT162b2 Covid-19 Vaccine over 6 Months. N Engl J Med [Internet]. 2021; Available from: http://dx.doi.org/10.1056/NEJMoa2114583 [0271] 6. Bian L, Gao F, Zhang J, He Q, Mao Q, Xu M, et al. Effects of SARS-CoV-2 variants on vaccine efficacy and response strategies. Expert Rev Vaccines. 2021; 20:365-73. [0272] 7. Twohig K A, Nyberg T, Zaidi A, Thelwall S, Sinnathamby M A, Aliabadi S, et al. Hospital admission and emergency care attendance risk for SARS-CoV-2 delta (B.1.617.2) compared with alpha (B.1.1.7) variants of concern: a cohort study. Lancet Infect Dis [Internet]. 2021; Available from: http://dx.doi.org/10.1016/S1473-3099(21)00475-8 [0273] 8. Tao K, Tzou P L, Nouhin J, Gupta R K, de Oliveira T, Kosakovsky Pond S L, et al. The biological and clinical significance of emerging SARS-CoV-2 variants. Nat Rev Genet [Internet]. 2021; Available from: http://dx.doi.org/10.1038/s41576-021-00408-x [0274] 9. Overmyer K A, Shishkova E, Miller I J, Balnis J, Bernstein M N, Peters-Clarke™, et al. Large-Scale Multi-omic Analysis of COVID-19 Severity. Cell Syst. 2021; 12:23-40.e7. [0275] 10. D'Alessandro A, Thomas T, Dzieciatkowska M, Hill R C, Francis R O, Hudson K E, et al. Serum Proteomics in COVID-19 Patients: Altered Coagulation and Complement Status as a Function of IL-6 Level. J Proteome Res. 2020; 19:4417-27. [0276] 11. Vllmy F, van den Toorn H, Zenezini Chiozzi R, Zucchetti O, Papi A, Volta C A, et al. A serum proteome signature to predict mortality in severe COVID-19 patients. Life Sci Alliance [Internet]. 2021; 4. Available from: http://dx.doi.org/10.26508/lsa.202101099 [0277] 12. Couzin-Frankel J. The mystery of the pandemic's “happy hypoxia.” Science. 2020; 368:455-6. [0278] 13. Demichev V, Tober-Lau P, Lemke O, Nazarenko T, Thibeault C, Whitwell H, et al. A time-resolved proteomic and prognostic map of COVID-19. Cell Syst. 2021; 12:780-94.e7. [0279] 14. Vegvari C, Caust E, Hadjichrysanthou C, Lawrence E, Weverling G-J, de Wolf F, et al. Using Clinical Trial Simulators to Analyse the Sources of Variance in Clinical Trials of Novel Therapies for Acute Viral Infections. PLoS One. 2016; 11:e0156622. [0280] 15. Shen C, Ferro E G, Xu H, Kramer D B, Patell R, Kazi D S. Underperformance of Contemporary Phase III Oncology Trials and Strategies for Improvement. J Natl Compr Canc Netw. 2021; 19:1072-8. [0281] 16. Gutmann C, Takov K, Burnap S A, Singh B, Ali H, Theofilatos K, et al. SARS-CoV-2 RNAemia and proteomic trajectories inform prognostication in COVID-19 patients admitted to intensive care. Nat Commun. 2021; 12:3406. [0282] 17. Wynants L, Van Calster B, Collins G S, Riley R D, Heinze G, Schuit E, et al. Prediction models for diagnosis and prognosis of covid-19: systematic review and critical appraisal. BMJ. 2020; 369:m1328. [0283] 18. Gupta R K, Marks M, Samuels T H A, Luintel A, Rampling T, Chowdhury H, et al. Systematic evaluation and external validation of 22 prognostic models among hospitalised adults with COVID-19: an observational cohort study. Eur Respir J [Internet]. 2020; 56. Available from: http://dx.doi.org/10.1183/13993003.03498-2020 [0284] 19. Park J, Kim H, Kim S Y, Kim Y, Lee J-S, Dan K, et al. In-depth blood proteome profiling analysis revealed distinct functional characteristics of plasma proteins between severe and non-severe COVID-19 patients. Sci Rep. 2020; 10:22418. [0285] 20. Ignjatovic V, Geyer P E, Palaniappan K K, Chaaban J E, Omenn G S, Baker M S, et al. Mass Spectrometry-Based Plasma Proteomics: Considerations from Sample Collection to Achieving Translational Data. J Proteome Res. 2019; 18:4085-97. [0286] 21. Filbin M R, Mehta A, Schneider A M, Kays K R, Guess J R, Gentili M, et al. Plasma proteomics reveals tissue-specific cell death and mediators of cell-cell interactions in severe COVID-19 patients. bioRxiv [Internet]. 2020; Available from: http://dx.doi.org/10.1101/2020.11.02.365536 [0287] 22. Messner C B, Demichev V, Wendisch D, Michalick L, White M, Freiwald A, et al. Ultra-high-throughput clinical proteomics reveals classifiers of COVID-19 infection. Cell Systems [Internet]. 2020; Available from: http://www.sciencedirect.com/science/article/pii/S2405471220301976 [0288] 23. Messner C B, Demichev V, Bloomfield N, Yu J S L, White M, Kreidl M, et al. Ultra-fast proteomics with Scanning SWATH. Nat Biotechnol [Internet]. 2021; Available from: http://dx.doi.org/10.1038/s41587-021-00860-4 [0289] 24. Keevil B G. LC-MS/MS analysis of steroids in the clinical laboratory. Clin Biochem. 2016; 49:989-97. [0290] 25. Gaudl A, Kratzsch J, Ceglarek U. Advancement in steroid hormone analysis by LC-MS/MS in clinical routine diagnostics—A three year recap from serum cortisol to dried blood 17α-hydroxyprogesterone. J Steroid Biochem Mol Biol. 2019; 192:105389. [0291] 26. Ma S, Guo Q, Zhang Z, He Z, Yue A, Song Z, et al. Expanded newborn screening for inborn errors of metabolism by tandem mass spectrometry in newborns from Xinxiang city in China. J Clin Lab Anal. 2020; 34:e23159. [0292] 27. Zhu Z, Gu J, Genchev G Z, Cai X, Wang Y, Guo J, et al. Improving the Diagnosis of Phenylketonuria by Using a Machine Learning-Based Screening Model of Neonatal MRM Data. Front Mol Biosci. 2020; 7:115. [0293] 28. Demichev V, Tober-Lau P, Nazarenko T, Aulakh S K, Whitwell H, Lemke O, et al. A proteomic survival predictor for COVID-19 patients in intensive care [Internet]. bioRxiv. medRxiv; 2021. Available from: http://medrxiv.org/lookup/doi/10.1101/2021.06.24.21259374 [0294] 29. Shen B, Yi X, Sun Y, Bi X, Du J, Zhang C, et al. Proteomic and Metabolomic Characterization of COVID-19 Patient Sera. Cell. 2020; 182:59-72.e15. [0295] 30. MacLean B, Tomazela D M, Shulman N, Chambers M, Finney G L, Frewen B, et al. Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics. 2010; 26:966-8. [0296] 31. Jones B R, Schultz G A, Eckstein J A, Ackermann B L. Surrogate matrix and surrogate analyte approaches for definitive quantitation of endogenous biomolecules. Bioanalysis. 2012; 4:2343-56. [0297] 32. Pino L K, Searle B C, Yang H-Y, Hoofnagle A N, Noble W S, MacCoss M J. Matrix-Matched Calibration Curves for Assessing Analytical Figures of Merit in Quantitative Proteomics. J Proteome Res. 2020; 19:1147-53. [0298] 33. Hall T G, Smukste I, Bresciano K R, Wang Y, McKearn D, Savage R E. Identifying and overcoming matrix effects in drug discovery and development. Tandem mass spectrometry—applications and principles. Intech Open, London, U K; 2012; 18:390-419. [0299] 34. Xu R N, Fan L, Rieser M J, E I-Shourbagy T A. Recent advances in high-throughput quantitative bioanalysis by LC-MS/MS. J Pharm Biomed Anal. 2007; 44:342-55. [0300] 35. Gerber S A, Rush J, Stemman O, Kirschner M W, Gygi S P. Absolute quantification of proteins and phosphoproteins from cell lysates by tandem M S. Proc Natl Acad Sci USA. 2003; 100:6940-5. [0301] 36. Yang J J, Han Y, Mah C H, Wanjaya E, Peng B, Xu T F, et al. Streamlined MRM method transfer between instruments assisted with HRMS matching and retention-time prediction. Anal Chim Acta. 2020; 1100:88-96. [0302] 37. Anderson L, Hunter C L. Quantitative Mass Spectrometric Multiple Reaction Monitoring Assays for Major Plasma Proteins*. Mol Cell Proteomics. 2006; 5:573-88. [0303] 38. Fountoulakis M, Juranville J-F, Jiang L, Avila D, Roder D, Jakob P, et al. Depletion of the high-abundance plasma proteins. Amino Acids. 2004; 27:249-59. [0304] 39. Krieg L, Schaffert A, Kern M, Landgraf K, Wabitsch M, Beck-Sickinger A G, et al. An MRM-Based Multiplexed Quantification Assay for Human Adipokines and Apolipoproteins. Molecules [Internet]. 2020; 25. Available from: http://dx.doi.org/10.3390/molecules25040775 [0305] 40. Brioschi M, Gianazza E, Agostoni P, Zoanni B, Mallia A, Banfi C. Multiplexed MRM-Based Proteomics Identified Multiple Biomarkers of Disease Severity in Human Heart Failure. Int J Mol Sci [Internet]. 2021; 22. Available from: http://dx.doi.org/10.3390/ijms22020838 [0306] 41. World Health Organization [Internet]. R&D blueprint novel coronavirus COVID-19 therapeutic trial synopsis (WHO). 2020. Available from: https://www.who.int/teams/blueprint/covid-19 [0307] 42. Bioanalytical-Method-Validation-Guidance-for-Industry.pdf. Available from: https://www.fda.gov/files/drugs/published/Bioanalytical-Method-Validation-Guidance-for-Industry.pdf [0308] 43. Benjamini Y, Hochberg Y. Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing. J R Stat Soc Series B Stat Methodol. [Royal Statistical Society, Wiley]; 1995; 57:289-300. [0309] 44. Pedregosa F, Varoquaux G, Gramfort A, Michel V, Thirion B, Grisel O, et al. Scikit-learn: Machine Learning in Python. J Mach Learn Res. 2011; 12:2825-30. [0310] 45. Davidson-Pilon C. lifelines: survival analysis in Python. J Open Source Softw. The Open Journal; 2019; 4:1317.
[0311] Reference is made herein to the peptides and proteins of Table 1 which are as follows:
TABLE-US-00006 TABLE 1 Corresponding heavy Protein Name Protein Uniprot Corresponding reference isotope-labelled peptide Protein Name Abbreviation Accesion Number peptide sequence internal standard sequence Proteoglycan 4 PRG4 Q92954 GLPNVVTSAISLPNIR SILWRGLPNVVTSAISLPNIR(U-13C6, 15N4) (SEQ ID No.: 1) (SEQ ID No.: 53) Inter-alpha-trypsin ITIH1 P19827 GHMLENHVER (SEQ ID GHMLENHVER(U-13C6, 15N4)LWAYL inhibitor heavy No.: 2) (SEQ ID No.: 54) chain H1 Plasminogen, PLG P00747 EAQLPVIENK (SEQ ID AGLLKEAQLPVIENK(U-13C6, 15N2) EC 3.4.21.7 No.: 3) (SEQ ID No.: 55) Plasminogen, PLG P00747 CQSWSSMTPHR (SEQ ID CQSWSSMTPHR(U-13C6, 15N4)HQK EC 3.4.21.7 No.: 4) (SEQ ID No.: 56) Actin, aortic smooth ACTA2; P62736; EITALAPSTMK (SEQ ID MQKEITALAPSTMK(U-13C6, 15N2) muscle; Actin, ACTB; P60709; No.: 5) (SEQ ID No.: 57) cytoplasmic 1; ACTG1; P63261; Actin, cytoplasmic 2; ACTG2 P63267 Actin, gamma-enteric smooth muscle Complement C1q C1QC P02747 FNAVLTNPQGDYDTSTGK NSLIRFNAVLTNPQGDYDTSTGK(U-13C6, subcomponent (SEQ ID No.: 6) 15N2) (SEQ ID No.: 58) subunit C Complement C1q C1QC P02747 TNQVNSGGVLLR (SEQ ID TNQVNSGGVLLR(U-13C6, 15N4)LQVGE subcomponent No.: 7) (SEQ ID No.: 59) subunit C Cystatin-C CST3 P01034 ALDFAVGEYNK (SEQ ID ALDFAVGEYNK(U-13C6, 15N2)ASNDM No.: 8) (SEQ ID No.: 60) Cystatin-C CST3 P01034 LVGGPMDASVEEEGVRR LVGGPMDASVEEEGVR(U-13C6, (SEQ ID No.: 9) 15N4)RALDFA (SEQ ID No.: 61) Protein ORM2 ORM2 Q06144 SDVMYTDWK (SEQ ID No.: SDVMYTDWK(U-13C6, 15N2)KDK 10) (SEQ ID No.: 62) Alpha-1- SERPINA3 P01011 WEMPFDPQDTHQSR (SEQ ID WEMPFDPQDTHQSR(U-13C6, 15N4)FYLSK antichymotrypsin No.: 11) (SEQ ID No.: 63) Alpha-1- SERPINA3 P01011 EQLSLLDR (SEQ ID No.: 12) EQLSLLDR(U-13C6, 15N4)FTEDA antichymotrypsin (SEQ ID No.: 64) Serotransferrin TF P02787 GDVAFVK (SEQ ID No.: 13) GDVAFVK(U-13C6, 15N2)HSTIF (SEQ ID No.: 65) Serotransferrin TF P02787 WCALSHHER (SEQ ID No.: PVKWCALSHHER(U-13C6, 15N4) (SEQ ID 14) No.: 66) Apolipoprotein B-100 APOB P04114 IAELSATAQEIIK (SEQ ID IAELSATAQEIIK(U-13C6, 15N2)SQAIA No.: 15) (SEQ ID No.: 67) Apolipoprotein B-100 APOB P04114 EQHLFLPFSYK (SEQ ID No.: EAICKEQHLFLPFSYK(U-13C6, 15N2) 16) (SEQ ID No.: 68) EGF-containing fibulin- EFEMP1 Q12805 ADQVCINLR (SEQ ID ADQVCINLR(U-13C6, 15N4)GSFAC like extracellular No.: 17) (SEQ ID No.: 69) matrix protein 1 von Willebrand factor VWF P04275 ILTSDVFQDCNK (SEQ ID ILTSDVFQDCNK(U-13C6, 15N2)LVDPE No.: 18) (SEQ ID No.: 70) von Willebrand factor VWF P04275 YAGSQVASTSEVLK (SEQ ID YAGSQVASTSEVLK(U-13C6, 15N2)YTLFQ No.: 19) (SEQ ID No.: 71) C-reactive protein CRP P02741 ESDTSYVSLK (SEQ ID ESDTSYVSLK(U-13C6, 15N2)APLTK No.: 20) (SEQ ID No.: 72) C-reactive protein CRP P02741 GYSIFSYATK (SEQ ID GYSIFSYATK(U-13C6, 15N2)RQDNE No.: 21) (SEQ ID No.: 73) Lysozyme C, LYZ P61626 STDYGIFQINSR (SEQ ID STDYGIFQINSR(U-13C6, 15N4)YWCND EC 3.2.1.17 No.: 22) (SEQ ID No.: 74) Lysozyme C, LYZ P61626 YWCNDGK (SEQ ID No.: 23) YWCNDGK(U-13C6, 15N2)TPGAV EC 3.2.1.17 (SEQ ID No.: 75) Apolipoprotein A-I APOA1 P02647 AHVDALR (SEQ ID No.: 24) AHVDALR(U-13C6, 15N4)THLAP (SEQ ID No.: 76) Apolipoprotein A-I APOA1 P02647 ATEHLSTLSEK (SEQ ID EYHAKATEHLSTLSEK(U-13C6, 15N2) No.: 25) (SEQ ID No.: 77) Alpha-2-HS-glycoprotein AHSG P02765 TVVQPSVGAAAGPVVPPCPGR TVVQPSVGAAAGPVVPPCPGR(U-13C6, (SEQ ID No.: 26) 15N4)IR (SEQ ID No.: 78) Alpha-2-HS-glycoprotein AHSG P02765 CNLLAEK (SEQ ID No.: 27) CNLLAEK(U-13C6, 15N2)QYGFC (SEQ ID No.: 79) Histidine-rich HRG P04196 GGEGTGYFVDFSVR (SEQ ID GGEGTGYFVDFSVR(U-13C6, 15N4)NCPR glycoprotein No.: 28) (SEQ ID No.: 80) Histidine-rich HRG P04196 IADAHLDR (SEQ ID No.: 29) IADAHLDR(U-13C6, 15N4)VENTT glycoprotein (SEQ ID No.: 81) Beta-2-microglobulin B2M P61769 VEHSDLSFSK (SEQ ID No.: VEHSDLSFSK(U-13C6, 15N2)DWSFY 30) (SEQ ID No.: 82) Beta-2-microglobulin B2M P61769 VNHVTLSQPK (SEQ ID No.: EYACRVNHVTLSQPK(U-13C6, 15N2) 31) (SEQ ID No.: 83) N-acetylmuramoyl-L- PGLYRP2 Q96PD5 GCPDVQASLPDAK (SEQ ID PDATKGCPDVQASLPDAK(U-13C6, 15N2) alanine amidase, EC No.: 32) (SEQ ID No.: 84) 3.5.1.28 N-acetylmuramoyl-L- PGLYRP2 Q96PD5 TFTLLDPK (SEQ ID TFTLLDPK(U-13C6, 15N2)ASLLT alanine amidase, EC No.: 33) (SEQ ID No.: 85) 3.5.1.28 Transthyretin TTR P02766 GSPAINVAVHVFR (SEQ ID GSPAINVAVHVFR(U-13C6, 15N4)KAADD No.: 34) (SEQ ID No.: 86) Transthyretin TTR P02766 AADDTWEPFASGK (SEQ ID AADDTWEPFASGK(U-13C6, 15N2)TSESG No.: 35) (SEQ ID No.: 87) Plasma kallikrein, EC KLKB1 P03952 DSVTGTLPK (SEQ ID GCFLKDSVTGTLPK(U-13C6, 15N2) 3.4.21.34 No.: 36) (SEQ ID No.: 88) Plasma kallikrein, EC KLKB1 P03952 IAYGTQGSSGYSLR (SEQ ID IAYGTQGSSGYSLR(U-13C6, 15N4)LCNTG 3.4.21.34 No.: 37) (SEQ ID No.: 89) Antithrombin-III SERPINC1 P01008 ANRPFLVFIR (SEQ ID ANRPFLVFIR(U-13C6, 15N4)EVPLN No.: 38) (SEQ ID No.: 90) Heparin cofactor 2 SERPIND1 P05546 TSCLLFMGR (SEQ ID TSCLLFMGR(U-13C6, 15N4)VANPS No.: 39) (SEQ ID No.: 91) Afamin AFM P43652 HFQNLGK (SEQ ID QQECKHFQNLGK(U-13C6, 15N2) No.: 40) (SEQ ID No.: 92) Afamin AFM P43652 TINPAVDHCCK (SEQ ID TINPAVDHCCK(U-13C6, 15N2)TNFAF No.: 41) (SEQ ID No.: 93) Plasma protease C1 SERPING1 P05155 LLDSLPSDTR (SEQ ID LLDSLPSDTR(U-13C6, 15N4)LVLLN inhibitor No.: 42) (SEQ ID No.: 94) Plasma protease C1 SERPING1 P05155 LVLLNAIYLSAK (SEQ ID PSDTRLVLLNAIYLSAK(U-13C6, 15N2) inhibitor No.: 43) (SEQ ID No.: 95) Transferrin receptor TFRC P02786 ILNIFGVIK (SEQ ID ILNIFGVIK(U-13C6, 15N2)GFVEP protein 1 No.: 44) (SEQ ID No.: 96) Transferrin receptor TFRC P02786 VSASPLLYTLIEK (SEQ ID VSASPLLYTLIEK(U-13C6, 15N2)TMQNV protein 1 No.: 45) (SEQ ID No.: 97) Low affinity FCGR3A P08637 DSGSYFCR (SEQ ID DSGSYFCR(U-13C6, 15N4)GLFGS immunoglobulin gamma No.: 46) (SEQ ID No.: 98) Fc region receptor III-A Monocyte differentiation CD14 P08571 VLDLSCNR (SEQ ID VLDLSCNR(U-13C6, 15N4)LNR antigen CD14 No.: 47) (SEQ ID No.: 99) Insulin-like growth IGFALS P35858 DFALQNPSAVPR (SEQ ID DFALQNPSAVPR(U-13C6, 15N4)FVQAI factor-binding No.: 48) (SEQ ID No.: 100) protein complex acid labile subunit Insulin-like growth IGFALS P35858 LAELPADALGPLQR (SEQ ID LAELPADALGPLQR(U-13C6, 15N4)AFWLD factor-binding No.: 49) (SEQ ID No.: 101) protein complex acid labile subunit Immunoglobulin heavy IGHV5-51 A0A0C4DH38 ASDTAMYYCAR (SEQ ID WSSLKASDTAMYYCAR(U-13C6, 15N4) variable 5-51 No.: 50) (SEQ ID No.: 102) Complement C3 C3 P01024 VEGTAFVIFGIQDGEQR (SEQ VEGTAFVIFGIQDGEQR(U-13C6, 15N4)ISLPE ID No.: 51) (SEQ ID No.: 103) Complement C3 C3 P01024 VHQYFNVELIQPGAVK (SEQ VHQYFNVELIQPGAVK(U-13C6, 15N2)VYAYY ID No.: 52) (SEQ ID No.: 104)
[0312] Table 2 shows experimentally optimised targeted LC-MS/MS conditions to monitor native peptides listed in Table 1. The data in Table 2 is based on AB Sciex triple quadruple LC-MS/MS platforms but is equally transferable to platforms manufactured by other companies. In Table 2, the following abbreviations are used: EP—Entrance Potential, CE—Collision Energy, CXP—Collision Cell Exit Potential, DP—Declustering Potential, and RT—retention time.
