IN VITRO PROCESS FOR THE QUANTIFICATION OF CARBOXYMETHYL AND CARBOXYETHYL LEVEL OF ALBUMIN IN A SAMPLE
20170168068 ยท 2017-06-15
Inventors
Cpc classification
G01N33/6851
PHYSICS
G01N2800/042
PHYSICS
G01N2560/00
PHYSICS
International classification
Abstract
An in vitro method for the identification and quantification of glycated human serum albumin to assess the extent of diabetic complications in diseased individuals. Further, a diagnostic kit for identifying the extent of diabetes in a diseased individual by estimating glycated serum albumin levels in such individuals.
Claims
1. An in vitro process for the identification, quantification or identification and quantification of carboxymethyl and carboxyethyl level of albumin in a sample comprising; (a) subjecting the sample to mass spectrometry to generate fragment ions; (b) identifying the specificity of fragment ions obtained in step (a) to advanced glycation end-product (AGE) modified glycation sites selected from carboxymethyl and carboxyethyl modified amino acid residues in human serum albumin by comparing said fragment ions with signature ions specific to modified carboxymethyl and carboxyethyl glycated sites selected from the group consisting of K36, K88, K160, K161, K183, K375, K438, K490 and K549 in Seq Id No. 1; and (c) further quantifying the levels of carboxymethyl and carboxyethyl modified peptide content wherein AGE modified glycation sites are situated at lysine (K) residues.
2. The process as claimed in claim 1, wherein AGE modified glycated sites are identified and quantified in human serum albumin represented by amino acid sequence having at least 80% similarity with Seq Id No. 1.
3. The process as claimed in claim 1, wherein AGE modified glycated sites are preferably selected from the group consisting of K549, K438, K490, K88, and K375.
4. A diagnostic kit for the identification, quantification or identification and quantification of carboxymethyl and carboxyethyl level of albumin in a sample withdrawn from a subject, said kit comprising; a sterilized device to draw out serum from said subject which serum is to be subjected to fragmentation by mass spectrometry methods; a chart, figure or representation of the signature fragment ion library specific to carboxymethyl and carboxyethyl modified glycated sites selected from the group consisting of K36, K88, K160, K161, K183, K375, K438, K490 and K549; and a catalogue or product information sheet containing instructions for use of the kit.
5. The diagnostic kit of claim 4 wherein said sterilized device is a minimally invasive device.
Description
BRIEF DESCRIPTION OF FIGURES
[0022]
[0023]
[0024]
[0025]
[0026]
DETAILED DESCRIPTION OF THE INVENTION
[0027] The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated.
[0028] The present invention provides a process for identifying and quantifying glycated level of albumin in human serum comprising;
(a) subjecting human serum sample to mass spectrometry to generate fragment ions;
(b) identifying the specificity of fragment ions obtained in step (a) to advanced glycation end-product (AGE) modified glycation sites selected from deoxyfructosyl, carboxymethyl and carboxyethyl modified amino acid residues in human serum albumin by comparing the said fragment ions with signature ions in the diagnostic fragment ion library; and
(c) further quantifying the AGE modified peptide content wherein AGE modified glycation sites are situated at lysine and valine residues.
[0029] In line with the above, the method to identify glycated peptides and quantify the glycemic status in individuals diagnosed with diabetes involves subjecting a human serum sample to fragmentation to generate diagnostic fragment ions. This fragmentation is brought about by mass spectrometry methods. The fragment ions resulting from the given biological fluid are determined for their specificity with deoxyfructosyl, carboxymethyl and carboxyethyl modified amino acid residues in human serum albumin, by comparing the said ions with the ions in a diagnostic ion library. A reference may be made to Table 1 of the present invention containing the said diagnostic fragment ions specific to standard glycated peptide sites.
[0030] Finally, the glycated albumin sites are quantified by target based mass spectrometry methods, by which the extent of diabetes and its associated complications in a diabetic or a person exhibiting hyperglycaemic symptoms can be assessed. The % carboxymethylated and carboxyethylated serum albumin levels is estimated by MRM (Multiple reaction monitoring)/PRM (Parallel reaction monitoring)/SWATH (Sequential Window Acquisition of all Theoretical Fragment Ion Spectra)/MS.sup.E methods.
[0031] Accordingly, the AGE modification of human serum albumin (HSA) is depicted in
[0032] In the present specification Amadori modified lysine i.e. AML and deoxyfructosyl modified lysine refer to the same modification and may be used interchangeably.
[0033] The levels of these AGE modified peptides have been quantified employing the present method. The glycated sites are represented in Seq Id No. 1 i.e. the amino acid sequence representing human serum albumin Glycated protein sequences having 80% similarity with Seq Id No. 1 are quantified by employing the present method.
[0034] In an embodiment, the present invention provides for the identification, characterization and quantification of thirteen glycated peptides in diabetic plasma comprising four AML modified residues, seven CML modified residues and two CEL modified residues representing nine lysine (K) sites of albumin.
[0035] In the present application, in accordance with the standard alphabetic letters used to represent amino acids, the alphabet K referred to in the following embodiments represents the amino acid lysine.
[0036] In another preferred embodiment, the present invention provides for the identification and quantification of AGE glycated sites of Seq Id No. 1 selected from the group consisting of K36, K88, K160, K161, K183, K375, K438, K490 and K549.