TABLE-US-00007 TABLE 2 Native Precursor Product Dwell Peptide (Q1) (Q3) time RT Sequence m/z (ms) (ms) EP CE CXP DP (min) VEHSDLSFSK 383.52 581.33 3.5 10 19 25 120 3.44 SEQ ID No.: 30) 383.52 468.25 3.5 10 19 25 120 3.44 VNHVTLSQPK 374.88 673.39 3.5 10 18.6 25 120 3.08 (SEQ ID No.: 31) 374.88 572.34 3.5 10 18.6 25 120 3.08 374.88 459.26 3.5 10 18.6 25 120 3.08 GLPNVVTSAISLPNIR 825.98 1170.68 3.5 10 43.3 25 176 6.58 (SEQ ID No.: 1) 825.98 1071.62 3.5 10 43.3 25 176 6.58 825.98 499.30 3.5 10 31.3 25 176 6.58 GHMLENHVER 407.86 783.37 3.5 10 20.2 25 132 2.9 (SEQ ID No.: 2) 407.86 654.33 3.5 10 24.2 25 132 2.9 407.86 403.23 3.5 10 24.2 25 132 2.9 EAQLPVIENK 570.82 699.40 3.5 10 26.5 25 142 4.38 (SEQ ID No.: 3) 570.82 503.28 3.5 10 34.5 25 142 4.38 570.82 390.20 3.5 10 34.5 25 142 4.38 CQSQSSMTPHR 459.53 815.38 3.5 10 22.6 25 124 3.81 (SEQ ID NO.: 4) 459.53 510.28 3.5 10 22.6 25 124 3.81 459.23 406.23 3.5 10 22.6 25 124 3.81 EITALAPSTMK 581.31 747.41 3.5 10 27.1 25 140 4.49 (SEQ ID No.: 5) 581.31 634.32 3.5 10 23.1 25 140 4.49 581.31 563.29 3.5 10 23.1 25 140 4.49 FNAVLTNPQGDYDTSTGK 643.31 1168.51 3.5 10 46.2 25 142 4.67 (SEQ ID No.: 6) 643.31 584.76 3.5 10 54.2 25 142 4.67 643.31 493.26 3.5 10 58.2 25 142 4.67 TNQVNSGGVLLR 629.35 914.54 3.5 10 33.5 25 152 4.16 (SEQ ID No.: 7) 629.35 815.47 3.5 10 33.5 25 152 4.16 629.35 701.43 3.5 10 33.5 25 152 4.16 ALDFAVGEYNK 613.81 610.28 3.5 10 24..7 25 140 5.05 (SEQ ID No.: 8) 613.81 709.35 3.5 10 28.7 25 140 5.05 613.81 610.28 3.5 10 24.7 25 140 5.05 LVGGPMDASVEEEGVRR 600.97 745.40 3.5 10 37.2 25 142 4.38 (SEQ ID No.: 9) 600.97 794.87 3.5 10 25.2 25 142 4.38 600.97 566.29 3.5 10 37.2 25 142 4.38 SDVMYTDWK 572.75 843.37 3.5 10 26.6 25 140 4.65 (SEQ ID No.: 10) 572.75 712.33 3.5 10 26.6 25 140 4.65 572.75 549.27 3.5 10 26.6 25 140 4.65 WEMPFDPQDTHQSR 591.93 968.45 3.5 10 32.8 25 138 5.34 (SEQ ID No.: 11) 591.93 628.32 3.5 10 40.8 25 138 5.34 591.93 729.83 3.5 10 24.8 25 138 5.34 EQLSLLDR 487.27 603.35 3.5 10 22.4 25 134 4.69 (SEQ ID No.: 12) 487.27 603.35 3.5 10 22.4 25 134 4.69 487.27 403.23 3.5 10 22.4 25 134 4.69 GDVAFK 368.21 563.36 3.5 10 16.4 25 112 3.69 (SEQ ID No.: 13) 368.21 464.29 3.5 10 16.4 25 112 3.69 368.21 393.25 3.5 10 20.4 25 112 3.69 WCALSHHER 399.19 665.31 3.5 10 23.8 25 118 3.16 (SEQ ID No.: 14) 399.19 578.28 3.5 10 27.8 25 118 3.16 399.19 441.22 3.5 10 27.8 25 118 3.16 IAELSATAQEIIK 693.90 960.54 3.5 10 32.7 25 164 5.32 (SEQ ID No.: 15) 693.90 873.50 3.5 10 32.7 25 164 5.32 693.90 802.47 3.5 10 32.7 25 164 5.32 EQHLFLPFSYK 470.25 641.33 3.5 10 19.1 25 120 5.97 (SEQ ID No. 16) 470.25 544.28 3.5 10 23.1 25 10 5.97 470.25 397.21 3.5 10 27.1 25 120 5.97 ADQVCINLR 544.78 774.43 3.5 10 29.2 25 134 4.32 (SEQ ID No.: 17) 544.78 675.36 3.5 10 29.2 25 134 4.32 544.78 515.33 3.5 10 29.2 25 134 4.32 ILTSDVFQDCNK 720.35 811.34 3.5 10 34 25 162 4.74 (SEQ ID No.: 18) 720.35 664.27 3.5 10 34 25 162 4.74 720.35 663.80 3.5 10 42 25 162 4.74 ESDTSYVSLK 564.77 696.39 3.5 10 26.2 25 144 3.89 (SEQ ID No.: 20) 564.77 609.36 3.5 10 26.2 25 144 3.89 564.77 446.30 3.5 10 26.2 25 144 3.89 GYSIFSYATK 568.78 716.36 3.5 10 26.4 25 136 5.4 (SEQ ID No.: 21) 568.78 569.29 3.5 10 18.4 25 136 5.4 568.78 482.26 3.5 10 38.4 25 136 5.4 DTSYGIFQINSR 700.84 934.51 3.5 10 37 25 86 5.37 (SEQ ID No.: 22) 700.84 764.40 3.5 10 33 25 86 5.37 700.84 617.34 3.5 10 33 25 86 5.37 YAGSQVASTSEVLK 720.37 933.53 3.5 10 38 25 172 4.11 (SEQ ID No.: 19) 720.37 834.46 3.5 10 38 25 172 4.11 720.37 763.42 3.5 10 38 25 172 4.11 YWCNDGK 471.69 779.31 3.5 10 21.6 25 132 2.88 (SEQ ID No.: 23) 471.69 593.23 3.5 10 21.6 25 132 2.88 471.69 433.20 3.5 10 25.6 25 132 2.88 AHVDALR 391.22 573.34 3.5 10 21.6 25 132 2.39 (SEQ ID No.: 24) 391.22 474.27 3.5 10 25.6 25 132 2.39 391.22 359.24 3.5 10 29.6 25 132 2.39 ATEHLSTLSEK 405.88 777.44 3.5 10 24.1 25 120 3.11 (SEQ ID No.: 25) 405.88 664.35 3.5 10 24.1 25 120 3.11 405.88 363.13 3.5 10 24.1 25 120 3.11 TVVQPSVGAAAGPVVPPCPGR 672.69 978.52 3.5 10 24.6 25 146 4.96 (SEQ ID No.: 26) 672.69 683.33 3.5 10 28.6 25 146 4.96 672.69 586.28 3.5 10 40.6 25 146 4.69 GGEGTGYFVDFSVR 745.85 1089.54 3.5 10 39.3 25 172 5.76 (SEQ ID No.: 28) 745.85 869.45 3.5 10 39.3 25 172 5.76 745.85 508.29 3.5 10 39.3 25 172 5.76 IADAHLDR 455.74 797.39 3.5 10 24.8 25 144 2.69 (SEQ ID No. 29) 455.74 726.35 3.5 10 24.8 25 144 2.69 455.74 611.33 3.5 10 28.58 25 144 2.69 CNLLAEK 424.22 687.40 3.5 10 23.2 25 128 3.55 (SEQ IS No.: 27) 424.22 573.36 3.5 10 23.2 25 128 3.55 424.22 460.28 3.5 10 23.2 25 128 3.55 GCPDVQASLPDAK 453.22 630.35 3.5 10 28 25 120 4.06 (SEQ ID No.: 32) 453.22 430.23 3.5 10 32 25 120 4.06 453.22 430.14 3.5 10 44 25 120 4.06 TFTLLDPK 467.77 686.41 3.5 10 21.4 25 132 5.26 (SEQ ID No.: 33) 467.77 585.36 3.5 10 21.4 25 132 5.26 467.77 472.28 3.5 10 21.4 25 132 5.26 GSPAINVAVHVFR 456.26 728.42 3.5 10 26.4 25 122 5.4 (SEQ ID No.: 34) 456.26 558.31 3.5 10 18.4 25 122 5.4 456.26 611.86 3.5 10 18.4 25 122 5.4 AADDTWEPFASGK 697.81 921.45 3.5 10 32.9 25 156 5.04 (SEQ ID No.: 35) 697.81 735.37 3.5 10 32.9 25 156 5.04 697.81 606.32 3.5 10 40.9 25 156 5.04 DSVTGTLPK 459.25 616.37 3.5 10 21 25 128 3.56 (SEQ ID No.: 36) 459.25 515.32 3.5 10 21 25 128 3.56 459.25 357.25 3.5 10 29 25 128 3.56 IATGTQGSSGYSLR 730.36 1112.53 3.5 10 38.5 25 166 4.19 (SEQ ID No.: 37) 730.36 954.46 3.5 10 38.5 25 166 4.19 730.36 826.41 3.5 10 42.5 25 166 4.19 ANRPFLVFIR 411.58 647.42 3.5 10 20.3 25 190 6 (SEQ ID No.: 38) 411.58 534.34 3.5 10 20.3 25 190 6 411.58 435.27 3.5 10 20.3 25 190 6 TSCLLFMGR 542.77 736.42 3.5 10 29.1 25 134 5.48 (SEQ ID No.: 39) 542.77 623.33 3.5 10 29.1 25 134 5.48 542.77 510.25 3.5 10 25.1 25 134 5.48 HFQNLGK 422.23 706.39 3.5 10 23.1 25 126 2.54 (SEQ ID No.: 40) 422.23 559.32 3.5 10 23.1 25 126 2.54 422.23 431.26 3.5 10 27.1 25 126 2.54 TINPAVDHCCK 438.87 719.26 3.5 10 25.6 25 120 2.92 (SEQ ID No.: 41) 438.87 604.23 3.5 10 25.6 25 120 2.92 438.87 467.17 3.5 10 33.6 25 120 2.92 LLDSLPSDTR 558.80 890.42 3.5 10 25.9 25 140 4.38 (SEQ ID No.: 42) 558.80 575.28 3.5 10 25.9 25 140 4.38 558.80 575.28 3.5 10 25.9 25 140 4.38 LVLLNAIYLSAK 659.41 879.49 3.5 10 31 25 148 6.82 (SEQ ID No.: 43) 659.41 765.45 3.5 10 31 25 148 6.82 659.41 694.41 3.5 10 43 25 148 6.82 ILNIFGVIK 508.83 790.48 3.5 10 23.4 25 132 6.8 (SEQ ID No.: 44) 508.83 676.44 3.5 10 23.4 25 132 6.8 508.83 563.36 3.5 10 23.4 25 132 6.8 VSASPLLYTLIEK 717.42 1089.66 3.5 10 29.9 25 156 6.66 (SEQ ID No.: 45) 717.42 879.52 3.5 10 37.9 25 156 6.66 717.42 766.43 3.5 10 33.9 25 156 6.66 DGSYFCR 496.20 789.33 3.5 10 22.8 25 84 3.58 (SEQ ID No.: 46) 496.20 645.28 3.5 10 22.8 25 84 3.58 496.20 482.22 3.5 10 22.8 25 84 3.58 VLDLSCNR 488.75 764.34 3.5 10 22.4 25 128 3.8 (SEQ ID No.: 47) 488.75 649.31 3.5 10 26.4 25 128 3.8 488.75 536.22 3.5 10 22.4 25 128 3.8 DFALQNPSAVPR 657.84 868.46 3.5 10 34.9 25 110 4.84 (SEQ ID No.: 48) 657.84 740.40 3.5 10 30.9 25 110 4.84 657.84 626.36 3.5 10 34.9 25 110 4.84 LAELPADALGLQR 732.41 10.37.57 3.5 10 34.6 25 88 5.77 (SEQ ID No.: 49) 732.41 754.46 3.5 10 50.6 25 88 5.77 732.41 570.34 3.5 10 34.6 25 88 5.77 ASDTAMYYCAR 654.77 934.39 3.5 10 34.7 25 148 5.18 (SEQ ID No.: 50) 654.77 863.35 3.5 10 30.7 25 148 5.18 654.77 732.31 3.5 10 30.7 25 148 5.18 VEGTAFVIFGIQDGEQR 622.65 732.33 3.5 10 26.3 25 142 6.46 (SEQ ID No.: 51) 622.65 604.27 3.55 10 30.3 25 142 6.46 622.65 525.25 3.5 10 22.3 25 142 6.46 VHQYFNVELIQPGAVK 614.67 712.44 3.5 10 21.9 25 132 5.7 (SEQ ID No.: 52) 614.67 471.29 3.5 10 33.9 25 132 5.7 614.67 509.24 3.5 10 21.9 25 132 5.7
[0313] Table 3 shows experimentally optimised targeted LC-MS/MS conditions to monitor heavy isotope-labelled peptide internal standards listed in Table 1. This is based on AB Sciex triple quadruple LC-MS/MS platforms.
TABLE-US-00008 TABLE 3 Internal standard Precusor Product Dwell RT peptide sequence (Q1) m/z (Q3) m/z time (ms) EP CE CXP DP (min) VEHSDLSFSK(U-13C6, 386.20 589.34 3.5 10 19 25 120 3.44 15N2)DWSFY 386.20 476.25 3.5 10 19 25 120 3.44 (SEQ ID No.: 82) EYACRVNHVTLSQPK 377.55 580.35 3.5 10 16.8 25 120 3.08 (U-13C6, 15N2) 377.55 467.27 3.5 10 18.6 25 120 3.08 (SEQ ID No.: 83) SILBRGLPNVVTSAISLPNIR 830.98 1180.69 3.5 10 43.3 25 176 6.58 (U-13C6, 15N4) 830.98 1081.62 3.5 10 43.3 25 176 6.58 (SEQ ID No.: 53) GHMLENHVER 411.20 493.38 3.5 10 20.2 25 132 2.9 (U-13C6, 15N4)LWAYL 411.20 413.23 3.5 10 24.2 25 132 2.9 (SEQ ID No.: 54) AGLLKEAQLPVIENK 574.84 707.41 3.5 10 26.5 25 142 4.38 (U-13C6, 15N4) 574.84 511.30 3.5 10 34.5 25 142 4.38 (SEQ ID No.: 55) CQSWSSMTPHR 462.86 520.28 3.5 10 22.6 25 124 3.81 (U-13C6, 15N4)HQK 462.86 419.23 3.5 10 22.6 25 124 3.81 (SEQ ID No.: 56) MQKEITALAPSTMK 585.31 755.42 3.5 10 27.1 25 140 4.49 (U-13C6, 15N4) 585.31 642.33 3.5 10 23.1 25 140 4.49 (SEQ ID No.: 57) NSLIRFNAVLTNPQGDYDTSTGK 645.97 1176.52 3.5 10 46.2 25 142 4.67 (U-13C6, 15N4) 645.97 588.76 3.5 10 54.2 25 142 4.67 (SEQ ID No.: 58) TNQVNSGGVLLR 634.35 924.55 3.5 10 33.5 25 152 4.16 (U-13C6, 15N4)LQVGE 634.35 825.48 3.5 10 33.5 25 152 4.16 (SEQ ID No.: 59) ALDFAVGEYNK 617.81 717.36 3.5 10 28.7 25 140 5.05 (U-13C6, 15N4)ASNDM 617.81 618.29 3.5 10 24.7 25 140 5.05 (SEQ ID No.: 60) LVGGPMDASVEEEGVR 604.30 799.88 3.5 10 25.2 25 142 4.38 (U-13C6, 15N4)RALDFA 604.30 571.30 3.5 10 37.2 25 142 4.38 (SEQ ID No.: SDVMYTDWK 576.75 851.38 3.5 10 26.6 25 140 4.65 (U-13C6, 15N4)KDK 576.75 720.34 3.5 10 26.6 25 140 4.65 (SEQ ID No.: WEMPFDPQDTHQSR 595.26 978.46 3.5 10 32.8 25 138 5.34 (U-13C6, 15N4)FYLSK 595.26 734.82 3.5 10 24.8 25 138 5.34 (SEQ ID No.: 63) EQLSLLDR 492.27 726.43 3.5 10 22.4 25 134 4.69 FTEDA 492.27 613.35 3.5 10 22.4 25 134 4.69 (SEQ ID No.: 64) GDVAFU 372.21 571.36 3.5 10 16.4 25 112 3.69 (U-13C6, 15N4)HSTIF 372.21 472.30 3.5 10 16.4 25 112 3.69 (SEQ ID No.: 65) PVKWCALSHHER 402.52 675.31 3.5 10 23.8 25 118 3.16 (U-13C6, 15N4) (SEQ ID No.: 66) 402.52 451.22 3.5 10 27.8 25 118 3.16 IAELSATAQEIIK 697.90 968.55 3.5 10 32.7 25 164 5.32 (U-13C6, 15N4)SQAIA 697.90 810.48 3.5 10 32.7 25 164 5.32 (SEQ ID No.: 67) EAICKEQHLFLPFSYK 472.91 649.34 3.5 10 19.1 25 120 5.97 (U-13C6, 15N4) (SEQ ID No.: 68) 472.91 552.29 3.5 10 23.1 25 120 5.97 ADQVCINLR 549.78 784.43 3.5 10 29.2 25 134 4.32 (U-13C6, 15N4)GSFAC 549.78 685.36 3.5 10 29.2 25 134 4.32 (SEQ ID No.: 69) ILTSDVFQDCNK 724.35 819.35 3.5 10 34 25 162 4.74 (U-13C6, 15N4)LVDPE 724.35 677.81 3.5 10 42 25 162 4.74 (SEQ ID No.: 70) ESDTSYVSLK 568.78 704.40 3.5 10 26.2 25 144 3.89 (U-13C6, 15N4)APLTK 568.78 617.37 3.5 10 26.2 25 144 3.89 (SEQ ID No.: 72) GYSIFSYATK 572.79 724.37 3.5 10 26.4 25 136 5.4 (U-13C6, 15N4)RQDNE 572.79 577.30 3.5 10 18.4 25 136 5.4 (SEQ ID No.: 73) STDYGIFQINSR 705.84 944.51 3.5 10 37 25 86 5.37 (U-13C6, 15N4)YWCND 705.84 774.41 3.5 10 33 25 86 5.37 (SEQ ID No.: 74) YAGSQVASTSEVLK 724.37 941.53 3.5 10 38 25 172 4.11 (U-13C6, 15N4)YTLFQ 724.37 842.47 3.5 10 38 25 172 4.11 (SEQ ID No.: 71) YWCNDGK 475.70 787.32 3.5 10 21.6 25 132 2.88 (U-13C6, 15N4)TPGAV 475.70 601.24 3.5 10 21.6 25 132 2.88 (SEQ ID No.: 75) AHVDAR 396.22 583.34 3.5 10 21.6 25 132 2.39 (U-13C6, 15N4)THLAP 396.22 484.27 3.5 10 25.6 25 132 2.39 (SEQ ID No.: 76) EYHAKATEHLSTLSEK 408.55 672.36 3.5 10 24.1 25 120 3.11 (U-13C6, 15N4) (SEQ ID No.: 77) TVVQPSVGAAAGPVVPPCPGR 676.03 988.52 3.5 10 24.6 25 146 4.96 (U-13C6, 15N4)IR 676.03 693.33 3.5 10 28.6 25 146 4.96 (SEQ ID No.: 78) GGEGTGYFVDFSVR 750.86 1099.54 3.5 10 39.3 25 172 5.76 (U-13C6, 15N4)NCPR 750.86 879.45 3.5 10 39.3 25 172 5.76 (SEQ ID No.: 80) IADAHLDR 460.74 807.40 3.5 10 24.8 25 144 2.69 (U-13C6, 15N4)VENTT 460.74 736.36 3.5 10 24.8 25 144 2.69 (SEQ ID No.: 81) CNLLAEK 428.22 581.37 3.5 10 23.2 25 128 3.55 (U-13C6, 15N4)QYGFC 428.22 468.30 3.5 10 23.2 25 128 3.55 (SEQ ID No.: 79) PDATKGCPDVQASLPDAK 455.89 638.35 3.5 10 28 25 120 4.06 (U-13C6, 15N4) 455.89 438.24 3.5 10 32 25 120 4.06 (SEQ ID No.: 84) TFTLLDPK 471.77 694.42 3.5 10 21.4 25 132 5.26 (U-13C6, 15N4)ASLLT 471.77 593.37 3.5 10 21.4 25 132 5.26 (SEQ ID No.: 85) GSPAINVAVHVFR 459.59 738.42 3.5 10 26.4 25 122 5.4 (U-13C6, 15N4)KAADD 459.59 616.86 3.5 10 18.4 25 122 5.4 (SEQ ID No.: 86) AADDTWEPFASGK 701.82 929.46 3.5 10 32.9 25 156 5.04 (U-13C6, 15N4)TSESG 701.82 614.33 3.5 10 40.9 25 156 5.04 (SEQ ID No.: 87) GCFLKDSVTGTLPK 463.25 624.38 3.5 10 21 25 128 3.56 (U-13C6, 15N4) 463.25 523.33 3.5 10 21 25 128 3.56 (SEQ ID No.: 88) IAYGTQGSSGYSLR 735.36 1122.54 3.5 10 38.5 25 166 4.19 (U-13C6, 15N4)LCNTG 735.36 836.41 3.5 10 42.5 25 166 4.19 (SEQ ID No.: 89) ANRPFLVFIR 414.91 544.34 3.5 10 20.3 25 190 6 (U-13C6, 15N4)EVPLN 414.91 445.27 3.5 10 20.3 25 190 6 (SEQ ID No.: 90) TSCLLFMGR 547.77 633.34 3.5 10 29.1 25 134 5.48 (U-13C6, 15N4)VANPS 547.77 520.25 3.5 10 25.1 25 134 5.48 (SEQ ID No.: 91) QQECKHFQNLGK 426.23 714.40 3.5 10 23.1 25 126 2.54 (U-13C6, 15N4) 426.23 567.33 3.5 10 23.1 25 126 2.54 (SEQ ID No.: 92) TINPAVDHCCK 441.54 727.27 3.5 10 25.6 25 120 2.92 (U-13C6, 15N4)TNFAF 441.54 612.24 3.5 10 25.6 25 120 2.92 (SEQ ID No.: 93) LLDSLPSDTR 563.80 900.42 3.5 10 25.9 25 140 4.38 (U-13C6, 15N4)LVLLN 563.80 585.28 3.5 10 25.9 25 140 4.38 (SEQ ID No.: 94) PSDTRLVLLNAIYLSAK 663.41 887.50 3.5 10 31 25 148 6.82 (U-13C6, 15N4) 663.42 773.46 3.55 10 31 25 148 6.82 (SEQ ID No.: 95) ILNIFGVIK 512.83 798.49 3.5 10 23.4 25 132 6.8 (U-13C6, 15N4)GFVEP 512.83 571.36 3.5 10 23.4 25 132 6.8 (SEQ ID No.: 96) VSASPLLTYLIEK 721.42 1097.66 3.5 10 29.9 25 156 6.66 (U-13C6, 15N4)TMQNV 721.42 774.44 3.5 10 33.9 25 1556 6.66 (SEQ ID No.: 97) DSGSYFCR 501.20 799.34 3.5 10 22.8 25 84 3.58 (U-13C6, 15N4)GLFGS 501.20 655.28 3.5 10 22.8 25 84 3.58 (SEQ ID No.: 98) VLDLSCNR 493.75 774.34 3.5 10 22.4 25 128 3.8 (U-13C6, 15N4)LNR 493.75 546.23 3.5 10 22.4 25 128 3.8 (SEQ ID No.: 99) DFALQNPSAVPR 662.84 750.41 3.5 10 30.9 25 110 4.84 (U-13C6, 15N4)FVQAI 662.84 636.37 3.5 10 34.9 25 110 4.84 (SEQ ID No.: 100) LAELPADALGPLQR 737.41 1047.52 3.5 10 34.6 25 88 5.77 (U-13C6, 15N4)AFWLD 737.41 764.46 3.5 10 50.6 25 88 5.77 (SEQ ID No.: 101) WSSLKASDTAMYYCAR 659.78 873.36 3.5 10 30.7 25 148 5.18 (U-13C6, 15N4) 659.78 742.32 3.5 10 30.7 25 148 5.18 (SEQ ID No.: 102) VEGTAFVIFGIQDGEQR 625.98 742.33 3.5 10 26.3 25 142 6.46 (U-13C6, 15N4)ISLPE 625.98 530.25 3.5 10 22.3 25 142 6.46 (SEQ ID No.: 103) VHQYFNVELIQPGAVK 617.33 720.44 3.5 10 21.9 25 132 5.7 (U-13C6, 15N4)VYAYY 617.33 479.30 3.5 10 33.9 25 132 5.7 (SEQ ID No.: 104)
[0314] Table 4 shows peptides in the targeted LC-MS/MS assay ranked by ANOVA p-value. Top 17 peptides are essential for the COVID-19 assay as they show statistically significant concentration changes at different severity grades of COVID-19 disease. The top 17 peptides can be further ranked according to their p-value where the smallest p-value indicates the most important contribution to COVID-19 disease severity classification and prediction. Other peptides in the table also contribute to the overall assay performance and can also be ranked based on their P-value.
TABLE-US-00009 TABLE 4 ANOVA Peptide p-value EQHLFLPFSYK (SEQ ID No.: 16) 0.0000 VLDLSCNR (SEQ ID No.: 47) 0.0000 GYSIFSYATK (SEQ ID No.: 21) 0.0000 LVLLNAIYLSAK (SEQ ID No.: 43) 0.0001 IADAHLDR (SEQ ID No.: 29) 0.0001 TFTLLDPK (SEQ ID No.: 33) 0.0005 GCPDVQASLPDAK (SEQ ID No.: 32) 0.0008 EITALAPSTMK (SEQ ID No.: 5) 0.0020 LLDSLPSDTR (SEQ ID No.: 42) 0.0020 EQLSLLDR (SEQ ID No.: 12) 0.0050 CNLLAEK (SEQ ID No.: 27) 0.0080 GLPNVVTSAISLPNIR (SEQ ID No.: 1) 0.0100 WCALSHHER (SEQ ID No.: 14) 0.0140 ADQVCINLR (SEQ ID No.: 17) 0.0180 GSPAINVAVHVFR (SEQ ID No.: 34) 0.0380 AHVDALR (SEQ ID No.: 24) 0.0450 TINPAVDHCCK (SEQ ID No.: 41) 0.0470 TVVQPSVGAAAGPVVPPCPGR (SEQ ID No.: 26) 0.0630 ATEHLSTLSEK (SEQ ID No.: 25) 0.0640 TSCLLFMGR (SEQ ID No.: 39) 0.0920 GGEGTGYFVDFSVR (SEQ ID No.: 28) 0.1190 YWCNDGK (SEQ ID No.: 23) 0.1400 ALDFAVGEYNK (SEQ ID No.: 8) 0.1890 HFQNLGK (SEQ ID No.: 40) 0.2470 DSVTGTLPK (SEQ ID No.: 36) 0.2840 ILNIFGVIK (SEQ ID No.: 44) 0.3130 ILTSDVFQDCNK (SEQ ID No.: 18) 0.3380 AADDTWEPFASGK (SEQ ID No.: 35) 0.3720 TNQVNSGGVLLR (SEQ ID No.: 7) 0.3740 DSGSYFCR (SEQ ID No.: 46) 0.4240 GHMLENHVER (SEQ ID No.: 2) 0.4730 SDVMYTDWK (SEQ ID No.: 0.4920 CQSWSSMTPHR (SEQ ID No.: 4) 0.6180 ANRPFLVFIR (SEQ ID No.: 38) 0.6510 EAQLPVIENK (SEQ ID No.: 3) 0.7330 FNAVLTNPQGDYDTSTGK (SEQ ID No.: 6) 0.8770
TABLE-US-00010 TABLE 5 Corresponding heavy Protein Uniprot Corresponding native isotope-labelled internal Protein Name Gene name Accesion Number peptide sequence standard peptide sequence Proteoglycan 4 PRG4 Q92954 GLPNVVTSAISLPNIR SILWRGLPNVVTSAISLPNIR(U-13C6, (SEQ ID No.: 1) 15N4) (SEQ ID No.: 53) Inter-alpha-trypsin ITIH1 P19827 GHMLENHVER (SEQ ID No.: GHMLENHVER(U-13C6, inhibitor heavy chain H1 2) 15N4)LWAYL (SEQ ID No.: 54) Plasminogen, EC 3.4.21.7 PLG P00747 EAQLPVIENK (SEQ ID No.: AGLLKEAQLPVIENK(U-13C6, 3) 15N2) (SEQ ID No.: 55) CQSWSSMTPHR (SEQ ID CQSWSSMTPHR(U-13C6, No.: 4) 15N4)HQK (SEQ ID No.: 56) Actin, aortic smooth ACTA2; P62736; EITALAPSTMK (SEQ ID MQKEITALAPSTMK(U-13C6, muscle; Actin, cytoplasmic ACTB; P60709; No.: 5) 15N2) (SEQ ID No.: 57) 1; Actin, cytoplasmic 2; ACTG1; P63261; Actin, gamma-enteric ACTG2 P63267 smooth muscle Complement C1q C1QC P02747 FNAVLTNPQGDYDTSTGK NSLIRFNAVLTNPQGDYDTSTGK(U- subcomponent subunit C (SEQ ID No.: 6) 13C6, 15N2) (SEQ ID No.: 58) TNQVNSGGVLLR (SEQ ID TNQVNSGGVLLR(U-13C6, No.: 7) 15N4)LQVGE (SEQ ID No.: 59) Cystatin-C CST3 P01034 ALDFAVGEYNK (SEQ ID ALDFAVGEYNK(U-13C6, No.: 8) 15N2)ASNDM (SEQ ID No.: 60) LVGGPMDASVEEEGVRR LVGGPMDASVEEEGVR(U-13C6, (SEQ ID No.: 9) 15N4)RALDFA (SEQ ID No.: 61) Protein ORM2 ORM2 Q06144 SDVMYTDWK (SEQ ID No.: SDVMYTDWK(U-13C6, 10) 15N2)KDK (SEQ ID No.: 62) Alpha-1-antichymotrypsin SERPINA3 P01011 WEMPFDPQDTHQSR (SEQ WEMPFDPQDTHQSR(U-13C6, ID No.: 11) 15N4)FYLSK (SEQ ID No.: 63) EQLSLLDR (SEQ ID No.: 12) EQLSLLDR(U-13C6, 15N4)FTEDA (SEQ ID No.: 64) Serotransferrin TF P02787 GDVAFVK (SEQ ID No.: 13) GDVAFVK(U-13C6, 15N2)HSTIF (SEQ ID No.: 65) WCALSHHER (SEQ ID No.: PVKWCALSHHER(U-13C6, 14) 15N4) (SEQ ID No.: 66) Apolipoprotein B-100 APOB P04114 IAELSATAQEIIK (SEQ ID No.: IAELSATAQEIIK(U-13C6, 15) 15N2)SQAIA (SEQ ID No.: 