[0037] In yet another preferred embodiment, the present invention provides highly sensitive glycation modification sites of amadori or deoxyfructosyl modified or carboxymethyl lysine modified sites selected from the group consisting of K549, K438, K490, K88 and K375 of SEQ ID No. 1.
[0038] On performing SWATH analysis, the fold change of the peptides were analysed. Their glycated sites selected from K549, K438, K490, K88 and K375 of Seq Id No. 1 were determined to have maximum fold change depending on their respective m/z intensities (
[0039] Accordingly, the aforementioned sites have both AML and CML modifications, and position K549 of SEQ ID No. 1 has an additional CEL modification. K375 (m/z 886.4) showed the highest significant fold change in prediabetes and diabetic plasma. Although it is intriguing that only one lysine site i.e. K549 was found to be CEL modified, this result strongly supports previous studies where no CEL modified peptide was identified.
[0040] Precursor ions appertaining to K549, K438, K490, K88, K549 were defined to serve as potential novel markers for assessing the degree of glycation or diabetic glycaemic status in general and
m/z 645.8 (K.sup.AML*QTALVELVK), m/z 593.8 (K.sup.CML*QTALVELVK), m/z 600.8 (K.sup.CEL*QTALVELVK),
m/z 601.3 (K.sup.AML*VPQVSTPTLVEVSR), m/z 528.3 (K.sup.CML*VPQVSTPTLVEVSRNLGK),
m/z 834.6 (MPC*AEDYLSVVLNQLC*VLHEK.SUP.AML.*TPVSDR),
m/z 808.6 (MPC*AEDYLSVVLNQLC*VLHEK.SUP.CML.*TPVSDR),
m/z 665.5 (TC*VADESAENC*DK.SUP.AML.*SLHTLFGDK),
m/z 639.5 (TC*VADESAENC*DK.SUP.CML.*SLHTLFGDK),
[0041] m/z 886.4 (QNC*ELFEQLGEYK.sup.CML*FQNALLVR) in particular.
[0042] In another embodiment, the present invention provides a diagnostic ion library comprising fragment ions specific to deoxyfructosyl, carboxymethyl and carboxyethyl modified glycation sites in human serum albumin.
[0043] This present diagnostic ion library is used for quantification of AGE modified peptides of albumin by targeted mass spectrometric approaches. The process for establishing the diagnostic ion library is shown in
[0044] In yet another embodiment, the present invention provides quantification of AGE modified peptides of human serum albumin by targeted SWATH workflow in clinical plasma to evaluate their use as diagnostic peptides.
[0045] In a preferred embodiment, the present invention provides a diagnostic kit for the identification and quantification of AGE modified glycated human serum level content comprising: [0046] (a) a sterilized device, preferably minimally invasive, to draw out serum from the diseased individual, which is to be subjected to fragmentation by mass spectrometry methods; [0047] (b) a chart, figure or representation of the signature fragment ion library specific to each glycated peptide; and [0048] (c) a catalogue or product information sheet containing instructions for use of the kit.
[0049] Accordingly, the method of employing the present diagnostic kit involves subjecting the human serum sample to fragmentation to generate diagnostic fragment ions. This fragmentation is brought about by target based mass spectrometry methods. The fragment ions resulting from the given biological fluid are determined for their specificity with deoxyfructosyl, carboxymethyl and carboxyethyl modified amino acid residues in human serum albumin, by comparing the said ions with the ions present in the diagnostic ion library. A reference is made by the subject or a pathologist to a chart, figure or representation containing the signature diagnostic fragment ions specific to standard glycated peptide sites accompanying the diagnostic kit, and the concentration of glycated peptides is quantified by mass spectrometric methods. The extent of diabetes is thus assessed using the present diagnostic kit.
[0050] In an embodiment, the present invention provides the use of the diagnostic kit in identifying the extent of diabetic complications in a subject, by classifying the diabetic stage to be pre-diabetic, current diabetic stage and also in the diagnosis of microalbuminuria in the said subject.
[0051] The following examples, which include preferred embodiments, will serve to illustrate the practice of this invention, it being understood that the particulars shown are by way of example and for purpose of illustrative discussion of preferred embodiments of the invention.
EXAMPLES
Example 1
Establishment of Diagnostic Fragment Ion Library for AML, CML and CEL Peptides of Human Serum Albumin
[0052] The present fragment ion library was constructed sequentially in five stages as depicted in
Example 2
Validation of Glycated Peptides of Human Serum Albumin by MS/MS Spectrum
[0053] The search algorithm used by Korwar A M et al (Eur J Mass Spectrom 2012; 18:475-81) was employed to identify glycated peptides which were manually validated by inspecting each MS/MS spectrum. Accordingly, at MS, each AGE modified peptide precursor was reviewed for the presence of unmodified peptide precursor ion.
[0054] CML modified precursor ion m/z 566.6 (1697.9 Da MH.sup.+) corresponding to peptide (K*VPQVSTPTLVEVSR) was inspected for the presence of its unmodified precursor ion m/z 547.31 (1639.9 Da). Such manual interrogation was performed for all glycated peptide precursor ions having an increased mass of 162.053, 58.005 and 72.021 Da corresponding to AML, CML and CEL modifications respectively. At MS/MS, glycated precursor fragment ions were carefully evaluated for glycated lysine ions of m/z 291.14 [128.09+162.051], m/z 187.09 [128.09+58.005], m/z 201.11 [128.09+72.021] corresponding to AML, CML and CEL respectively, followed by increment shift in mass across b or y ion series depending on the site of modification in peptide.
[0055] Example of modification at N-terminal lysine, CML modified K*VPQVSTPTLVEVSR (m/z566.652) peptide spawned signature diagnostic b series fragment ions b.sub.1.sup.+187.10, b.sub.2.sup.+286.17, b.sub.3.sup.+383.22, b.sub.4.sup.+511.28, b.sub.5.sup.+610.35, b.sub.6.sup.+697.38, b.sub.7.sup.+798.43, b.sub.9.sup.+996.53 bearing 58 Da mass shift across b ion series.
[0056] Example of modifications at C-terminal, CML modified RHPYFYAPELLFFAK*R (m/z705.037) peptide spawned signature diagnostic y series fragment ions y.sub.2.sup.+361.21, y.sub.3.sup.+432.25, y.sub.4.sup.+579.32, y.sub.5.sup.+726.39, y.sub.6.sup.+839.47, y.sub.7.sup.+952.56, y.sub.8.sup.+1081.60, y.sub.9.sup.+1178.65 and y.sub.10.sup.+1249.69 bearing 58 Da mass shift across y ion series.
[0057] Example modification in the middle of the peptide, CML modified FGERAFK*AWAVAR (m/z783.910) peptide spawned signature diagnostic fragment ions of both b and y series b.sub.7.sup.+894.44, b.sub.8.sup.+965.48, b.sub.9.sup.2+576.28, b.sub.10.sup.+1222.60, b.sub.11.sup.+1321.66, b.sub.12.sup.+1392.70, y.sub.7.sup.+859.47, y.sub.8.sup.+1006.54, y.sub.9.sup.+1077.58, y.sub.10.sup.2+617.34.
[0058] In certain instances, poor MS/MS techniques result in identification of false positives. Therefore to rule out any such identification, presence of three consecutive fragment ion bearing modification was adopted as an important criterion.
[0059] All such glucose, GA and MG induced glycated peptides are disclosed in Table 1 (Corresponding fragment ions and MS/MS spectra specific to the glycated peptides are not shown). In disagreement with the rule of consecutive ions, some of the peptides were consented as true modifications for their better MS/MS fragmentation pattern, where either y or b series ion were bearing the shift of modified mass, (related data not shown, as well as data related to corresponding peptide fragment ions and MS/MS spectra and corresponding un-glycated peptides is not produced in the present patent application).
[0060] Together 50 glycated peptides were identified for AML, CML and CEL modified HSA. Each of the glycated protein showed 20, 17, 13 modified peptides induced by glucose GA and MG respectively. These 50 modified peptides represented 23 lysine modified sites. K549, K438 and K183 sites were modified 6 times followed by K375, K490 (each 4 times) suggesting that these sites were more sensitive to glycation modification than others (Table 1). MS and corresponding XICs of all 50 glycated peptides along with unmodified control peptides were also performed.
[0061] The reliability of diagnostic fragment ion library for AML, CML and CEL modified peptides of HSA was validated by parallel reaction monitoring of selected glycated peptide ion m/z. The resultant ion chromatograms were extracted with a mass tolerance of 5 ppm and presence of at least 2 corresponding diagnostic fragment ions were confirmed manually.
Example 3
Validation of the Present Fragment Ion Library
[0062] Signature ions specific to glycated peptides determined by the present method of AGE quantification was validated by quantifying and validating the said glycated sites in human serum albumin in clinical plasma samples of healthy subjects and patients supposedly diagnosed having diabetes.
[0063] Accordingly, subjects were recruited from the Madras Diabetes Research Foundation (MDRF). The study was approved by the institutional ethics committee of MDRF and prior written informed consent was obtained from all the study subjects. Descriptive characters and diagnostic parameters including fasting blood glucose, HbA1c, oral glucose tolerance test, postprandial blood Sugar, lipids, urea, creatinine, and microalbumin were measured (data not shown). The plasma samples were classified into four group namely healthy control [Normal glucose tolerance (NGT)], prediabetes [impaired glucose tolerance (IGT)], diabetic[T2DM], microalbuminuria [MIC]. Two plasma samples with similar HbA1c (deviation of <0.2%) were pooled and three such pooled plasma in each group were used for mass spectrometric analysis.
[0064] First, The AGE-HSA MS/MS fragment ion library with diagnostic ions was established for glycation modifications AML, CML and CEL. Diagnostic signature ions were validated for glycated peptides in diabetic plasma using targeted parallel reaction monitoring (PRM) workflow. Further, based on the fragment ion library information, glycated peptides in healthy control, prediabetic, diabetic and microalbuminuria plasma were quantified by using SWATH to discover candidate peptide biomarkers to assess the extent of glycation in diabetes. The results of SWATH were verified by MS.sup.E, another label free quantification method. Two-way analysis of variance (ANOVA) was performed to assess the statistical significance of quantified glycated peptides.