67) EQHLFLPFSYK (SEQ ID No.: EAICKEQHLFLPFSYK(U-13C6, 16) 15N2) (SEQ ID No.: 68) EGF-containing fibulin-like EFEMP1 Q12805 ADQVCINLR (SEQ ID No.: ADQVCINLR(U-13C6, extracellular matrix 17) 15N4)GSFAC (SEQ ID No.: 69) protein 1 von Willebrand factor VWF P04275 ILTSDVFQDCNK (SEQ ID ILTSDVFQDCNK(U-13C6, No.: 18) 15N2)LVDPE (SEQ ID No.: 70) YAGSQVASTSEVLK (SEQ ID YAGSQVASTSEVLK(U-13C6, No.: 19) 15N2)YTLFQ (SEQ ID No.: 71) C-reactive protein CRP P02741 ESDTSYVSLK (SEQ ID No.: ESDTSYVSLKAPLTK(U-13C6, 20) 15N2) (SEQ ID No.: 72) GYSIFSYATK (SEQ ID No.: GYSIFSYATK(U-13C6, 21) 15N2)RQDNE (SEQ ID No.: 73) Lysozyme C, EC 3.2.1.17 LYZ P61626 STDYGIFQINSR (SEQ ID No.: STDYGIFQINSR(U-13C6, 22) 15N4)YWCND (SEQ ID No.: 74) YWCNDGK (SEQ ID No.: 23) YWCNDGK(U-13C6, 15N2)TPGAV (SEQ ID No.: 75) Apolipoprotein A-I APOA1 P02647 AHVDALR (SEQ ID No.: 24) AHVDALR(U-13C6, 15N4)THLAP (SEQ ID No.: 76) ATEHLSTLSEK (SEQ ID No.: EYHAKATEHLSTLSEK(U-13C6, 25) 15N2) (SEQ ID No.: 77) Alpha-2-HS-glycoprotein AHSG P02765 TVVQPSVGAAAGPVVPPCPGR TVVQPSVGAAAGPVVPPCPGR(U- (SEQ ID No.: 26) 13C6, 15N4)IR (SEQ ID No.: 78) CNLLAEK (SEQ ID No.: 27) CNLLAEK(U-13C6, 15N2)QYGFC (SEQ ID No.: 79) Histidine-rich glycoprotein HRG P04196 GGEGTGYFVDFSVR (SEQ ID GGEGTGYFVDFSVR(U-13C6, No.: 28) 15N4)NCPR (SEQ ID No.: 80) IADAHLDR (SEQ ID No.: 29) IADAHLDR(U-13C6, 15N4)VENTT (SEQ ID No.: 81) N-acetylmuramoyl-L- PGLYRP2 Q96PD5 GCPDVQASLPDAK (SEQ ID PDATKGCPDVQASLPDAK(U-13C6, alanine amidase, EC No.: 32) 15N2) (SEQ ID No.: 84) 3.5.1.28 TFTLLDPK (SEQ ID No.: 33) TFTLLDPK(U-13C6, 15N2)ASLLT (SEQ ID No.: 85) Transthyretin TTR P02766 GSPAINVAVHVFR (SEQ ID GSPAINVAVHVFR(U-13C6, No.: 34) 15N4)KAADD (SEQ ID No.: 86) AADDTWEPFASGK (SEQ ID AADDTWEPFASGK(U-13C6, No.: 35) 15N2)TSESG (SEQ ID No.: 87) Plasma kallikrein, EC KLKB1 P03952 DSVTGTLPK (SEQ ID No.: GCFLKDSVTGTLPK(U-13C6, 3.4.21.34 36) 15N2) (SEQ ID No.: 88) IAYGTQGSSGYSLR (SEQ ID IAYGTQGSSGYSLR(U-13C6, No.: 37) 15N4)LCNTG (SEQ ID No.: 89) Antithrombin-III SERPINC1 P01008 ANRPFLVFIR (SEQ ID No.: ANRPFLVFIR(U-13C6, 38) 15N4)EVPLN (SEQ ID No.: 90) Heparin cofactor 2 SERPIND1 P05546 TSCLLFMGR (SEQ ID No.: TSCLLFMGR(U-13C6, 39) 15N4)VANPS (SEQ ID No.: 91) Afamin AFM P43652 HFQNLGK (SEQ ID No.: 40) QQECKHFQNLGK(U-13C6, 15N2) (SEQ ID No.: 92) TINPAVDHCCK (SEQ ID No.: TINPAVDHCCK(U-13C6, 41) 15N2)TNFAF (SEQ ID No.: 93) Plasma protease C1 SERPING1 P05155 LLDSLPSDTR (SEQ ID No.: LLDSLPSDTR(U-13C6, inhibitor 42) 15N4)LVLLN (SEQ ID No.: 94) LVLLNAIYLSAK (SEQ ID No.: PSDTRLVLLNAIYLSAK(U-13C6, 43) 15N2) (SEQ ID No.: 95) Transferrin receptor TFRC P02786 ILNIFGVIK (SEQ ID No.: 44) ILNIFGVIK(U-13C6, protein 1 15N2)GFVEP (SEQ ID No.: 96) VSASPLLYTLIEK (SEQ ID No.: VSASPLLYTLIEK(U-13C6, 45) 15N2)TMQNV (SEQ ID No.: 97) Low affinity FCGR3A P08637 DSGSYFCR (SEQ ID No.: 46) DSGSYFCR(U-13C6, immunoglobulin gamma Fc 15N4)GLFGS (SEQ ID No.: 98) region receptor III-A Monocyte differentiation CD14 P08571 VLDLSCNR (SEQ ID No.: 47) VLDLSCNR(U-13C6, antigen CD14 15N4)LNR (SEQ ID No.: 99) Insulin-like growth factor- IGFALS P35858 DFALQNPSAVPR (SEQ ID DFALQNPSAVPR(U-13C6, binding protein complex No.: 48) 15N4)FVQAI (SEQ ID No.: 100) acid labile subunit LAELPADALGPLQR (SEQ ID LAELPADALGPLQR(U-13C6, No.: 49) 15N4)AFWLD (SEQ ID No.: 101) Immunoglobulin heavy IGHV5-51 A0A0C4DH38 ASDTAMYYCAR (SEQ ID WSSLKASDTAMYYCAR(U-13C6, variable 5-51 No.: 50) 15N4) (SEQ ID No.: 102) Complement C3 C3 P01024 VEGTAFVIFGIQDGEQR (SEQ VEGTAFVIFGIQDGEQR(U-13C6, ID No.: 51) 15N4)ISLPE (SEQ ID No.: 103) VHQYFNVELIQPGAVK (SEQ VHQYFNVELIQPGAVK(U-13C6, ID No.: 52) 15N2)VYAYY (SEQ ID No.: 104)
[0315] Table 6 shows native and labelled peptide MRM transitions on 6495c (Agilent) LC-MS/MS platform. Internal standard (labelled) peptide sequences are shown in their post-digestion form (without tryptic tags). In table 6 the following abbreviation is used: CE—collision energy.
TABLE-US-00011 TABLE 6 Peptide Dwell Cell Standard Standard Precursor Product time CE Accelerator RT Sequence type (m/z) (m/z) (ms) (eV) Fragmentor Voltage (min) GLPNVVTSAISLPNIR Native 825.98 740.93 12.92 26.60 166.00 5.00 1.60 (SEQ ID No.: 1) 825.98 499.30 12.92 26.60 166.00 5.00 1.60 825.98 1170.68 12.92 26.60 166.00 5.00 1.60 GLPNVVTSAISLPNIR Labelled 830.98 745.93 12.92 26.60 166.00 5.00 1.60 (U-13C6, 15N4) 830.98 509.31 12.92 26.60 166.00 5.00 1.60 (SEQ ID No.: 53) GHMLENHVER Native 407.86 514.25 14.75 9.90 166.00 5.00 1.66 (SEQ ID No.: 2) 407.86 448.73 14.75 9.90 166.00 5.00 1.66 407.86 410.18 14.75 9.90 166.00 5.00 1.66 GHMLENHVER(U-13C6, 15N4) Labelled 411.20 519.26 14.75 9.90 166.00 5.00 1.66 (SEQ ID No.: 54) 411.20 453.74 14.75 9.90 166.00 5.00 1.67 EAQLPVIENK (SEQ ID No.: 3) Native 570.82 699.40 2.12 18.70 166.00 5.00 1.67 570.82 503.28 2.12 18.70 166.00 5.00 1.67 570.82 812.49 2.12 18.70 166.00 5.00 1.67 EAQLPVIENK(U-13C6, 15N2) Labelled 574.82 707.42 2.12 18.70 166.00 5.00 1.67 (SEQ ID No.: 55) 574.82 820.50 2.12 18.70 166.00 5.00 1.95 CQSWSSMTPHR Native 459.53 510.28 3.61 11.70 166.00 5.00 1.95 (SEQ ID No.: 4) 459.53 641.32 3.61 11.70 166.00 5.00 1.95 459.53 409.23 3.61 11.70 166.00 5.00 1.95 CQSWSSMTPHR Labelled 462.87 520.29 3.61 11.70 166.00 5.00 2.09 (U-13C6, 15N4) 462.87 419.24 3.61 11.70 166.00 5.00 2.09 (SEQ ID No.: 56) EITALAPSTMK Native 581.31 634.32 1.99 19.00 166.00 5.00 2.09 (SEQ ID No.: 5) 581.31 919.49 1.99 19.00 166.00 5.00 2.09 581.31 563.29 1.99 19.00 166.00 5.00 2.12 EITALAPSTMK(U-13C6, 15N2) Labelled 585.32 642.34 1.99 19.00 166.00 5.00 2.12 (SEQ ID No.: 57) 585.32 927.51 1.99 19.00 166.00 5.00 2.12 FNAVLTNPQGDYDTSTGK Native 964.46 1383.60 2.03 30.90 166.00 5.00 2.12 (SEQ ID No.: 6) 964.46 1282.55 2.03 30.90 166.00 5.00 2.12 964.46 1168.51 2.03 30.90 166.00 5.00 2.13 FNAVLTNPQGDYDTSTGK Labelled 968.46 1290.57 2.03 30.90 166.00 5.00 2.13 (U-13C6, 15N2) 968.46 1176.53 2.03 30.90 166.00 5.00 2.13 (SEQ ID No.: 58) TNQVNSGGVLLR Native 629.35 914.54 2.55 20.50 166.00 5.00 2.13 (SEQ ID No.: 7) 629.35 815.47 2.55 20.50 166.00 5.00 2.23 629.35 701.43 2.55 20.50 166.00 5.00 2.23 TNQVNSGGVLLR Labelled 634.35 825.48 2.55 20.50 166.00 5.00 2.23 (U-13C6, 15N4) 634.35 711.44 2.55 20.50 166.00 5.00 2.23 (SEQ ID No.: 59) ALDFAVGEYNK Native 613.81 780.39 2.07 20.00 166.00 5.00 2.82 (SEQ ID No.: 8) 613.81 709.35 2.07 20.00 166.00 5.00 2.82 166.00 5.00 ALDFAVGEYNK Labelled 617.81 788.40 2.07 20.00 166.00 5.00 2.82 (U-13C6, 15N2) 617.81 717.37 2.07 20.00 166.00 5.00 2.82 (SEQ ID No.: 60) LVGGPMDASVEEEGVRR Native 600.97 745.40 2.18 16.80 166.00 5.00 2.82 (SEQ ID No.: 9) 600.97 616.35 2.18 16.80 166.00 5.00 2.85 600.97 737.85 2.18 16.80 166.00 5.00 2.85 600.97 689.32 2.18 16.80 166.00 5.00 2.85 LVGGPMDASVEEEGVRR Labelled 607.64 765.41 2.18 16.80 166.00 5.00 2.85 (U-13C6, 15N4)R 607.64 636.37 2.18 16.80 166.00 5.00 2.85 (SEQ ID No.: 61) 607.64 747.86 2.18 16.80 166.00 5.00 2.89 607.64 699.33 2.18 16.80 166.00 5.00 2.89 SDVMYTDWK Native 572.75 942.44 1.98 18.80 166.00 5.00 2.89 (SEQ ID No.: 10) 572.75 843.37 1.98 18.80 166.00 5.00 2.89 572.75 712.33 1.98 18.80 166.00 5.00 2.89 SDVMYTDWK Labelled 576.76 851.38 1.98 18.80 166.00 5.00 2.93 (U-13C6, 15N2) 576.76 720.34 1.98 18.80 166.00 5.00 2.93 (SEQ ID No.: 62) WEMPFDPQDTHQSR Native 591.93 968.45 2.49 16.50 166.00 5.00 2.93 (SEQ ID No.: 11) 591.93 729.83 2.49 16.50 166.00 5.00 2.93 591.93 664.30 2.49 16.50 166.00 5.00 2.93 WEMPFDPQDTHQSR Labelled 595.26 734.83 2.49 16.50 166.00 5.00 2.93 (U-13C6, 15N4) 595.26 669.31 2.49 16.50 166.00 5.00 2.93 (SEQ ID No.: 63) EQLSLLDR (SEQ ID No.: 12) Native 487.27 716.43 1.99 16.10 166.00 5.00 2.93 487.27 603.35 1.99 16.10 166.00 5.00 2.93 EQLSLLDR Labelled 492.27 726.44 1.99 16.10 166.00 5.00 2.93 (U-13C6, 15N4) 492.27 613.35 1.99 16.10 166.00 5.00 3.01 (SEQ ID No.: 64) GDVAFVK (SEQ ID No.: 13) Native 368.21 563.36 3.99 12.40 166.00 5.00 3.01 368.21 464.29 3.99 12.40 166.00 5.00 3.01 368.21 393.25 3.99 12.40 166.00 5.00 3.01 368.21 246.18 3.99 12.40 166.00 5.00 3.01 368.21 17.11 3.99 12.40 166.00 5.00 3.02 GDVAFVK Labelled 372.21 571.37 3.99 12.40 166.00 5.00 3.02 (U-13C6, 15N2) 372.21 472.30 3.99 12.40 166.00 5.00 3.02 (SEQ ID No.: 65) 372.21 401.26 3.99 12.40 166.00 5.00 3.02 372.21 254.20 3.99 12.40 166.00 5.00 3.02 372.21 155.13 3.99 12.40 166.00 5.00 3.04 WCALSHHER (SEQ ID No.: 14) Native 399.19 505.24 8.15 9.60 166.00 5.00 3.04 399.19 425.22 8.15 9.60 166.00 5.00 3.04 WCALSHHER Labelled 402.52 510.24 8.15 9.60 166.00 5.00 3.04 (U-13C6, 15N4) 402.52 430.22 8.15 9.60 166.00 5.00 3.04 (SEQ ID No.: 66) IAELSATAQEIIK Native 693.90 960.54 3.43 22.50 166.00 5.00 3.28 (SEQ ID No.: 15) 693.90 873.50 3.43 22.50 166.00 5.00 3.28 693.90 802.47 3.43 22.50 166.00 5.00 3.28 IAELSATAQEIIK Labelled 697.90 968.55 3.43 22.50 166.00 5.00 3.28 (U-13C6, 15N2) 697.90 881.52 3.43 22.50 166.00 5.00 3.28 (SEQ ID No.: 67) 697.90 810.48 3.43 22.50 166.00 5.00 3.39 EQHLFLPFSYK Native 470.25 641.33 7.21 12.10 166.00 5.00 3.39 (SEQ ID No.: 16) 470.25 544.28 7.21 12.10 166.00 5.00 3.39 470.25 655.32 7.21 12.10 166.00 5.00 3.39 EQHLFLPFSYK Labelled 472.92 649.34 7.21 12.10 166.00 5.00 3.39 (U-13C6, 15N2) 472.92 552.29 7.21 12.10 166.00 5.00 3.39 (SEQ ID No.: 68) ADQVCINLR (SEQ ID No.: 17) Native 544.78 774.43 2.12 17.90 166.00 5.00 3.50 544.78 675.36 2.12 17.90 166.00 5.00 3.50 544.78 402.25 2.12 17.90 166.00 5.00 3.50 ADQVCINLR Labelled 549.78 784.44 2.12 17.90 166.00 5.00 3.50 (U-13C6, 15N4) 549.78 685.37 2.12 17.90 166.00 5.00 3.50 (SEQ ID No.: 69) ILTSDVFQDCNK Native 720.35 1112.47 2.09 23.30 166.00 5.00 3.52 (SEQ ID No.: 18) 720.35 1025.44 2.09 23.30 166.00 5.00 3.52 720.35 664.27 2.09 23.30 166.00 5.00 3.52 ILTSDVFQDCNK Labelled 724.35 1120.48 2.09 23.30 166.00 5.00 3.52 (U-13C6, 15N2) 724.35 1033.45 2.09 23.30 166.00 5.00 3.52 (SEQ ID No.: 70) YAGSQVASTSEVLK Native 720.37 933.53 2.38 23.30 166.00 5.00 3.54 (SEQ ID No.: 19) 720.37 834.46 2.38 23.30 166.00 5.00 3.54 720.37 763.42 2.38 23.30 166.00 5.00 3.54 YAGSQVASTSEVLK Labelled 724.38 941.54 2.38 23.30 166.00 5.00 3.54 (U-13C6, 15N2) 724.38 842.47 2.38 23.30 166.00 5.00 3.54 (SEQ ID No.: 71) 724.38 771.43 2.38 23.30 166.00 5.00 3.56 ESDTSYVSLK Native 564.77 696.39 3.38 18.50 166.00 5.00 3.56 (SEQ ID No.: 20) 564.77 609.36 3.38 18.50 166.00 5.00 3.56 564.77 446.30 3.38 18.50 166.00 5.00 3.56 ESDTSYVSLK Labelled 568.78 617.37 3.38 18.50 166.00 5.00 3.56 (U-13C6, 15N2) 568.78 454.31 3.38 18.50 166.00 5.00 3.59 (SEQ ID No.: 72) GYSIFSYATK Native 568.78 916.48 2.49 18.60 166.00 5.00 3.59 (SEQ ID No.: 21) 568.78 829.45 2.49 18.60 166.00 5.00 3.59 568.78 716.36 2.49 18.60 166.00 5.00 3.59 GYSIFSYATK Labelled 572.79 924.49 2.49 18.60 166.00 5.00 3.59 (U-13C6, 15N2) 572.79 724.38 2.49 18.60 166.00 5.00 3.59 (SEQ ID No.: 73) STDYGIFQINSR Native 700.84 934.51 2.72 22.70 166.00 5.00 3.59 (SEQ ID No. 22) 700.84 764.40 2.72 22.70 166.00 5.00 3.59 700.84 617.34 2.72 22.70 166.00 5.00 3.69 STDYGIFQINSR Labelled 705.85 944.52 2.72 22.70 166.00 5.00 3.69 (U-13C6, 15N4) 705.85 774.41 2.72 22.70 166.00 5.00 3.69 (SEQ ID No.: 74) 705.85 627.34 2.72 22.70 166.00 5.00 3.69 YWCNDGK (SEQ ID No.: 23) Native 471.69 779.31 11.56 15.60 166.00 5.00 3.69 471.69 593.23 11.56 15.60 166.00 5.00 3.69 YWCNDGK Labelled 475.70 787.33 11.56 15.60 166.00 5.00 3.69 (U-13C6, 15N2) 475.70 601.25 11.56 15.60 166.00 5.00 3.69 (SEQ ID No.: 75) AHVDALR (SEQ ID No.: 24) Native 261.15 288.20 21.50 4.60 166.00 5.00 3.69 261.15 423.20 21.50 4.60 166.00 5.00 3.69 AHVDALR Labelled 264.48 298.21 21.50 4.60 166.00 5.00 3.82 (U-13C6, 15N4) 264.48 423.20 21.50 4.60 166.00 5.00 3.82 (SEQ ID No.: 76) ATEHLSTLSEK Native 405.88 522.27 6.70 9.80 166.00 5.00 3.82 (SEQ ID No.: 25) 405.88 457.75 6.70 9.80 166.00 5.00 3.82 ATEHLSTLSEK Labelled 408.55 526.28 6.70 9.80 166.00 5.00 3.82 (U-13C6, 15N2) 408.55 461.76 6.70 9.80 166.00 5.00 3.85 (SEQ ID No.: 77) TVVQPSVGAAAGPVVPPCPGR Native 672.69 683.33 2.02 19.40 166.00 5.00 3.85 (SEQ ID No.: 26) 672.69 489.76 2.02 19.40 166.00 5.00 3.85 672.69 1038.56 2.02 19.40 166.00 5.00 3.85 TVVQPSVGAAAGPVVPPCPGR Labelled 676.03 693.34 2.02 19.40 166.00 5.00 3.95 (U-13C6, 15N4) 676.03 494.77 2.02 19.40 166.00 5.00 3.95 (SEQ ID No.: 78) CNLLAEK (SEQ ID No.: 27) Native 424.22 573.36 5.01 14.20 166.00 5.00 3.95 424.22 460.28 5.01 14.20 166.00 5.00 3.95 424.22 347.19 5.01 14.20 166.00 5.00 3.95 CNLLAEK Labelled 428.23 581.37 5.01 14.20 166.00 5.00 4.01 (U-13C6, 15N2) 428.23 355.21 5.01 14.20 166.00 5.00 4.01 (SEQ ID No.: 79) GGEGTGYFVDFSVR Native 497.57 623.31 5.59 13.10 166.00 5.00 4.01 (SEQ ID No.: 28) 497.57 508.29 5.59 13.10 166.00 5.00 4.01 497.57 624.31 5.59 13.10 166.00 5.00 4.01 GGEGTGYFVDFSVR Labelled 500.90 518.30 5.59 13.10 166.00 5.00 4.08 (U-13C6, 15N4) 500.90 629.31 5.59 13.10 166.00 5.00 4.08 (SEQ ID No.: 80) IADAHLDR (SEQ ID No.: 29) Native 304.16 403.23 9.24 6.10 166.00 5.00 4.08 304.16 399.20 9.24 6.10 166.00 5.00 4.08 304.16 363.68 9.24 6.10 166.00 5.00 4.08 IADAHLDR Labelled 307.50 404.20 9.24 6.10 166.00 5.00 4.15 (U-13C6, 15N4) 307.50 368.68 9.24 6.10 166.00 5.00 4.15 (SEQ ID No.: 81) GCPDVQASLPDAK Native 679.32 1140.59 2.40 22.10 166.00 5.00 4.15 (SEQ ID No.: 32) 679.32 430.23 2.40 22.10 166.00 5.00 4.15 679.32 570.80 2.40 22.10 166.00 5.00 4.15 GCPDVQASLPDAK Labelled 683.33 1148.60 2.40 22.10 166.00 5.00 4.18 (U-13C6, 15N2) 683.33 574.81 2.40 22.10 166.00 5.00 4.18 (SEQ ID No.: 84) 2.40 22.10 TFTLLDPK (SEQ ID No.: 33) Native 467.77 686.41 2.32 15.50 166.00 5.00 4.18 467.77 585.36 2.32 15.50 166.00 5.00 4.18 467.77 472.28 2.32 15.50 166.00 5.00 4.18 TFTLLDPK Labelled 471.77 694.42 2.32 15.50 166.00 5.00 4.19 (U-13C6, 15N2) 471.77 593.37 2.32 15.50 166.00 5.00 4.19 (SEQ ID No.: 85) GSPAINVAVHVFR Native 456.26 558.31 2.80 11.60 166.00 5.00 4.19 (SEQ ID No.: 34) 456.26 563.33 2.80 11.60 166.00 5.00 4.19 456.26 527.81 2.80 11.60 166.00 5.00 4.19 GSPAINVAVHVFR Labelled 459.59 568.33 2.80 11.60 166.00 5.00 4.19 (U-13C6, 15N4) 459.59 532.82 2.80 11.60 166.00 5.00 4.19 (SEQ ID No.: 86) AADDTWEPFASGK Native 697.81 921.45 2.02 22.60 166.00 5.00 4.19 (SEQ ID No.: 35) 697.81 735.37 2.02 22.60 166.00 5.00 4.30 AADDTWEPFASGK Labelled 701.82 929.46 2.02 22.60 166.00 5.00 4.30 (U-13C6, 15N2) 701.82 614.34 2.02 22.60 166.00 5.00 4.30 (SEQ ID No.: 87) DSVTGTLPK (SEQ ID No.: 36) Native 459.25 715.43 4.31 15.20 166.00 5.00 4.43 459.25 616.37 4.31 15.20 166.00 5.00 4.43 459.25 515.32 4.31 15.20 166.00 5.00 4.43 DSVTGTLPK Labelled 463.26 624.38 4.31 15.20 166.00 5.00 4.43 (U-13C6, 15N2) 463.26 523.33 4.31 15.20 166.00 5.00 4.43 (SEQ ID No.: 88) IAYGTQGSSGYSLR Native 730.36 954.46 2.60 23.60 166.00 5.00 4.51 (SEQ ID No.: 37) 730.36 826.41 2.60 23.60 166.00 5.00 4.51 730.36 682.34 2.60 23.60 166.00 5.00 4.51 IAYGTQGSSGYSLR Labelled 735.37 964.47 2.60 23.60 166.00 5.00 4.51 (U-13C6, 15N4) 735.37 692.36 2.60 23.60 166.00 5.00 4.51 (SEQ ID No.: 89) ANRPFLVFIR (SEQ ID No.: 38) Native 411.58 534.34 5.80 10.00 166.00 5.00 4.51 411.58 435.27 5.80 10.00 166.00 5.00 4.51 ANRPFLVFIR Labelled 414.92 544.35 5.80 10.00 166.00 5.00 4.51 (U-13C6, 15N4) 414.92 445.28 5.80 10.00 166.00 5.00 4.51 (SEQ ID No.: 90) TSCLLFMGR (SEQ ID No.: 39) Native 542.77 896.45 2.90 17.80 166.00 5.00 4.51 542.77 736.42 2.90 17.80 166.00 5.00 4.52 542.77 623.33 2.90 17.80 166.00 5.00 4.52 TSCLLFMGR Labelled 547.77 906.46 2.90 17.80 166.00 5.00 4.52 (U-13C6, 15N4) 547.77 633.34 2.90 17.80 166.00 5.00 4.52 (SEQ ID No.: 91) HFQNLGK (SEQ ID No.: 40) Native 281.82 317.22 13.01 5.30 166.00 5.00 4.52 281.82 413.19 13.01 5.30 166.00 5.00 4.54 281.82 527.24 13.01 5.30 166.00 5.00 4.54 HFQNLGK Labelled 284.49 413.19 13.01 5.30 166.00 5.00 4.54 (U-13C6, 15N2) 284.49 527.24 13.01 5.30 166.00 5.00 4.54 (SEQ ID No.: 92) TINPAVDHCCK Native 438.87 550.73 7.32 11.00 166.00 5.00 4.54 (SEQ ID No.: 41) 438.87 493.71 7.32 11.00 166.00 5.00 4.54 TINPAVDHCCK Labelled 441.54 554.74 7.32 11.00 166.00 5.00 4.63 (U-13C6, 15N2) 441.54 497.72 7.32 11.00 166.00 5.00 4.63 (SEQ ID No.: 93) LLDSLPSDTR (SEQ ID No.: 42) Native 558.80 775.39 2.06 18.30 166.00 5.00 4.63 558.80 575.28 2.06 18.30 166.00 5.00 4.63 558.80 542.32 2.06 18.30 166.00 5.00 4.63 LLDSLPSDTR Labelled 563.80 785.40 2.06 18.30 166.00 5.00 4.63 (U-13C6, 15N4) 563.80 585.29 2.06 18.30 166.00 5.00 4.64 (SEQ ID No.: 94) LVLLNAIYLSAK Native 659.41 879.49 17.14 21.40 166.00 5.00 4.64 (SEQ ID No.: 43) 659.41 694.41 17.14 21.40 166.00 5.00 4.64 659.41 581.33 17.14 21.40 166.00 5.00 4.64 LVLLNAIYLSAK Labelled 663.42 887.51 17.14 21.40 166.00 5.00 4.64 (U-13C6, 15N2) 663.42 589.34 17.14 21.40 166.00 5.00 4.91 (SEQ ID No.: 95) ILNIFGVIK (SEQ ID No.: 44) Native 508.83 790.48 29.95 16.80 166.00 5.00 4.91 508.83 676.44 29.95 16.80 166.00 5.00 4.91 508.83 563.36 29.95 16.80 166.00 5.00 4.91 ILNIFGVIK Labelled 512.84 798.50 29.95 16.80 166.00 5.00 4.91 (U-13C6, 15N2) 512.84 571.37 29.95 16.80 166.00 5.00 4.97 (SEQ ID No.: 96) VSASPLLYTLIEK Native 717.42 1089.66 10.80 23.20 166.00 5.00 4.97 (SEQ ID No.: 45) 717.42 879.52 10.80 23.20 166.00 5.00 4.97 717.42 545.33 10.80 23.20 166.00 5.00 4.97 VSASPLLYTLIEK Labelled 721.42 887.53 10.80 23.20 166.00 5.00 4.97 (U-13C6, 15N2) 721.42 549.34 10.80 23.20 166.00 5.00 5.01 (SEQ ID No.: 97) DSGSYFCR (SEQ ID No.: 46) Native 496.20 789.33 5.56 16.40 166.00 5.00 5.01 496.20 732.31 5.56 16.40 166.00 5.00 5.01 496.20 645.28 5.56 16.40 166.00 5.00 5.01 DSGSYFCR Labelled 501.20 799.34 5.56 16.40 166.00 5.00 5.03 (U-13C6, 15N4) 501.20 655.29 5.56 16.40 166.00 5.00 5.03 (SEQ ID No.: 98) VLDLSCNR (SEQ D No.: 47) Native 488.75 877.42 3.68 16.20 166.00 5.00 5.03 488.75 764.34 3.68 16.20 166.00 5.00 5.03 488.75 649.31 3.68 16.20 166.00 5.00 5.03 VLDLSCNR Labelled 493.75 774.34 3.68 16.20 166.00 5.00 5.20 (U-13C6, 15N4) 493.75 659.32 3.68 16.20 166.00 5.00 5.20 (SEQ ID No.: 99) DFALQNPSAVPR Native 657.84 981.55 2.07 21.40 166.00 5.00 5.20 (SEQ ID No.: 48) 657.84 740.40 2.07 21.40 166.00 5.00 5.20 657.84 626.36 2.07 21.40 166.00 5.00 5.20 DFALQNPSAVPR Labelled 662.85 750.41 2.07 21.40 166.00 5.00 5.61 (U-13C6, 15N4) 662.85 636.37 2.07 21.40 166.00 5.00 5.61 (SEQ ID No.: 100) LAELPADALGPLQR Native 732.41 1037.57 6.28 23.70 166.00 5.00 5.61 (SEQ ID No.: 49) 732.41 570.34 6.28 23.70 166.00 5.00 5.61 732.41 519.29 6.28 23.70 166.00 5.00 5.61 LAELPADALGPLQR Labelled 737.42 1047.58 6.28 23.70 166.00 5.00 5.61 (U-13C6, 15N4) 737.42 524.29 6.28 23.70 166.00 5.00 5.84 (SEQ ID No.: 101) ASDTAMYYCAR Native 654.77 934.39 2.14 21.30 166.00 5.00 5.84 (SEQ ID No.: 50) 654.77 863.35 2.14 21.30 166.00 5.00 5.84 654.77 732.31 2.14 21.30 166.00 5.00 5.84 654.77 569.25 2.14 21.30 166.00 5.00 5.84 ASDTAMYYCAR Labelled 659.78 944.40 2.14 21.30 166.00 5.00 5.88 (U-13C6, 15N4) 659.78 873.36 2.14 21.30 166.00 5.00 5.88 (SEQ ID No.: 102) 659.78 742.32 2.14 21.30 166.00 5.00 5.88 659.78 579.26 2.14 21.30 166.00 5.00 5.88 VEGTAFVIFGIQDGEQR Native 622.65 732.33 9.89 17.60 166.00 5.00 5.88 (SEQ ID No.: 51) 622.65 604.27 9.89 17.60 166.00 5.00 5.91 622.65 489.24 9.89 17.60 166.00 5.00 5.91 VEGTAFVIFGIQDGEQR Labelled 625.99 742.34 9.89 17.60 166.00 5.00 5.91 (U-13C6, 15N4) 625.99 614.28 9.89 17.60 166.00 5.00 5.91 (SEQ ID No.: 103) 625.99 499.25 9.89 17.60 166.00 5.00 5.91 VHQYFNVELIQPGAVK Native 614.67 825.52 5.42 17.30 166.00 5.00 5.99 (SEQ ID No.: 52) 614.67 712.44 5.42 17.30 166.00 5.00 5.99 614.67 471.29 5.42 17.30 166.00 5.00 5.99 VHQYFNVELIQPGAVK Labelled 617.34 720.45 5.42 17.30 166.00 5.00 5.99 (U-13C6, 15N2) 617.34 479.31 5.42 17.30 166.00 5.00 5.99 (SEQ ID No.: 104)
[0316] Table 7 shows native and labelled peptide MRM transitions on 7500 (Sciex) LC-MS/MS platform. Internal standard (labelled) peptide sequences are shown in their post-digestion form (without tryptic tags). The following abbreviations are used in table 7: EP—entrance potential; CE—collision energy; CXP—collision cell exit potential; DP—declustering potential; RT—retention time