Example 4
Materials and Method Employed for Clinical Sample Preparation and Subsequent Analysis
[0065] All the chemicals were procured from Sigma-Aldrich (Sigma-Aldrich, MO, USA). MS-grade solvents (acetonitrile (ACN), water, and methanol) were procured from J T. Baker (J T. Baker, Pa., USA). RapiGest was procured from Waters (Waters Corporation, MA, USA). Membrane filters of 3 and 30 KDa cut off were procured from Millipore (Millipore, Mass., USA).
Sample Preparation for MS Analysis
[0066] In-solution tryptic digestion: Equal amount of protein (100 g) of AGE-modified HSA and clinical plasma proteins were diluted with 100 L of ammonium bicarbonate buffer (50 mM) containing 0.1% RapiGest followed by incubation at 80 C. for complete proteome solubilization. The denatured proteins were then reduced with DTT (0.100 M) at 60 C. for 15 mM, followed by alkylation with iodoacetamide (0.200 M) at room temperature in the dark for 30 mM. The proteins were digested with proteomic grade trypsin at 1:50 enzyme to substrate ratio overnight at 37 C. The digestion reaction was stopped by adding concentrated HCL and incubated for 10 mM at 37 C. before centrifuged. The peptides were desalted by using C18 Zip tip (Millipore, Mass., USA) and concentrated by vacuum centrifuge and stored at 20 C. until further use.
Liquid Chromatography-Mass Spectrometric Analysis
[0067] Instrument-specific methods and settings [(LC-HR/AM Q-ExactiveOrbitrap and PRM), (Triple TOF 5600 (DDA and SWATH-MS), (label free LC-MSE on SYNAPT HDMS)] were used for the construction of fragment ion library and quantification of glycated peptides.
Structure Analysis
[0068] Three dimensional structure of albumin highlighting lysine residues image was generated by using PyMOL Molecular Graphics System (Schrodinger, LLC). Electrostatic surface potentials were calculated using Bluues with default values as implemented in the Bluues web server (Walsh I, et al Bioinformatics 2012; 28: 2189-90).
Statistical Analysis
[0069] Significant differences between glycated peptide m/z within disease condition and across m/z were determined by using two-way ANOVA followed by Tukey's multiple comparison test. NS represents non-significant difference within the conditions and/or across m/z.
Example 5
Quantification of HSA Glycated Peptides in Clinical Plasma
[0070] AGE modified HSA peptides from pooled plasma samples of healthy control, prediabetic, diabetic and microalbuminuria were quantified by using label-free targeted SWATH workflow. In SWATH analysis, the intensity fold change of 13 glycated peptides listed in
[0071] Further two-way ANOVA was performed to assess the statistical significance of intensity fold change of glycated HSA peptides amongst prediabetes, diabetes and microalbuminuria. Two-way ANOVA results showed statistically significant interaction with a total variance of 29.63% at p, 0.0001 between glycated peptides m/z and different diabetic conditions (prediabetic, diabetic and microalbuminuria). Tukey's multiple comparisons test suggested that, out of 13 consistent glycated peptides, fold expression of four m/z viz. 600.8, 808.6, 665.5 and 886.4 was significant when compared across three diabetic conditions(i) m/z600.8, SiteK549, sequenceK.sup.CEL*QTALVELVK was found to be significant in diabetes (p=0.01) and microalbuminuria (p=0.02) as compared to prediabetes; (ii) m/z808.6, SiteK490, sequence MPC*AEDYLSVVLNQLC*VLHEK.sup.CML*TPVSDR was found to be significant in diabetes (p=0.01) as compared to prediabetes and microalbuminuria; (iii) m/z665.5, SiteK88, sequenceTC*VADESAENC*DK.sup.AML*SLHTLFGDK was found to be more significant in diabetes (p=0.0005) as compared to microalbuminuria and significant in prediabetes (p=0.03) as compared to microalbuminuria; (iv) m/z886.4, SiteK426, sequenceQNC*ELFEQLGEYK.sup.CML*FQNALLVR was found to be more significant in diabetes (p=0.0007) and prediabetes (p=0.003) as compared to microalbuminuria. Further, to determine the significance of intensity fold change of each glycated peptide was compared across all glycated peptides. Glycated peptide ion m/z 886.4 and m/z 433.5 were found to be significant in prediabetic; while m/z 886.4, m/z 433.5 and m/z 665.5 were found to be significant in diabetes; and m/z 601.3 and m/z 593.8 were found to be significant in microalbuminuria condition.
[0072] Further, the results of SWATH analysis were verified by MS.sup.E workflow. Out of 13 glycated peptides, 9 peptides intensity fold change was found to be higher in prediabetes, diabetes and microalbuminuria in comparison with control, and showed similar trend with the SWATH results (
Example 6
Glycated Sites 3D Structures of Human Serum Albumin
[0073] In vitro glucose, GA and MG induced glycated HSA representing glycation sites were studied in three dimensional structure of HSA. From the surface electrostatic distribution calculation it was clear that most of the modified lysine residues were situated near the positive or neutral groove. CML modifications by GA were predominantly found in higher electropositive grooves suggesting CML modification requires highly positive local surface environment.
[0074] In this context, a fragment ion library for synthetically AML, CML, and CEL modified peptides of albumin by using high resolution accurate mass spectrometer followed with rigorous inspection and validation of MS/MS spectra. Furthermore, using the ion library, AML, CML and CEL modified albumin peptides were quantified by targeted SWATH analysis in the clinical plasma.