TABLE-US-00012 TABLE 7 Peptide Product Standard Standard Precursor (Q3) Dwell RT Sequence type (Q1) m/z m/z time (ms) EP CE CXP (min) GLPNVVTSAISLPNIR Native 825.98 1170.68 3.50 10.00 43.30 25.00 6.58 (SEQ ID No.: 1) 825.98 1071.62 3.50 10.00 43.30 25.00 6.58 825.98 499.30 3.50 10.00 31.30 25.00 6.58 GLPNVVTSAISLPNIR Labelled 830.98 1180.69 3.50 10.00 43.30 25.00 6.58 (U-13C6, 15N4) (SEQ ID No.: 53) 830.98 1081.62 3.50 10.00 43.30 25.00 6.58 GHMLENHVER (SEQ ID No.: 2) Native 407.86 783.37 3.50 10.00 20.20 25.00 2.90 407.86 654.33 3.50 10.00 24.20 25.00 2.90 407.86 403.23 3.50 10.00 24.20 25.00 2.90 GHMLENHVER(U-13C6, 15N4) Labelled 411.20 793.38 3.50 10.00 20.20 25.00 2.90 (SEQ ID No.: 54) 411.20 413.23 3.50 10.00 24.20 25.00 2.90 EAQLPVIENK (SEQ ID No.: 3) Native 570.82 699.40 3.50 10.00 26.50 25.00 4.38 570.82 503.28 3.50 10.00 34.50 25.00 4.38 570.82 390.20 3.50 10.00 34.50 25.00 4.38 EAQLPVIENK(U-13C6, 15N2) Labelled 574.84 707.41 3.50 10.00 26.50 25.00 4.38 (SEQ ID No.: 55) 574.84 511.30 3.50 10.00 34.50 25.00 4.38 CQSWSSMTPHR (SEQ ID No.: 4) Native 459.53 815.38 3.50 10.00 22.60 25.00 3.81 459.53 510.28 3.50 10.00 22.60 25.00 3.81 459.53 409.23 3.50 10.00 22.60 25.00 3.81 CQSWSSMTPHR(U-13C6, 15N4) Labelled 462.86 520.28 3.50 10.00 22.60 25.00 3.81 (SEQ ID No.: 56) 462.86 419.23 3.50 10.00 22.60 25.00 3.81 EITALAPSTMK (SEQ ID No.: 5) Native 581.31 747.41 3.50 10.00 27.10 25.00 4.49 581.31 634.32 3.50 10.00 23.10 25.00 4.49 581.31 563.29 3.50 10.00 23.10 25.00 4.49 EITALAPSTMK(U-13C6, 15N2) Labelled 585.31 755.42 3.50 10.00 27.10 25.00 4.49 (SEQ ID No.: 57) 585.31 642.33 3.50 10.00 23.10 25.00 4.49 FNAVLTNPQGDYDTSTGK Native 643.31 1168.51 3.50 10.00 46.20 25.00 4.67 (SEQ ID No.: 6) 643.31 584.76 3.50 10.00 54.20 25.00 4.67 643.31 493.26 3.50 10.00 58.20 25.00 4.67 FNAVLTNPQGDYDTSTGK Labelled 645.97 1176.52 3.50 10.00 46.20 25.00 4.67 (U-13C6, 15N2) 645.97 588.76 3.50 10.00 54.20 25.00 4.67 (SEQ ID No.: 58) TNQVNSGGVLLR Native 629.35 914.54 3.50 10.00 33.50 25.00 4.16 (SEQ ID No.: 7) 629.35 815.47 3.50 10.00 33.50 25.00 4.16 629.35 701.43 3.50 10.00 33.50 25.00 4.16 TNQVNSGGVLLR(U-13C6, 15N4) Labelled 634.35 924.55 3.50 10.00 33.50 25.00 4.16 (SEQ ID No.: 59) 634.35 825.48 3.50 10.00 33.50 25.00 4.16 ALDFAVGEYNK Native 613.81 780.39 3.50 10.00 28.70 25.00 5.05 (SEQ ID No.: 8) 613.81 709.35 3.50 10.00 28.70 25.00 5.05 613.81 610.28 3.50 10.00 24.70 25.00 5.05 ALDFAVGEYNK(U-13C6, 15N2) Labelled 617.81 717.36 3.50 10.00 28.70 25.00 5.05 (SEQ ID No.: 60) 617.81 618.29 3.50 10.00 24.70 25.00 5.05 LVGGPMDASVEEEGVRR Native 600.97 745.40 3.50 10.00 37.20 25.00 4.38 (SEQ ID No.: 9) 600.97 794.87 3.50 10.00 25.20 25.00 4.38 600.97 566.29 3.50 10.00 37.20 25.00 4.38 LVGGPMDASVEEEGVRR Labelled 604.30 799.88 3.50 10.00 25.20 25.00 4.38 (U-13C6, 15N4)R 604.30 571.30 3.50 10.00 37.20 25.00 4.38 (SEQ ID No.: 61) SDVMYTDWK (SEQ ID No.: 10) Native 572.75 843.37 3.50 10.00 26.60 25.00 4.65 572.75 712.33 3.50 10.00 26.60 25.00 4.65 572.75 549.27 3.50 10.00 26.60 25.00 4.65 SDVMYTDWK Labelled 576.75 851.38 3.50 10.00 26.60 25.00 4.65 (U-13C6, 15N2) 576.75 720.34 3.50 10.00 26.60 25.00 4.65 (SEQ ID No.: 62) WEMPFDPQDTHQSR Native 591.93 968.45 3.50 10.00 32.80 25.00 5.34 (SEQ ID No.: 11) 591.93 628.32 3.50 10.00 40.80 25.00 5.34 591.93 729.83 3.50 10.00 24.80 25.00 5.34 WEMPFDPQDTHQSR Labelled 595.26 978.46 3.50 10.00 32.80 25.00 5.34 (U-13C6, 15N4) 595.26 734.82 3.50 10.00 24.80 25.00 5.34 (SEQ ID No.: 63) EQLSLLDR (SEQ ID No.: 12) Native 487.27 716.43 3.50 10.00 22.40 25.00 4.69 487.27 603.35 3.50 10.00 22.40 25.00 4.69 487.27 403.23 3.50 10.00 22.40 25.00 4.69 EQLSLLDR Labelled 492.27 726.43 3.50 10.00 22.40 25.00 4.69 (U-13C6, 15N4) 492.27 613.35 3.50 10.00 22.40 25.00 4.69 (SEQ ID No.: 64) GDVAFVK (SEQ ID No.: 13) Native 368.21 563.36 3.50 10.00 16.40 25.00 3.69 368.21 464.29 3.50 10.00 16.40 25.00 3.69 368.21 393.25 3.50 10.00 20.40 25.00 3.69 GDVAFVK Labelled 372.21 571.36 3.50 10.00 16.40 25.00 3.69 (U-13C6, 15N2) 372.21 472.30 3.50 10.00 16.40 25.00 3.69 (SEQ ID No.: 65) WCALSHHER (SEQ ID No.: 14) Native 399.19 665.31 3.50 10.00 23.80 25.00 3.16 399.19 578.28 3.50 10.00 27.80 25.00 3.16 399.19 441.22 3.50 10.00 27.80 25.00 3.16 WCALSHHER Labelled 402.52 675.31 3.50 10.00 23.80 25.00 3.16 (U-13C6, 15N4) 402.52 451.22 3.50 10.00 27.80 25.00 3.16 (SEQ ID No.: 66) IAELSATAQEIIK Native 693.90 960.54 3.50 10.00 32.70 25.00 5.32 (SEQ ID No.: 15) 693.90 873.50 3.50 10.00 32.70 25.00 5.32 693.90 802.47 3.50 10.00 32.70 25.00 5.32 IAELSATAQEIIK Labelled 697.90 968.55 3.50 10.00 32.70 25.00 5.32 (U-13C6, 15N2) 697.90 810.48 3.50 10.00 32.70 25.00 5.32 (SEQ ID No.: 67) EQHLFLPFSYK (SEQ ID No.: 16) Native 470.25 641.33 3.50 10.00 19.10 25.00 5.97 470.25 544.28 3.50 10.00 23.10 25.00 5.97 470.25 397.21 3.50 10.00 27.10 25.00 5.97 EQHLFLPFSYK Labelled 472.91 649.34 3.50 10.00 19.10 25.00 5.97 (U-13C6, 15N2) 472.91 552.29 3.50 10.00 23.10 25.00 5.97 (SEQ ID No.: 68) ADQVCINLR (SEQ ID No.: 17) Native 544.78 774.43 3.50 10.00 29.20 25.00 4.32 544.78 675.36 3.50 10.00 29.20 25.00 4.32 544.78 515.33 3.50 10.00 29.20 25.00 4.32 ADQVCINLR Labelled 549.78 784.43 3.50 10.00 29.20 25.00 4.32 (U-13C6, 15N4) 549.78 685.36 3.50 10.00 29.20 25.00 4.32 (SEQ ID No.: 69) ILTSDVFQDCNK Native 720.35 811.34 3.50 10.00 34.00 25.00 4.74 (SEQ ID No.: 18) 720.35 664.27 3.50 10.00 34.00 25.00 4.74 720.35 663.80 3.50 10.00 42.00 25.00 4.74 ILTSDVFQDCNK Labelled 724.35 819.35 3.50 10.00 34.00 25.00 4.74 (U-13C6, 15N2) 724.35 667.81 3.50 10.00 42.00 25.00 4.74 (SEQ ID No.: 70) YAGSQVASTSEVLK Native 564.77 696.39 3.50 10.00 26.20 25.00 3.89 (SEQ ID No.: 19) 564.77 609.36 3.50 10.00 26.20 25.00 3.89 564.77 446.30 3.50 10.00 26.20 25.00 3.89 YAGSQVASTSEVLK Labelled 568.78 704.40 3.50 10.00 26.20 25.00 3.89 (U-13C6, 15N2) 568.78 617.37 3.50 10.00 26.20 25.00 3.89 (SEQ ID No.: 71) ESDTSYVSLK (SEQ ID No.: 20) Native 568.78 716.36 3.50 10.00 26.40 25.00 5.40 568.78 569.29 3.50 10.00 18.40 25.00 5.40 568.78 482.26 3.50 10.00 38.40 25.00 5.40 ESDTSYVSLK Labelled 572.79 724.37 3.50 10.00 26.40 25.00 5.40 (U-13C6, 15N2) 572.79 577.30 3.50 10.00 18.40 25.00 5.40 (SEQ ID No.: 72) GYSIFSYATK (SEQ ID No.: 21) Native 700.84 934.51 3.50 10.00 37.00 25.00 5.37 700.84 764.40 3.50 10.00 33.00 25.00 5.37 700.84 617.34 3.50 10.00 33.00 25.00 5.37 GYSIFSYATK Labelled 705.84 944.51 3.50 10.00 37.00 25.00 5.37 (U-13C6, 15N2) 705.84 774.41 3.50 10.00 33.00 25.00 5.37 (SEQ ID No.: 73) STDYGIFQINSR Native 720.37 933.53 3.50 10.00 38.00 25.00 4.11 (SEQ ID No. 22) 720.37 834.46 3.50 10.00 38.00 25.00 4.11 720.37 763.42 3.50 10.00 38.00 25.00 4.11 STDYGIFQINSR Labelled 724.37 941.53 3.50 10.00 38.00 25.00 4.11 (U-13C6, 15N4) 724.37 842.47 3.50 10.00 38.00 25.00 4.11 (SEQ ID No.: 74) YWCNDGK (SEQ ID No.: 23) Native 471.69 779.31 3.50 10.00 21.60 25.00 2.88 471.69 593.23 3.50 10.00 21.60 25.00 2.88 471.69 433.20 3.50 10.00 25.60 25.00 2.88 YWCNDGK Labelled 475.70 787.32 3.50 10.00 21.60 25.00 2.88 (U-13C6, 15N2) 475.70 601.24 3.50 10.00 21.60 25.00 2.88 (SEQ ID No.: 75) AHVDALR (SEQ ID No.: 24) Native 391.22 573.34 3.50 10.00 21.60 25.00 2.39 391.22 474.27 3.50 10.00 25.60 25.00 2.39 391.22 359.24 3.50 10.00 29.60 25.00 2.39 AHVDALR Labelled 396.22 583.34 3.50 10.00 21.60 25.00 2.39 (U-13C6, 15N4) 396.22 484.27 3.50 10.00 25.60 25.00 2.39 (SEQ ID No.: 76) ATEHLSTLSEK (SEQ ID No.: 25) Native 405.88 777.44 3.50 10.00 24.10 25.00 3.11 405.88 664.35 3.50 10.00 24.10 25.00 3.11 405.88 363.19 3.50 10.00 24.10 25.00 3.11 ATEHLSTLSEK Labelled 408.55 672.36 3.50 10.00 24.10 25.00 3.11 (U-13C6, 15N2) 408.55 371.20 3.50 10.00 24.10 25.00 3.11 (SEQ ID No.: 77) TVVQPSVGAAAGPVVPPCPGR Native 672.69 978.52 3.50 10.00 24.60 25.00 4.96 (SEQ ID No.: 26) 672.69 683.33 3.50 10.00 28.60 25.00 4.96 672.69 586.28 3.50 10.00 40.60 25.00 4.96 TVVQPSVGAAAGPVVPPCPGR Labelled 676.03 988.52 3.50 10.00 24.60 25.00 4.96 (U-13C6, 15N4) 676.03 693.33 3.50 10.00 28.60 25.00 4.96 (SEQ ID No.: 78) CNLLAEK (SEQ ID No.: 27) Native 745.85 1089.54 3.50 10.00 39.30 25.00 5.76 745.85 869.45 3.50 10.00 39.30 25.00 5.76 745.85 508.29 3.50 10.00 39.30 25.00 5.76 CNLLAEK Labelled 750.85 1099.54 3.50 10.00 39.30 25.00 5.76 (U-13C6, 15N2) 750.85 879.45 3.50 10.00 39.30 25.00 5.76 (SEQ ID No.: 79) GGEGTGYFVDFSVR Native 455.74 797.39 3.50 10.00 24.80 25.00 2.69 (SEQ ID No.: 28) 455.74 726.35 3.50 10.00 24.80 25.00 2.69 455.74 611.33 3.50 10.00 28.80 25.00 2.69 GGEGTGYFVDFSVR Labelled 460.74 807.40 3.50 10.00 24.80 25.00 2.69 (U-13C6, 15N4) 460.74 736.36 3.50 10.00 24.80 25.00 2.69 (SEQ ID No.: 80) IADAHLDR (SEQ ID No.: 29) Native 424.22 687.40 3.50 10.00 23.20 25.00 3.55 424.22 573.36 3.50 10.00 23.20 25.00 3.55 424.22 460.28 3.50 10.00 23.20 25.00 3.55 IADAHLDR Labelled 428.22 581.37 3.50 10.00 23.20 25.00 3.55 (U-13C6, 15N4) 428.22 468.30 3.50 10.00 23.20 25.00 3.55 (SEQ ID No.: 81) GCPDVQASLPDAK Native 453.22 630.35 3.50 10.00 28.00 25.00 4.06 (SEQ ID No.: 32) 453.22 430.23 3.50 10.00 32.00 25.00 4.06 453.22 430.14 3.50 10.00 44.00 25.00 4.06 GCPDVQASLPDAK Labelled 455.89 638.35 3.50 10.00 28.00 25.00 4.06 (U-13C6, 15N2) 455.89 438.24 3.50 10.00 32.00 25.00 4.06 (SEQ ID No.: 84) TFTLLDPK (SEQ ID No.: 33) Native 467.77 686.41 3.50 10.00 21.40 25.00 5.26 467.77 585.36 3.50 10.00 21.40 25.00 5.26 467.77 472.28 3.50 10.00 21.40 25.00 5.26 TFTLLDPK Labelled 471.77 694.42 3.50 10.00 21.40 25.00 5.26 (U-13C6, 15N2) 471.77 593.37 3.50 10.00 21.40 25.00 5.26 (SEQ ID No.: 85) GSPAINVAVHVFR Native 456.26 728.42 3.50 10.00 26.40 25.00 5.40 (SEQ ID No.: 34) 456.26 558.31 3.50 10.00 18.40 25.00 5.40 456.26 611.86 3.50 10.00 18.40 25.00 5.40 GSPAINVAVHVFR Labelled 459.59 738.42 3.50 10.00 26.40 25.00 5.40 (U-13C6, 15N4) 459.59 616.86 3.50 10.00 18.40 25.00 5.40 (SEQ ID No.: 86) AADDTWEPFASGK Native 697.81 921.45 3.50 10.00 32.90 25.00 5.04 (SEQ ID No.: 35) 697.81 735.37 3.50 10.00 32.90 25.00 5.04 697.81 606.32 3.50 10.00 40.90 25.00 5.04 AADDTWEPFASGK Labelled 701.82 929.46 3.50 10.00 32.90 25.00 5.04 (U-13C6, 15N2) 701.82 614.33 3.50 10.00 40.90 25.00 5.04 (SEQ ID No.: 87) DSVTGTLPK (SEQ ID No.: 36) Native 459.25 616.37 3.50 10.00 21.00 25.00 3.56 459.25 515.32 3.50 10.00 21.00 25.00 3.56 459.25 357.25 3.50 10.00 29.00 25.00 3.56 DSVTGTLPK Labelled 463.25 624.38 3.50 10.00 21.00 25.00 3.56 (U-13C6, 15N2) 463.25 523.33 3.50 10.00 21.00 25.00 3.56 (SEQ ID No.: 88) IAYGTQGSSGYSLR Native 730.36 1112.53 3.50 10.00 38.50 25.00 4.19 (SEQ ID No.: 37) 730.36 954.46 3.50 10.00 38.50 25.00 4.19 730.36 826.41 3.50 10.00 42.50 25.00 4.19 IAYGTQGSSGYSLR Labelled 735.36 1122.54 3.50 10.00 38.50 25.00 4.19 (U-13C6, 15N4) 735.36 836.41 3.50 10.00 42.50 25.00 4.19 (SEQ ID No.: 89) ANRPFLVFIR (SEQ ID No.: 38) Native 411.58 647.42 3.50 10.00 20.30 25.00 6.00 411.58 534.34 3.50 10.00 20.30 25.00 6.00 411.58 435.27 3.50 10.00 20.30 25.00 6.00 ANRPFLVFIR Labelled 414.91 544.34 3.50 10.00 20.30 25.00 6.00 (U-13C6, 15N4) 414.91 445.27 3.50 10.00 20.30 25.00 6.00 (SEQ ID No.: 90) TSCLLFMGR (SEQ ID No.: 39) Native 542.77 736.42 3.50 10.00 29.10 25.00 5.48 542.77 623.33 3.50 10.00 29.10 25.00 5.48 542.77 510.25 3.50 10.00 25.10 