[0075] The present invention has led to the identification, characterization and quantification of 13 glycated peptides in the clinical diabetic plasma comprising 4-AML, 7-CML and 2-CEL modification representing 9 lysine sites of albumin (K36, K88, K160, K161, K183, K375, K438, K490 and K549). Amongst these sites K549, K438, K490, K88 and K375 were highly sensitive for glycation modification as they had both AML and CML modifications and K549 had additional CEL modification. While K375 (m/z 886.4) showed the highest significant fold change in prediabetes and diabetic plasma. Although it is intriguing that only one lysine site i.e. K549 was found to be CEL modified, this result strongly supports a recent study where no CEL modified peptide was identified.
[0076] The development of fragment ion library for AML, CML and CEL modified peptides of albumin will be very useful for quantification of AGE modified peptides of albumin by targeted mass spectrometric approaches. Using this fragment ion library novel AGE modified peptides were quantified. Precursor ions appertaining to K549, K438, K490, K88, K549 could serve as potential novel markers for assessing the degree of glycation or diabetic glycemic status in general and m/z 645.8 (K.sup.AML*QTALVELVK), m/z 593.8 (K.sup.CML*QTALVELVK), m/z 600.8 (K.sup.CEL*QTALVELVK), m/z 601.3 (K.sup.AML*VPQVSTPTLVEVSR), m/z 528.3 (K.sup.CML*VPQVSTPTLVEVSRNLGK), m/z 834.6 (MPC*AEDYLSVVLNQLC*VLHEK.sup.AML*TPVSDR), m/z 808.6 (MPC*AEDYLSVVLNQLC*VLHEK.sup.CML*TPVSDR), m/z 665.5 (TC*VADESAENC*DK.sup.AML*SLHTLFGDK), m/z 639.5 (TC*VADESAENC*DK.sup.CML*SLHTLFGDK), m/z 886.4 (QNC*ELFEQLGEYK.sup.CML*FQNALLVR) in particular.
Advantages of the Invention
[0077] The present fragment ion library established specific to AML, CML and CEL modified peptides of human serum albumin is useful for quantification of AGE modified peptides of albumin by targeted mass spectrometric approaches.
[0078] The peptides involving these lysine sites can be potential novel markers to assess the degree of glycation in diabetes.
[0079] Precursor ions appertain to K549, K438, K490, K88, K549 could serve as potential novel markers for assessing the degree of glycation or diabetic glycemic status in general.
TABLE-US-00001 TABLE 1 Diagnostic fragment ion library for AML, CML and CEL peptides of albumin Monoisotopic Modified AGE-modified peptide Peptide m/z Da Type No site Peptide start-end sequence MH + Da (mmu/ppm) CS RT XC MC of Glycation Diagnostic fragment ions Glucose induced glycation modifications 1 490 470-496 MPC*AEDYLSVVLNQ 3231.54702 808.64 +4 32.61 5.36 1 CML 674.34 860.44 989.48 1239.62 LC*VLHEK*TPVSDR (0.35/0.43) [y.sub.6.sup.+] [y.sub.7.sup.+] [y.sub.8.sup.+] [y.sub.10.sup.+] 704.23 980.38 1067.41 1166.48 [b.sub.6.sup.+] [b.sub.8.sup.+] [b.sub.9.sup.+] [b.sub.10.sup.+] 2 183 169-184 RHPYFYAPELLFFAK*R 2113.09951 529.03 +4 27.16 4.82 2 CML 175.11 726.39 839.47 952.56 (0.59/1.11) [y.sub.1.sup.+] [y.sub.5.sup.+] [y.sub.6.sup.+] [y.sub.7.sup.+] 3 490 469-496 RMPC*AEDYLSVVLN 3387.64687 847.66 +4 31.43 4.05 2 CML 573.34 989.48 [y.sub.8.sup.+] 1239.62 1498.72 QLC*VLHEK*TPVSDR (0.67/0.79) [y.sub.5.sup.+] [y.sub.10.sup.+] [y.sub.12.sup.+] 288.14 860.44 [b.sub.7.sup.+] 1023.40 1223.50 [b.sub.2.sup.+] [b.sub.8.sup.+] [b.sub.10.sup.+] 4 337 311-341 SHC*IAEVENDEMPA 3534.57338 884.39 +4 27.78 3.98 1 CML 521.23 707.33 923.41 1355.61 DLPSLAADFVESK*DV (+0.57/+0.64) [y.sub.4.sup.+] [y.sub.5.sup.+] [y.sub.7.sup.+] [y.sub.11.sup.+] C*K 5 183 170-184 HPYFYAPELLFFAK*R 1957.00174 653.00 +3 28.86 3.66 1 CML 175.11 726.39 839.47 952.56 (+0.33/+0.51) [y.sub.1.sup.