25.00 5.48 TSCLLFMGR Labelled 547.77 633.34 3.50 10.00 29.10 25.00 5.48 (U-13C6, 15N4) 547.77 520.25 3.50 10.00 25.10 25.00 5.48 (SEQ ID No.: 91) HFQNLGK (SEQ ID No.: 40) Native 422.23 706.39 3.50 10.00 23.10 25.00 2.54 422.23 559.32 3.50 10.00 23.10 25.00 2.54 422.23 431.26 3.50 10.00 27.10 25.00 2.54 HFQNLGK Labelled 426.23 714.40 3.50 10.00 23.10 25.00 2.54 (U-13C6, 15N2) 426.23 567.33 3.50 10.00 23.10 25.00 2.54 (SEQ ID No.: 92) TINPAVDHCCK Native 438.87 719.26 3.50 10.00 25.60 25.00 2.92 (SEQ ID No.: 41) 438.87 604.23 3.50 10.00 25.60 25.00 2.92 438.87 467.17 3.50 10.00 33.60 25.00 2.92 TINPAVDHCCK Labelled 441.54 727.27 3.50 10.00 25.60 25.00 2.92 (U-13C6, 15N2) 441.54 612.24 3.50 10.00 25.60 25.00 2.92 (SEQ ID No.: 93) LLDSLPSDTR (SEQ ID No.: 42) Native 558.80 890.42 3.50 10.00 25.90 25.00 4.38 558.80 775.39 3.50 10.00 33.90 25.00 4.38 558.80 575.28 3.50 10.00 25.90 25.00 4.38 LLDSLPSDTR Labelled 563.80 900.42 3.50 10.00 25.90 25.00 4.38 (U-13C6, 15N4) 563.80 585.28 3.50 10.00 25.90 25.00 4.38 (SEQ ID No.: 94) LVLLNAIYLSAK Native 659.41 879.49 3.50 10.00 31.00 25.00 6.82 (SEQ ID No.: 43) 659.41 765.45 3.50 10.00 31.00 25.00 6.82 659.41 694.41 3.50 10.00 43.00 25.00 6.82 LVLLNAIYLSAK Labelled 663.41 887.50 3.50 10.00 31.00 25.00 6.82 (U-13C6, 15N2) 663.41 773.46 3.50 10.00 31.00 25.00 6.82 (SEQ ID No.: 95) ILNIFGVIK (SEQ ID No.: 44) Native 508.83 790.48 3.50 10.00 23.40 25.00 6.80 508.83 676.44 3.50 10.00 23.40 25.00 6.80 508.83 563.36 3.50 10.00 23.40 25.00 6.80 ILNIFGVIK Labelled 512.83 798.49 3.50 10.00 23.40 25.00 6.80 (U-13C6, 15N2) 512.83 571.36 3.50 10.00 23.40 25.00 6.80 (SEQ ID No.: 96) VSASPLLYTLIEK Native 717.42 1089.66 3.50 10.00 29.90 25.00 6.66 (SEQ ID No.: 45) 717.42 879.52 3.50 10.00 37.90 25.00 6.66 717.42 766.43 3.50 10.00 33.90 25.00 6.66 VSASPLLYTLIEK Labelled 721.42 1097.66 3.50 10.00 29.90 25.00 6.66 (U-13C6, 15N2) 721.42 774.44 3.50 10.00 33.90 25.00 6.66 (SEQ ID No.: 97) DSGSYFCR (SEQ ID No.: 46) Native 496.20 789.33 3.50 10.00 22.80 25.00 3.58 496.20 645.28 3.50 10.00 22.80 25.00 3.58 496.20 482.22 3.50 10.00 22.80 25.00 3.58 DSGSYFCR Labelled 501.20 799.34 3.50 10.00 22.80 25.00 3.58 (U-13C6, 15N4) 501.20 655.28 3.50 10.00 22.80 25.00 3.58 (SEQ ID No.: 98) VLDLSCNR (SEQ D No.: 47) Native 488.75 764.34 3.50 10.00 22.40 25.00 3.80 488.75 649.31 3.50 10.00 26.40 25.00 3.80 488.75 536.22 3.50 10.00 22.40 25.00 3.80 VLDLSCNR Labelled 493.75 774.34 3.50 10.00 22.40 25.00 3.80 (U-13C6, 15N4) 493.75 546.23 3.50 10.00 22.40 25.00 3.80 (SEQ ID No.: 99) DFALQNPSAVPR Native 657.84 868.46 3.50 10.00 34.90 25.00 4.84 (SEQ ID No.: 48) 657.84 740.40 3.50 10.00 30.90 25.00 4.84 657.84 626.36 3.50 10.00 34.90 25.00 4.84 DFALQNPSAVPR Labelled 662.84 750.41 3.50 10.00 30.90 25.00 4.84 (U-13C6, 15N4) 662.84 636.37 3.50 10.00 34.90 25.00 4.84 (SEQ ID No.: 100) LAELPADALGPLQR Native 732.41 1037.57 3.50 10.00 34.60 25.00 5.77 (SEQ ID No.: 49) 732.41 754.46 3.50 10.00 50.60 25.00 5.77 732.41 570.34 3.50 10.00 34.60 25.00 5.77 LAELPADALGPLQR Labelled 737.41 1047.52 3.50 10.00 34.60 25.00 5.77 (U-13C6, 15N4) 737.41 764.46 3.50 10.00 50.60 25.00 5.77 (SEQ ID No.: 101) ASDTAMYYCAR Native 654.77 934.39 3.50 10.00 34.70 25.00 5.18 (SEQ ID No.: 50) 654.77 863.35 3.50 10.00 30.70 25.00 5.18 654.77 732.31 3.50 10.00 30.70 25.00 5.18 ASDTAMYYCAR Labelled 659.78 873.36 3.50 10.00 30.70 25.00 5.18 (U-13C6, 15N4) 659.78 742.32 3.50 10.00 30.70 25.00 5.18 (SEQ ID No.: 102) VEGTAFVIFGIQDGEQR Native 622.65 732.33 3.50 10.00 26.30 25.00 6.46 (SEQ ID No.: 51) 622.65 604.27 3.50 10.00 30.30 25.00 6.46 622.65 525.25 3.50 10.00 22.30 25.00 6.46 VEGTAFVIFGIQDGEQR Labelled 625.98 742.33 3.50 10.00 26.30 25.00 6.46 (U-13C6, 15N4) 625.98 530.25 3.50 10.00 22.30 25.00 6.46 (SEQ ID No.: 103) VHQYFNVELIQPGAVK Native 614.67 712.44 3.50 10.00 21.90 25.00 5.70 (SEQ ID No.: 52) 614.67 471.29 3.50 10.00 33.90 25.00 5.70 614.67 509.24 3.50 10.00 21.90 25.00 5.70 VHQYFNVELIQPGAVK Labelled 617.33 720.44 3.50 10.00 21.90 25.00 5.70 (U-13C6, 15N2) 617.33 479.30 3.50 10.00 33.90 25.00 5.70 (SEQ ID No.: 104)
[0317] Table 8 shows a summary of analytical validation. The following abbreviations are used in table 8 are: LLOQ—lower limit of quantitation; ULOQ—upper limit of quantification; CV—coefficient of variation
TABLE-US-00013 TABLE 8 Medium Low concentration concentration (LLOQ + LLOQ ULOQ CV (LLOQ) ULOQ)/2 (ng/ (ng/ (%) CV Accuracy CV Accuracy Peptide ml) ml) limit Validation (%) (%) (%) (%) AADDTWEPFASGK 6.37 1630.0 20 Pass 19.2 90.7 7.2 98.4 (SEQ ID No.: 35) ADQVCINLR 0.10 1630.0 20 Pass 12.5 96.9 11.2 97.9 (SEQ ID No.: 17) AHVDALR 0.10 1630.0 20 Pass 2.7 99.4 2.1 99.2 (SEQ ID No.: 24) ALDFAVGEYNK 1.59 1630.0 20 Pass 15.2 89.5 4.4 97.8 (SEQ ID No.: 8) ANRPFLVFIR 0.10 1630.0 20 Pass 10.7 87.6 2.9 97.7 (SEQ ID No.: 38) ASDTAMYYCAR 25.47 1630.0 20 Pass 18.3 90.4 11.8 94.7 (SEQ ID No.: 50) ATEHLSTLSEK 0.40 1630.0 20 Pass 17.5 93.9 2.0 99.1 (SEQ ID No.: 25) CNLLAEK 25.47 1630.0 20 Pass 11.3 99.1 11.3 97.6 (SEQ ID No.: 27) CQSWSSMTPHR 25.47 1630.0 20 Pass 18.4 95.6 19.2 88.7 (SEQ ID No.: 4) DFALQNPSAVPR 6.37 407.5 20 Pass 11.1 95.9 8.9 94.9 (SEQ ID No.: 48) DSGSYFCR 1.59 25.5 20 Pass 15.7 96.9 9.8 97.3 (SEQ ID No.: 46) DSVTGTLPK 0.10 407.5 40 Pass 18.3 93.8 5.7 97.8 (SEQ ID No.: 36) EAQLPVIENK 0.02 1630.0 20 Pass 19.5 90.5 2.7 98.3 (SEQ ID No.: 3) EITALAPSTMK 6.37 1630.0 40 Pass 3.2 96.7 2.5 98.5 (SEQ ID No.: 5) EQHLFLPFSYK 6.37 407.5 20 Pass 6.8 93.6 4.0 98.8 (SEQ ID No.: 16) EQLSLLDR 6.37 407.5 20 Pass 6.8 94.4 5.2 96.4 (SEQ ID No.: 12) ESDTSYVSLK 1.59 407.5 20 Pass 17.2 94.1 7.7 98.8 (SEQ ID No.: 20) FNAVLTNPQGDYDTSTGK 25.47 1630.0 20 Pass 28.0 91.1 15.3 94.0 (SEQ ID No.: 6) GCPDVQASLPDAK 1.59 1630.0 40 Pass 25.5 95.8 19.4 98.7 (SEQ ID No.: 32) GDVAFVK 0.40 1630.0 20 Pass 20.3 87.5 1.9 99.9 (SEQ ID No.: 13) GGEGTGYFVDFSVR 6.37 407.5 20 Pass 17.0 92.0 12.1 91.1 (SEQ ID No.: 28) GHMLENHVER 25.47 1630.0 20 Pass 13.3 99.3 1.7 98.8 (SEQ ID No.: 2) GLPNVVTSAISLPNIR 0.40 1630.0 20 Pass 29.8 96.0 36.5 96.9 (SEQ ID No.: 1) GSPAINVAVHVFR 6.37 1630.0 40 Pass 24.6 79.6 16.5 87.8 (SEQ ID No.: 34) GYSIFSYATK 1.59 1630.0 20 Pass 8.1 98.4 3.1 98.7 (SEQ ID No.: 21) HFQNLGK 6.37 1630.0 40 Pass 29.6 96.4 21.8 98.0 (SEQ ID No.: 40) IADAHLDR 0.10 407.5 40 Pass 9.3 96.7 7.3 97.9 (SEQ ID No.: 29) IAELSATAQEIIK 0.02 1630.0 40 Pass 12.2 92.8 7.5 97.0 (SEQ ID No.: 15) IAYGTQGSSGYSLR 6.37 1630.0 20 Pass 11.6 97.4 7.4 94.1 (SEQ ID No.: 37) ILNIFGVIK 0.02 407.5 20 Pass 34.2 97.9 42.1 99.8 (SEQ ID No.: 44) ILTSDVFQDCNK not pass 85.8 94.5 25.9 97.0 (SEQ ID No.: 18) LAELPADALGPLQR 0.10 1630.0 20 Pass 10.9 95.5 8.8 95.1 (SEQ ID No.: 49) LLDSLPSDTR 0.10 1630.0 20 Pass 14.2 88.6 3.6 98.7 (SEQ ID No.: 42) LVGGPMDASVEEEGVRR not pass 82.4 75.7 44.1 87.5 (SEQ ID No.: 9) LVLLNAIYLSAK 0.40 1630.0 20 Pass 80.6 76.4 70.8 97.2 (SEQ ID No.: 43) SDVMYTDWK 6.37 1630.0 20 Pass 18.6 93.9 4.5 96.5 (SEQ ID No.: 10) STDYGIFQINSR 1.59 1630.0 20 Pass 14.3 85.7 6.5 98.7 (SEQ ID No. 22) TFTLLDPK 6.37 1630.0 20 Pass 4.8 98.7 3.8 99.4 (SEQ ID No.: 33) TINPAVDHCCK 1.59 1630.0 40 Pass 22.8 97.8 36.4 98.4 (SEQ ID No.: 41) TNQVNSGGVLLR 1.59 1630.0 20 Pass 7.4 96.8 6.4 99.1 (SEQ ID No.: 7) TSCLLFMGR 25.47 1630.0 20 Pass 15.5 99.3 10.4 97.8 (SEQ ID No.: 39) TVVQPSVGAAAGPVVPPCPGR 6.37 1630.0 20 Pass 10.2 93.7 9.5 99.2 (SEQ ID No.: 26) VEGTAFVIFGIQDGEQR not pass 20.3 94.5 17.0 89.0 (SEQ ID No.: 51) VHQYFNVELIQPGAVK 0.81 13300.0 40 Pass 45.2 87.8 30.4 98.4 (SEQ ID No.: 52) VLDLSCNR 0.02 1630.0 20 Pass 10.7 94.4 7.2 99.6 (SEQ D No.: 47) VSASPLLYTLIEK 0.10 1630.0 20 Pass 11.1 96.4 5.9 97.6 (SEQ ID No.: 45) WCALSHHER 6.37 1630.0 20 Pass 15.0 99.2 10.8 99.6 (SEQ ID No.: 14) WEMPFDPQDTHQSR 25.47 1630.0 20 Pass 11.2 93.0 7.6 95.1 (SEQ ID No.: 11) YAGSQVASTSEVLK 1.59 1630.0 20 Pass 16.1 91.2 5.6 95.8 (SEQ ID No.: 19) YWCNDGK (SEQ ID No.: 23) 6.37 407.5 20 Pass 8.5 97.7 19.8 98.7 Median 1.6 1630.0 — — 15.3 94.5 7.6 97.9 High concentration Matrix effects (ULOQ) Significant CV Accuracy P- matrix Matrix slope slope % (%) (%) value effects? factor BSA plasma deviation AADDTWEPFASGK 10.0 94.5 0.004 Yes X 0.03+/−0.006 0.019+/−0.001 157.9 (SEQ ID No.: 35) ADQVCINLR 6.5 97.8 0.06 No — 0.068+/−0.005 0.062+/−0.002 109.7 (SEQ ID No.: 17) AHVDALR 4.5 99.7 0.98 No — 0.033+/−0 0.031+/−0 106.5 (SEQ ID No.: 24) ALDFAVGEYNK 5.4 94.4 0.6 No — 0.024+/−0.001 0.024+/−0.002 100 (SEQ ID No.: 8) ANRPFLVFIR 5.0 99.3 0.41 No — 0.044+/−0.002 0.042+/−0.003 104.8 (SEQ ID No.: 38) ASDTAMYYCAR 11.7 93.2 0.01 Yes X 0.013+/−0.002 0.009+/−0 144.4 (SEQ ID No.: 50) ATEHLSTLSEK 5.4 97.5 0.02 Yes X 0.024+/−0 0.023+/−0.001 104.3 (SEQ ID No.: 25) CNLLAEK 7.6 96.7 0.83 No — 0.007+/−0.001 0.006+/−0 116.7 (SEQ ID No.: 27) CQSWSSMTPHR 6.5 98.0 0.73 No — 0.029+/−0.003 0.019+/−0.003 152.6 (SEQ ID No.: 4) DFALQNPSAVPR 6.6 96.3 0.83 No — 0.146+/−0.009 0.391+/−0.046 37.3 (SEQ ID No.: 48) DSGSYFCR 10.6 98.2 0.45 No — 0.061+/−0.003 0.054+/−0.004 113 (SEQ ID No.: 46) DSVTGTLPK 5.1 98.7 0.99 No — 0.114+/−0.003 0.133+/−0.004 85.7 (SEQ ID No.: 36) EAQLPVIENK 1.8 99.8 0.53 No — 0.022+/−0 0.024+/−0 91.7 (SEQ ID No.: 3) EITALAPSTMK 17.9 98.6 0.07 No — 0.027+/−0.001 0.035+/−0 77.1 (SEQ ID No.: 5) EQHLFLPFSYK 4.4 98.4 0.58 No — 0.02+/−0 0.021+/−0.001 95.2 (SEQ ID No.: 16) EQLSLLDR 4.5 98.2 0.02 Yes X 0.074+/−0.002 0.135+/−0.01 54.8 (SEQ ID No.: 12) ESDTSYVSLK 4.2 99.1 0.11 No — 0.116+/−0.009 0.198+/−0.033 58.6 (SEQ ID No.: 20) FNAVLTNPQGDYDTSTGK 12.1 95.3 0.38 No — 0.013+/−0.004 0.013+/−0.006 100 (SEQ ID No.: 6) GCPDVQASLPDAK 32.3 97.2 0.72 No — 0.1+/−0.006 0.12+/−0.01 83.3 (SEQ ID No.: 32) GDVAFVK 6.6 97.8 0.04 Yes X 0.02+/−0.001 0.02+/−0 100 (SEQ ID No.: 13) GGEGTGYFVDFSVR 14.5 91.1 0.11 No — 0.069+/−0.009 0.076+/−0.024 90.8 (SEQ ID No.: 28) GHMLENHVER 4.9 99.1 0.72 No — 0.01+/−0 0.01+/−0 100 (SEQ ID No.: 2) GLPNVVTSAISLPNIR 38.5 99.5 0.45 No — 0.038+/−0.002 0.026+/−0.001 146.2 (SEQ ID No.: 1) GSPAINVAVHVFR 19.6 79.3 0.98 No — 0.011+/−0.002 0.015+/−0.002 73.3 (SEQ ID No.: 34) GYSIFSYATK 5.8 99.6 0.75 No — 0.036+/−0.002 0.036+/−0.002 100 (SEQ ID No.: 21) HFQNLGK 3.3 98.8 0.31 No — 0.039+/−0.001 0.039+/−0.001 100 (SEQ ID No.: 40) IADAHLDR 3.5 99.0 0.01 Yes X 0.187+/−0.003 0.374+/−0.018 50 (SEQ ID No.: 29) IAELSATAQEIIK 7.5 98.1 0.56 No — 0.024+/−0.001 0.024+/−0.001 100 (SEQ ID No.: 15) IAYGTQGSSGYSLR 12.7 85.3 0.27 No — 0.12+/−0.027 0.106+/−0.017 113.2 (SEQ ID No.: 37) ILNIFGVIK 38.7 97.9 0.23 No — 0.053+/−0.001 0.05+/−0.001 106 (SEQ ID No.: 44) ILTSDVFQDCNK 41.6 76.9 0.05 No — 0.281+/−0.047 0.443+/−0.16 63.4 (SEQ ID No.: 18) LAELPADALGPLQR 12.5 96.5 0.08 No — 0.099+/−0.004 0.159+/−0.011 62.3 (SEQ ID No.: 49) LLDSLPSDTR 4.1 98.9 0.19 No — 0.024+/−0.001 0.025+/−0 96 (SEQ ID No.: 42) LVGGPMDASVEEEGVRR 29.0 75.4 — (SEQ ID No.: 9) LVLLNAIYLSAK 71.4 98.3 0.49 No — 0.022+/−0.001 0.023+/−0.001 95.7 (SEQ ID No.: 43) SDVMYTDWK 11.8 97.4 0.17 No — 0.077+/−0.004 0.084+/−0.009 91.7 (SEQ ID No.: 10) STDYGIFQINSR 5.6 98.4 0.86 No — 0.011+/−0.001 0.011+/−0.001 100 (SEQ ID No. 22) TFTLLDPK 5.7 97.1 0.55 No — 0.01+/−0.001 0.009+/−0 111.1 (SEQ ID No.: 33) TINPAVDHCCK 20.5 98.5 0.11 No — 0.141+/−0.009 0.107+/−0.003 131.8 (SEQ ID No.: 41) TNQVNSGGVLLR 8.8 98.4 0.23 No — 0.038+/−0.002 0.036+/−0.003 105.6 (SEQ ID No.: 7) TSCLLFMGR 7.5 99.0 0.69 No — 0.052+/−0.003 0.043+/−0.001 120.9 (SEQ ID No.: 39) TVVQPSVGAAAGPVVPPCPGR 10.1 96.3 0.93 No — 0.15+/−0.014 0.127+/−0.011 118.1 (SEQ ID No.: 26) VEGTAFVIFGIQDGEQR 25.4 98.3 0.58 No — (SEQ ID No.: 51) VHQYFNVELIQPGAVK 25.8 95.1 0.15 No — 0.032+/−0.002 0.032+/−0.004 100 (SEQ ID No.: 52) VLDLSCNR 15.8 99.7 0.51 No — 0.067+/−0.001 0.065+/−0.001 103.1 (SEQ D No.: 47) VSASPLLYTLIEK 9.0 99.1 0.49 No — 0.083+/−0.003 0.081+/−0.004 102.5 (SEQ ID No.: 45) WCALSHHER 12.6 97.7 0.4 No — 0.057+/−0.002 0.045+/−0.001 126.7 (SEQ ID No.: 14) WEMPFDPQDTHQSR 6.2 97.7 0.61 No — 0.011+/−0.001 0.011+/−0.001 100 (SEQ ID No.: 11) YAGSQVASTSEVLK 6.1 96.1 0.38 No — 0.03+/−0.002 0.029+/−0.004 103.4 (SEQ ID No.: 19) YWCNDGK (SEQ ID No.: 23) 10.9 99.0 0.09 No — 0.439+/−0.015 0.569+/−0.041 77.2 Median 8.2 98.0 — — —
TABLE-US-00014 TABLE 9 Cohort 1 - Statistical analysis results. Kendall's Tau trend test between peptide quantities and COVID19 treatment escalation score (WHO 0, 3, 4, 5, 6, 7) Peptide Peptide.Genes pval.kt p.adjust.bh.kt slope.kt WEMPFDPQDTHQSR (SEQ ID WEMPFDPQDTHQSR (SEQ ID No.: 11); 5.92E−08 1.33E−06 1332.332 No.: 11) SERPINA3 EQLSLLDR (SEQ ID No.: 12) EQLSLLDR (SEQ ID No.: 12); SERPINA3 6.63E−08 1.33E−06 264.684 CNLLAEK (SEQ ID No.: 27) CNLLAEK (SEQ ID No.: 27); AHSG 1.80E−07 1.80E−06 −1442.73 GYSIFSYATK (SEQ ID No.: 21) GYSIFSYATK (SEQ ID No.: 21); CRP 1.80E−07 1.80E−06 208.8282 TVVQPSVGAAAGPVVPPCPGR TVVQPSVGAAAGPVVPPCPGR (SEQ ID No.: 2.78E−07 2.06E−06 −746.215 (SEQ ID No.: 26) 26); AHSG GDVAFVK (SEQ ID No.: 13) GDVAFVK (SEQ ID No.: 13); TF 3.09E−07 2.06E−06 −2079.83 WCALSHHER (SEQ ID No.: 14) WCALSHHER (SEQ ID No.: 14); TF 7.99E−07 4.56E−06 −2224.65 ATEHLSTLSEK (SEQ ID No.: 25) ATEHLSTLSEK (SEQ ID No.: 25); APOA1 9.82E−07 4.91E−06 −5266.75 AHVDALR (SEQ ID No.: 24) AHVDALR (SEQ ID No.: 24); APOA1 1.21E−06 5.36E−06 −4172.75 LVLLNAIYLSAK (SEQ ID No.: 43) LVLLNAIYLSAK (SEQ ID No.: 43); SERPING1 2.97E−06 1.19E−05 689.3288 ALDFAVGEYNK (SEQ ID No.: 8) ALDFAVGEYNK (SEQ ID No.: 8); CST3 3.62E−06 1.32E−05 14.27221 SDVMYTDWK (SEQ ID No.: 10) SDVMYTDWK (SEQ ID No.: 10); ORM2 5.33E−06 1.78E−05 299.6885 LLDSLPSDTR (SEQ ID No.: 42) LLDSLPSDTR (SEQ ID No.: 42); SERPING1 9.42E−06 2.90E−05 167.6913 YWCNDGK (SEQ ID No.: 23) YWCNDGK (SEQ ID No.: 23); LYZ 1.25E−05 3.56E−05 0.530168 VHQYFNVELIQPGAVK (SEQ VHQYFNVELIQPGAVK (SEQ ID No.: 52); C3 1.80E−05 4.80E−05 675.1894 ID No.: 52) GSPAINVAVHVFR (SEQ ID GSPAINVAVHVFR (SEQ ID No.: 34); TTR 1.21E−04 3.03E−04 −251.965 No.: 34) GLPNVVTSAISLPNIR (SEQ ID GLPNVVTSAISLPNIR (SEQ ID No.: 1); PRG4 1.55E−04 3.65E−04 8.66864 No.: 1) FNAVLTNPQGDYDTSTGK FNAVLTNPQGDYDTSTGK (SEQ ID No.: 6); 1.98E−04 4.16E−04 150.7118 (SEQ ID No.: 6) C1QC TFTLLDPK (SEQ ID No.: 33) TFTLLDPK (SEQ ID No.: 33); PGLYRP2 1.98E−04 4.16E−04 −32.7362 ILTSDVFQDCNK (SEQ ID No.: 18) ILTSDVFQDCNK (SEQ ID No.: 18); VWF 2.14E−04 4.28E−04 0.842321 LAELPADALGPLQR (SEQ ID LAELPADALGPLQR (SEQ ID No.: 49); IGFALS 2.51E−04 4.78E−04 −58.0734 No.: 49) EQHLFLPFSYK (SEQ ID No.: 16) EQHLFLPFSYK (SEQ ID No.: 16); APOB 5.03E−04 9.14E−04 52.61636 ADQVCINLR (SEQ ID No.: 17) ADQVCINLR (SEQ ID No.: 17); EFEMP1 1.88E−03 3.27E−03 4.809207 TNQVNSGGVLLR (SEQ ID TNQVNSGGVLLR (SEQ ID No.: 7); C1QC 4.34E−03 7.24E−03 31.51522 No.: 7) AADDTWEPFASGK (SEQ ID AADDTWEPFASGK (SEQ ID No.: 35); TTR 7.58E−03 1.21E−02 −34.0661 No.: 35) GCPDVQASLPDAK (SEQ ID GCPDVQASLPDAK (SEQ ID No.: 32); PGLYRP2 8.04E−03 0.012368 −5.77165 No.: 32) ANRPFLVFIR (SEQ ID No.: 38) ANRPFLVFIR (SEQ ID No.: 38); SERPINC1 0.010811 0.016016 −125.755 GHMLENHVER (SEQ ID No.: 2) GHMLENHVER (SEQ ID No.: 2); ITIH1 0.011454 0.016363 −107.945 TINPAVDHCCK (SEQ ID No.: 41) TINPAVDHCCK (SEQ ID No.: 41); AFM 0.014381 0.019835 −74.1523 HFQNLGK (SEQ ID No.: 40) HFQNLGK (SEQ ID No.: 40); AFM 0.01699 0.022653 −16.8833 IAYGTQGSSGYSLR (SEQ ID IAYGTQGSSGYSLR (SEQ ID No.: 37); KLKB1 0.017948 0.023158 −16.5593 No.: 37) ESDTSYVSLK (SEQ ID No.: 20) ESDTSYVSLK (SEQ ID No.: 20); CRP 0.02111 0.026387 87.19703 IAELSATAQEIIK (SEQ ID No.: 15) IAELSATAQEIIK (SEQ ID No.: 15); APOB 0.065656 0.079583 65.92091 STDYGIFQINSR (SEQ ID No.: 22) STDYGIFQINSR (SEQ ID No.: 22); LYZ 0.292861 0.344543 −12.2537 TSCLLFMGR (SEQ ID No.: 39) TSCLLFMGR (SEQ ID No.: 39); SERPIND1 0.352114 0.402416 5.528197 ILNIFGVIK (SEQ ID No.: 44) ILNIFGVIK (SEQ ID No.: 44); TFRC 0.504434 0.560482 −0.33407 DFALQNPSAVPR (SEQ ID No.: 48) DFALQNPSAVPR (SEQ ID No.: 48); IGFALS 0.734001 0.785476 3.771921 EAQLPVIENK (SEQ ID No.: 3) EAQLPVIENK (SEQ ID No.: 3); PLG 0.746202 0.785476 −14.3991 GGEGTGYFVDFSVR (SEQ ID GGEGTGYFVDFSVR (SEQ ID No.: 28); HRG 0.823917 0.845043 −19.6409 No.: 28) CQSWSSMTPHR (SEQ ID No.: 4) CQSWSSMTPHR (SEQ ID No.: 4); PLG 0.871443 0.871443 −6.77322 Cohort 2 - Statistical analysis results. Kendall's Tau trend test between peptide quantities and COVID19 treatment escalation score (WHO 3, 4, 5, 6, 7) adjusted Peptide Peptide.Genes p.palue p.value slope.kt TFTLLDPK (SEQ ID No.: 33) TFTLLDPK (SEQ ID No.: 33); PGLYRP2 1.73E−16 8.29E−15 −232.59 GCPDVQASLPDAK (SEQ ID GCPDVQASLPDAK (SEQ ID No.: 32); 5.21E−16 1.25E−14 −171.366 No.: 32) PGLYRP2 ESDTSYVSLK (SEQ ID No.: 20) ESDTSYVSLK (SEQ ID No.: 20); CRP 2.22E−14 2.66E−13 2388.399 GYSIFSYATK (SEQ ID No.: 21) GYSIFSYATK (SEQ ID No.: 21); CRP 2.22E−14 2.66E−13 317.1732 CNLLAEK (SEQ ID No.: 27) CNLLAEK (SEQ ID No.: 27); AHSG 5.18E−13 4.97E−12 −20167.6 TVVQPSVGAAAGPVVPPCPGR TVVQPSVGAAAGPVVPPCPGR (SEQ ID No.: 1.43E−12 1.14E−11 −1445.22 (SEQ ID No.: 26) 26); AHSG GDVAFVK (SEQ ID No.: 13) GDVAFVK (SEQ ID No.: 13); TF 3.70E−11 2.54E−10 −3235.65 DSVTGTLPK (SEQ ID No.: 36) DSVTGTLPK (SEQ ID No.: 36); KLKB1 5.47E−11 3.28E−10 −59.9985 AHVDALR (SEQ ID No.: 24) AHVDALR (SEQ ID No.: 24); APOA1 7.50E−10 3.73E−09 −6118.33 ATEHLSTLSEK (SEQ ID No.: 25) ATEHLSTLSEK (SEQ ID No.: 25); APOA1 7.78E−10 3.73E−09 −8823.07 WCALSHHER (SEQ ID No.: 14) WCALSHHER (SEQ ID No.: 14); TF 1.63E−09 7.13E−09 −5238.31 LAELPADALGPLQR (SEQ ID LAELPADALGPLQR (SEQ ID No.: 49); 4.32E−09 1.73E−08 −55.1319 No.: 49) IGFALS DFALQNPSAVPR (SEQ ID No.: 48) DFALQNPSAVPR (SEQ ID No.: 48); IGFALS 6.33E−09 2.34E−08 −29.6012 TINPAVDHCCK (SEQ ID No.: 41) TINPAVDHCCK (SEQ ID No.: 41); AFM 5.50E−08 1.89E−07 −550.3 EQLSLLDR (SEQ ID No.: 12) EQLSLLDR (SEQ ID No.: 12); SERPINA3 1.31E−07 4.18E−07 2625.823 HFQNLGK (SEQ ID No.: 40) HFQNLGK (SEQ ID No.: 40); AFM 2.37E−07 6.90E−07 −54.3341 GHMLENHVER (SEQ ID No.: 2) GHMLENHVER (SEQ ID No.: 2); ITIH1 2.44E−07 6.90E−07 −517.84 WEMPFDPQDTHQSR (SEQ ID WEMPFDPQDTHQSR (SEQ ID No.: 11); 7.50E−07 2.00E−06 1039.275 No.: 11) SERPINA3 EITALAPSTMK (SEQ ID No.: 5) EITALAPSTMK (SEQ ID No.: 5); ACTA1; 9.37E−07 2.37E−06 23.97025 ACTA2; ACTB; ACTC1; ACTG1; ACTG2 ALDFAVGEYNK (SEQ ID No.: 8) ALDFAVGEYNK (SEQ ID No.: 8); CST3 1.45E−06 3.49E−06 31.13371 VLDLSCNR (SEQ ID No.: 47) VLDLSCNR (SEQ ID No.: 47); CD14 2.37E−06 5.41E−06 13.53262 ANRPFLVFIR (SEQ ID No.: 38) ANRPFLVFIR (SEQ ID No.: 38); SERPINC1 4.28E−06 8.92E−06 −314.623 IADAHLDR (SEQ ID No.: 29) IADAHLDR (SEQ ID No.: 29); HRG 4.28E−06 8.92E−06 −51.6363 IAYGTQGSSGYSLR (SEQ ID IAYGTQGSSGYSLR (SEQ ID No.: 37); KLKB1 1.20E−05 2.40E−05 −37.7417 No.: 37) VEGTAFVIFGIQDGEQR (SEQ VEGTAFVIFGIQDGEQR (SEQ ID No.: 51); C3 1.85E−05 3.56E−05 798.2789 ID No.: 51) YAGSQVASTSEVLK (SEQ ID YAGSQVASTSEVLK (SEQ ID No.: 19); VWF 2.06E−05 3.79E−05 25.56539 No.: 19) TSCLLFMGR (SEQ ID No.: 39) TSCLLFMGR (SEQ ID No.: 39); SERPIND1 6.30E−05 0.000112 −84.4991 EAQLPVIENK (SEQ ID No.: 3) EAQLPVIENK (SEQ ID No.: 3); PLG 0.000861 0.001476 −222.069 GGEGTGYFVDFSVR (SEQ ID GGEGTGYFVDFSVR (SEQ ID No.: 28); HRG 0.002483 0.004109 −171.783 No.: 28) IAELSATAQEIIK (SEQ ID No.: 15) IAELSATAQEIIK (SEQ ID No.: 15); APOB 0.005025 0.00804 −80.0036 ADQVCINLR (SEQ ID No.: 17) ADQVCINLR (SEQ ID No.: 17); EFEMP1 0.005745 0.008895 7.558618 AADDTWEPFASGK (SEQ ID AADDTWEPFASGK (SEQ ID No.: 35); TTR 0.008423 0.012635 −27.1604 No.: 35) ILTSDVFQDCNK (SEQ ID No.: 18) ILTSDVFQDCNK (SEQ ID No.: 18); VWF 0.017321 0.025194 10.14308 GSPAINVAVHVFR (SEQ ID GSPAINVAVHVFR (SEQ ID No.: 34); TTR 0.039824 0.056222 −104.475 No.: 34) EQHLFLPFSYK (SEQ ID No.: 16) EQHLFLPFSYK (SEQ ID No.: 16); APOB 0.055657 0.076206 286.7285 CQSWSSMTPHR (SEQ ID No.: 4) CQSWSSMTPHR (SEQ ID No.: 4); PLG 0.057154 0.076206 −713.482 LLDSLPSDTR (SEQ ID No.: 42) LLDSLPSDTR (SEQ ID No.: 42); SERPING1 0.059463 0.077108 116.5034 YWCNDGK (SEQ ID No.: 23) YWCNDGK (SEQ ID No.: 23); LYZ 0.061044 0.077108 6.794503 LVLLNAIYLSAK (SEQ ID No.: 43) LVLLNAIYLSAK (SEQ ID No.: 43); SERPING1 0.253996 0.31261 448.8475 FNAVLTNPQGDYDTSTGK FNAVLTNPQGDYDTSTGK (SEQ ID No.: 6); 0.280187 0.336224 −124.192 (SEQ ID No.: 6) C1QC STDYGIFQINSR (SEQ ID No.: 22) STDYGIFQINSR (SEQ ID No.: 22); LYZ 0.301334 0.352781 19.95125 VHQYFNVELIQPGAVK (SEQ VHQYFNVELIQPGAVK (SEQ ID No.: 52); C3 0.425932 0.486779 −341.517 ID No.: 52) VSASPLLYTLIEK (SEQ ID No.: 45) VSASPLLYTLIEK (SEQ ID No.: 45); TFRC 0.608057 0.678762 0.295307 ILNIFGVIK (SEQ ID No.: 44) ILNIFGVIK (SEQ ID No.: 44); TFRC 0.736992 0.797797 −0.16742 SDVMYTDWK (SEQ ID No.: 10) SDVMYTDWK (SEQ ID No.: 10); ORM2 0.747934 0.797797 86.59714 GLPNVVTSAISLPNIR (SEQ ID GLPNVVTSAISLPNIR (SEQ ID No.: 1); PRG4 0.848462 0.885351 0.976178 No.: 1) TNQVNSGGVLLR (SEQ ID TNQVNSGGVLLR (SEQ ID No.: 7); C1QC 0.891763 0.907806 −4.61482 No.: 7) DSGSYFCR (SEQ ID No.: 46) DSGSYFCR (SEQ ID No.: 46); FCGR3A 0.907806 0.907806 −0.1716