+] [y.sub.5.sup.+] [y.sub.6.sup.+] [y.sub.7.sup.+] 235.11 545.25 708.31 779.35 [b.sub.2.sup.+] [b.sub.4.sup.+] [b.sub.5.sup.+] [b.sub.6.sup.+] 6 426 414-434 QNC*ELFEQLGEYK*F 2657.30820 886.44 +3 28.13 3.61 1 CML 960.56 [y.sub.8.sup.+] 1146.66 1309.72 1495.78 QNALLVR (+1.76/+1.99) [y.sub.9.sup.+] [y.sub.10.sup.+] [y.sub.12.sup.+] 7 337 311-341 SHC*IAEVENDEMPA 3638.61074 910.40 +4 27.03 3.60 1 AML 521.23 [y.sub.4.sup.+] 300.15 [y.sub.6.sup.3+] 1388.60 1459.64 DLPSLAADFVESK*DV (1.93/2.12) [y.sub.10.sup.+] [y.sub.11.sup.+] C*K 8 438 438-452 K*VPQVSTPTLVEVSR 1697.94284 566.65 +3 19.35 3.53 1 CML 187.10 [b.sub.1.sup.+] 511.28 [b.sub.4.sup.+] 610.35 798.43 (0.15/0.27) [b.sub.5.sup.+] [b.sub.7.sup.+] 9 549 549-558 K*QTALVELVK 1186.70268 593.85 +2 18.97 3.46 1 CML 187.10 [b.sub.1.sup.+] 600.33 [b.sub.5.sup.+] 699.40 828.44 (0.77/1.29) [b.sub.6.sup.+] [b.sub.7.sup.+] 10 88 88-109 TC*VADESAENC*DK* 2555.09878 639.53 +4 19.86 3.39 1 CML 1017.53 [y.sub.9.sup.+] 1203.62 1318.66 385.39 SLHTLFGDK (0.95/1.48) [y.sub.10.sup.+] [y.sub.11.sup.+] [b.sub.13.sup.4+] 11 490 470-496 MPC*AEDYLSVVLNQ 3335.59267 834.65 +4 31.89 3.10 1 AML 674.34 [y.sub.6.sup.+] 274.12 [y.sub.8.sup.4+] 1343.66 1442.73 LC*VLHEK*TPVSDR (0.77/0.93) [y.sub.10.sup.+] [y.sub.11.sup.+] 12 88 88-109 TC*VADESAENC*DK* 2659.15249 665.54 +4 18.87 2.96 1 AML 821.87 [b.sub.13.sup.2+] 921.87 948.91 1114.48 SLHTLFGDK (0.88/1.33) [b.sub.15.sup.2+] [y.sub.15.sup.2+] [y.sub.18.sup.2+] Glyoxylic acid induced glycation modifications 1 160 139-168 LVRPEVDVMC*TAFH 3744.84463 936.96 +4 29.34 7.33 2 CML 927.49 [y.sub.7.sup.+] 1055.58 1241.68 1602.88 DNEETFLK*KYLYEIAR (+1.11/+1.19) [y.sub.8.sup.+] [y.sub.9.sup.+] [y.sub.12.sup.+] 2 160 & 139-168 LVRPEVDVMC*TAFH 3802.84170 951.46 +4 29.83 6.67 2 CML 927.49 [y.sub.7.sup.+] 1299.69 1412.77 1789.93 161 DNEETFLK*K*YLYEI (0.99/1.04) [y.sub.9.sup.+] [y.sub.10.sup.+] [y.sub.13.sup.+] AR 3 402 397-434 VFDEFK*PLVEEPQNL 4683.37995 937.48 +5 33.42 6.33 2 CML 638.28 [b.sub.5.sup.+] 921.43 [b.sub.7.sup.+] 1133.58 1262.63 IKQNC*ELFEQLGEYK (0.12/0.13) [b.sub.9.sup.+] [b.sub.10.sup.+] FQNALLVR 4 183 169-184 RHPYFYAPELLFFAK*R 2113.09842 705.03 +3 27.02 5.92 2 CML 175.11 [y.sub.1.sup.+] 726.39 [y.sub.5.sup.+] 839.47 1178.65 (1.15/1.63) [y.sub.6.sup.+] [y.sub.9.sup.+] 554.28 [b.sub.4.sup.+] 701.35 [b.sub.5.sup.+] 864.41 1274.63 [b.sub.6.sup.+] [b.sub.10.sup.+] 5 437 435-452 YTK*KVPQVSTPTLVE 2090.14560 697.38 +3 18.55 4.90 2 CML 265.11 [b.sub.2.sup.+] 226.11 [b.sub.3.sup.2+] 579.31 678.38 VSR (1.22/1.75) [b.sub.4.sup.+] [b.sub.5.sup.+] 6 437 & 438 435-452 YTK*K*VPQVSTPTLV 2148.14902 716.72 +3 19.01 4.70 2 CML 265.11 [b.sub.2.sup.+] 226.11 [b.sub.3.sup.2+] 637.31 736.38 EVSR (1.91/2.67) [b.sub.4.sup.+] [b.sub.5.sup.+] 7 438 438-456 K*VPQVSTPTLVEVSR 2110.18496 528.30 +4 21.49 4.67 2 CML 187.10 [b.sub.1.sup.+] 286.17 [b.sub.2.sup.+] 511.28 798.44 NLGK (0.45/0.84) [b.sub.4.sup.+] [b.sub.7.sup.+] 674.39 [y.sub.6.sup.+] 773.46 [y.sub.7.sup.+] 902.50 1001.57 [y.sub.8.sup.+] [y.sub.9.sup.+] 8 438 438-452 K*VPQVSTPTLVEVSR 1697.94413 566.65 +3 19.27 4.09 1 CML 187.10 [b.sub.1.sup.+] 383.26 [b.sub.3.sup.+] 511.28 610.35 (+0.28/+0.49) [b.sub.4.sup.+] [b.sub.5.sup.+] 589.33 [y.sub.5.sup.+] 702.41 [y.sub.6.sup.+] 803.46 900.51 [y.sub.7.sup.+] [y.sub.8.sup.+] 9 183 170-184 HPYFYAPELLFFAK*R 1956.99973 653.00 +3 28.80 4.51 1 CML 175.11 [y.sub.1.sup.+] 432.25 [y.sub.3.sup.+] 579.32 726.39 (0.34/0.52) [y.sub.4.sup.+] [y.sub.5.sup.+] 10 236 230-242 FGERAFK*AWAVAR 1566.81462 783.91 +2 20.40 4.19 2 CML 894.44 [b.sub.7.sup.+] 965.48 [b.sub.8.sup.+] 859.47 1006.54 (1.52/1.94) [y.sub.7.sup.+] [y.sub.8.sup.+] 11 426 414-434 QNC*ELFEQLGEYK*F 2657.30143 886.43 +3 28.08 4.00 1 CML 960.56 [y.sub.8.sup.+] 1146.66 1309.72 1495.78 QNALLVR (0.50/0.56) [y.sub.9.sup.+] [y.sub.10.sup.+] [y.sub.12.sup.+] 12 549 549-558 K*QTALVELVK 1186.70354 593.85 +2 18.85 3.48 1 CML 187.10 [b.sub.1.sup.+] 315.16 [b.sub.2.sup.+] 600.33 699.40 (0.34/0.57) [b.sub.5.sup.+] [b.sub.6.sup.+] 13 229 224-233 C*ASLQK*FGER 1253.59375 418.53 +3 13.97 2.43 1 CML 508.25 [y.sub.4.sup.+] 694.35 [y.sub.5.sup.+] 822.41 935.49 (0.20/0.48) [y.sub.6.sup.+] [y.sub.7.sup.+] Methylglyoxal induced glycation modifications 1 543 509-545 RPC*FSALEVDETYVP 4509.12122 902.62 +5 26.77 6.13 2 CEL 304.16 504.27 720.35 [y.sub.5.sup.+] 1094.51 [y.sub.8.sup.+] KEFNAETFTFHADIC* (+3.94/+4.37) [y.sub.2.sup.+] [y.sub.3.sup.+] TLSEK*ER 2 183 169-184 RHPYFYAPELLFFAK*R 2127.11709 532.53 +4 27.21 5.58 2 CEL 175.11 375.23 593.34 [y.sub.4.sup.+] 740.40 (0.11/0.20) [y.sub.1.sup.+] [y.sub.2.sup.+] [y.sub.5.sup.+] 3 490 469-496 RMPC*AEDYLSVVLN 3401.67127 681.14 +5 31.37 5.46 2 CEL 573.29 874.46 1140.56 [y.sub.9.sup.+] 1253.64 QLC*VLHEK*TPVSDR (+1.22/+1.79) [y.sub.7.sup.+] [y.sub.8.sup.+] [y.sub.10.sup.+] 4 426 414-434 QNC*ELFEQLGEYK*F 2671.31943 891.11 +3 28.15 5.26 1 CEL 960.56 1160.67 1323.74 1509.80 QNALLVR (+0.29/+0.32) [y.sub.8.sup.+] [y.sub.9.sup.+] [y.sub.10.sup.+] [y.sub.12.sup.+] 5 97 89-105 SLHTLFGDK*LC*TVA 2004.05752 501.76 +4 24.04 4.48 1 CEL 871.43 1071.54 592.81 395.54 [b.sub.10.sup.3+] TLR (0.21/0.42) [b.sub.8.sup.+] [b.sub.9.sup.+] [b.sub.10.sup.2+] 6 438 438-452 K*VPQVSTPTLVEVSR 1711.95920 571.32 +3 19.37 4.04 1 CEL 201.12 300.19 525.30 [b.sub.4.sup.+] 624.37 (+0.08/+0.15) [b.sub.1.sup.+] [b.sub.2.sup.+] [b.sub.5.sup.+] 7 549 549-558 K*QTALVELVK 1200.71855 600.86 +2 19.12 3.90 1 CEL 201.12 329.18 614.35 [b.sub.5.sup.+] 842.46 (0.66/1.10) [b.sub.1.sup.+] [b.sub.2.sup.+] [b.sub.7.sup.+] 8 183 170-184 HPYFYAPELLFFAK*R 1971.01664 657.67 +3 28.99 3.43 1 CEL 175.11 375.23 740.40 [y.sub.5.sup.+] 853.49 (+0.08/+0.12) [y.sub.1.sup.+] [y.sub.2.sup.+] [y.sub.6.sup.+] 235.11 545.25 708.31 [b.sub.5.sup.+] 1005.44 [b.sub.8.sup.+] [b.sub.2.sup.+] [b.sub.4.sup.+] 9 598 598-609 K*LVAASQAALGL 1213.71477 607.36 +2 21.30 3.20 1 CEL 201.12 484.31 770.44 [b.sub.7.sup.+] 841.47 (0.18/0.30) [b.sub.1.sup.+] [b.sub.4.sup.+] [b.sub.8.sup.+] 10 36 35-44 FK*DLGEENFK 1298.62470 433.54 +3 16.26 2.62 1 CEL 148.07 348.19 463.21 [b.sub.3.sup.+] 633.32 (0.56/1.29) [b.sub.1.sup.+] [b.sub.2.sup.+] [b.sub.5.sup.+] Static modification: *Carbamidomethyl (57.02146 Da), CSCharge state, RTRetention time, XCXcorr, MCMissed cleavage
TABLE-US-00002 Sequenceofhumanserumalbumin SEQIDNo.1 MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIA FAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCT VATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTA FHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAA CLLPKLDELRDEGKASSAKQGLKCASLQKFGERAFKAWAVARLSQRFPKA EFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLK ECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVGSKDVCKNYAEAKDVF LGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDE FKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEV SRNLGKVGSKCCKHPEAKRMPCAEDCLSVFLNQLCVLHEKTPVSDRVTKC CTESLVNGRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQ TALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLV AASQAALGL [0080] The above sequence depicts the amino acid sequence of human serum albumin [0081] length of the sequence: 609 [0082] It is naturally occurring.
TABLE-US-00003 SEQUENCELISTING <110> CSIR,IN <120> Aninvitroprocessfortheidentificationandquantification ofcarboxymethylandcarboxyethyllevelofalbumininasample <130> 0221NF2015 <160> 1 <170> PatentInversion3.5 <210> 1 <211> 609 <212> PRT <213> Homosapiens <400> 1 MetLysTrpValThrPheIleSerLeuLeuPheLeuPheSerSerAla 151015 TyrSerArgGlyValPheArgArgAspAlaHisLysSerGluValAla 202530 HisArgPheLysAspLeuGlyGluGluAsnPheLysAlaLeuValLeu 354045 IleAlaPheAlaGlnTyrLeuGlnGlnCysProPheGluAspHisVal 505560 LysLeuValAsnGluValThrGluPheAlaLysThrCysValAlaAsp 65707580 GluSerAlaGluAsnCysAspLysSerLeuHisThrLeuPheGlyAsp 859095 LysLeuCysThrValAlaThrLeuArgGluThrTyrGlyGluMetAla 100105110 AspCysCysAlaLysGlnGluProGluArgAsnGluCysPheLeuGln 115120125 HisLysAspAspAsnProAsnLeuProArgLeuValArgProGluVal 130135140145 AspValMetCysThrAlaPheHisAspAsnGluGluThrPheLeuLys 150155160 LysTyrLeuTyrGluIleAlaArgArgHisProTyrPheTyrAlaPro 165170175 GluLeuLeuPhePheAlaLysArgTyrLysAlaAlaPheThrGluCys 180185190 CysGlnAlaAlaAspLysAlaAlaCysLeuLeuProLysLeuAspGlu 195200205 LeuArgAspGluGlyLysAlaSerSerAlaLysGlnGlyLeuLysCys 210215220225 AlaSerLeuGlnLysPheGlyGluArgAlaPheLysAlaTrpAlaVal 230235240 AlaArgLeuSerGlnArgPheProLysAlaGluPheAlaGluValSer 245250255 LysLeuValThrAspLeuThrLysValHisThrGluCysCysHisGly 260265270 AspLeuLeuGluCysAlaAspAspArgAlaAspLeuAlaLysTyrIle 275280285 CysGluAsnGlnAspSerIleSerSerLysLeuLysGluCysCysGlu 290295300 LysProLeuLeuGluLysSerHisCysIleAlaGluValGluAsnAsp 305310315320 GluMetProAlaAspLeuProSerLeuAlaAlaAspPheValGlySer 325330335 LysAspValCysLysAsnTyrAlaGluAlaLysAspValPheLeuGly 340345350 MetPheLeuTyrGluTyrAlaArgArgHisProAspTyrSerValVal 355360365 LeuLeuLeuArgLeuAlaLysThrTyrGluThrThrLeuGluLysCys 370375380385 CysAlaAlaAlaAspProHisGluCysTyrAlaLysValPheAspGlu 390395400 PheLysProLeuValGluGluProGlnAsnLeuIleLysGlnAsnCys 405410415 GluLeuPheGluGlnLeuGlyGluTyrLysPheGlnAsnAlaLeuLeu 420425430 ValArgTyrThrLysLysValProGlnValSerThrProThrLeuVal 435440445 GluValSerArgAsnLeuGlyLysValGlySerLysCysCysLysHis 450455460465 ProGluAlaLysArgMetProCysAlaGluAspCysLeuSerValPhe 470475480 LeuAsnGlnLeuCysValLeuHisGluLysThrProValSerAspArg 485490495 ValThrLysCysCysThrGluSerLeuValAsnGlyArgProCysPhe 500505510 SerAlaLeuGluValAspGluThrTyrValProLysGluPheAsnAla 515520525 GluThrPheThrPheHisAlaAspIleCysThrLeuSerGluLysGlu 530535540545 ArgGlnIleLysLysGlnThrAlaLeuValGluLeuValLysHisLys 550555560 ProLysAlaThrLysGluGlnLeuLysAlaValMetAspAspPheAla 565570575 AlaPheValGluLysCysCysLysAlaAspAspLysGluThrCysPhe 580585590 AlaGluGluGlyLysLysLeuValAlaAlaSerGlnAlaAlaLeuGly 595600605 Leu 609