PROCESS FOR PREPARING A PEPTIDE SAMPLE

Abstract

A process for preparing a peptide sample from a biological sample, and a process for detecting or quantifying proteins including the process for preparing a sample. Also disclosed is the use of these processes for the detection or monitoring of a condition or a disease.

Claims

1. A process for the in vitro preparation of a peptide sample from a biological sample, comprising the following successive steps: a) denaturation of the proteins present in said sample; b) reduction and alkylation of the proteins resulting from step a); c) cleaning of the proteins resulting from step b) by reversed-phase chromatography on a solid polymeric support, said solid support comprising at least one polystyrene-divinylbenzene polymer; and d) digestion of the proteins resulting from step c) by a protease.

2. The process according to claim 1, characterized in that said biological sample is a blood sample in liquid form selected from the group consisting of: whole blood, serum and plasma.

3. The process according to claim 1, characterized in that said biological sample is a blood sample selected from the group consisting of: whole blood, serum and plasma, in that said sample is in solid or dried form, and in that said process comprises, prior to the step of denaturation of the proteins, a step of extraction of said proteins from said sample in solid or dried form.

4. The process according to claim 3, characterized in that said blood sample in solid or dried form is a sample of the DBS (Dried Blood Spot) type deposited on a collection paper, preferably a paper of the blotting paper type.

5. The process according to claim 1, characterized in that the step of digestion of the proteins by a protease is carried out in the presence of trypsin/endoproteinase Lys-C with a quantity of enzyme/quantity of substrate protein ratio comprised between 1/10 and 1/200, for a duration greater than or equal to 2 hours.

6. The process according to claim 1, wherein the DBS-type sample is treated during step d) in the presence of trypsin/endoproteinase Lys-C with a quantity of enzyme/quantity of substrate protein ratio comprised between 1/50 and 1/100, for a duration greater than or equal to 2 hours.

7. A process for detecting or quantifying proteins in a biological sample, comprising a process for the in vitro preparation of a peptide sample, according to claim 1, followed by a step of detection or quantification of at least one protein by means of an analysis technique, preferably by means of mass spectrometry, preferably the Liquid Chromatography Mass Spectrometry (LC-MS) technique.

8. The process according to claim 7, characterized in that said at least one protein is selected from the following proteins: afamin, alpha-1-antichymotrypsin, alpha-1B-glycoprotein (A1BG), alpha-1-acid glycoprotein, albumin (ALBU), alpha-2-HS-glycoprotein, alpha-2-macroglobulin (A2MG), antithrombin-3 (ANT3), apolipoprotein B100 (Apo B100), apolipoprotein C2 (Apo C2), apolipoprotein D, apolipoprotein E (Apo E), apolipoprotein M, apolipoprotein (a), apolipoprotein al (Apo A1), apolipoprotein A2 (Apo A2), apolipoprotein a4, beta-2-glycoprotein 1, beta-2-microglobulin (B2M), beta-Ala-His-dipeptidase, C4b-binding protein (alpha chain), CD5 antigen-like, cDNA-FLJ53327, ceruloplasmin (CERU), cholinesterase, clusterin, coagulation factor X (CF-X), coagulation factor XI, coagulation factor XII, complement C1q subcomponent subunit B, subunit C of complement C1q, complement C1r subcomponent, complement C1s subcomponent, complement C2 (C2), complement C3 (C3), complement C4B (C4B), complement C5, complement component C8 (beta chain), complement component C9, complement factor B, complement factor D, complement factor I, cystatin C (CysC), corticoid-binding globulin, C-reactive protein (CRP), fibrinogen (alpha chain) (FIBA), fibrinogen (beta chain) (FIBB), fibronectin, fibulin-1, gelsolin, haptoglobin, haemoglobin subunit alpha, haemopexin, heparin cofactor 2, the acid-labile subunit of insulin-like growth factor binding protein, insulin-like growth factor binding protein 3, haptoglobin (HPT), inter-alpha-trypsin inhibitor heavy chain H1, inter-alpha-trypsin inhibitor heavy chain H2, insulin-like growth factor binding protein 3 (IGFB3), lipopolysaccharide-binding protein, lumican, neuropilin-2, orosomucoid (ORM), PEDF, plasminogen (PLMN), protein AMBP, prothrombin, retinol-binding protein 4, serotransferrin (TRANSF), serum amyloid A4 protein, thyroxine-binding globulin, transthyretin (prealbumin) (TTHY), vasorin, vitamin D-binding protein, vitamin K-dependent protein C, vitamin K-dependent protein S, retinol-binding protein 4 (RET4).

9. A use of a process according to claim 1 for the preparation of a peptide sample for the detection or monitoring of the progression of a condition selected from: neurodegeneration, in particular Alzheimer's disease, a neurological or psychiatric disease, and in particular multiple sclerosis and autism, a state of deficiency, infection, inflammation or malnutrition, and a chronic or progressive disease, in particular cancer, hepatitis or a metabolic disease.

Description

[0056] Other advantages and characteristics of the invention will become apparent on examining the detailed description of an embodiment, which is in no way limitative, and the attached drawings.

[0057] FIG. 1 diagrammatically represents an embodiment of a process for preparing a peptide sample according to the invention from a DBS sample (FIG. 1A) and an embodiment of a process for preparing a peptide sample according to the state of the art from plasma (FIG. 1B).

[0058] FIG. 2 represents a histogram showing the compared results of the LC-MS analysis of 26 different proteins, carried out on samples prepared according to three different protocols: a) process according to the invention applied to a dried blood sample (“DBS RPW”), b) process according to the invention applied to a plasma sample (“plasma”), c) process for preparing DBS samples according to the state of the art (“standard DBS preparation”). For each of the proteins, the values of the areas obtained during the analysis by mass spectrometry under the “DBS RPW” and “plasma” conditions are divided, respectively, by the corresponding values of the areas obtained with the standard process for preparing the sample. LOG (area/area after standard DBS preparation) is represented on the y axis, the results for each of the 26 proteins are represented on the x axis, with “DBS RPW” as a light histogram and “plasma” as a dark histogram.

[0059] FIG. 3 diagrammatically represents the comparison of the quantification, in 95 plasma samples, of two serum proteins: C-reactive protein (CRP) (FIG. 3A) and serotransferrin (TRANSF) (FIG. 3B). For each of the proteins, the result of the quantification by clinically validated immunoassay is expressed on the x axis (in mg/L for CRP and g/L for TRANSF) and the result of the quantification by mass spectrometry on samples prepared using a process according to the invention is expressed on the y axis (arbitrary units).

[0060] The present invention will be better understood on reading the following examples which are given to illustrate the invention and not to limit its scope.

EXAMPLE

Example 1: Preparation of a Peptide Sample from a Dried Blood Sample (DBS) and Comparative Analysis by LC-MS

[0061] The efficiency of the process according to the invention was evaluated by detecting and quantifying 26 proteins by LC-MS, by comparing the analysis of a DBS sample treated using a process according to the invention (“DBS-RPW”) and, respectively, the analysis of a plasma sample treated using a process according to the state of the art (“plasma”) with an analysis carried out directly from a DBS sample (“standard DBS”). The steps of the process according to the invention are represented diagrammatically in FIG. 1A, the steps of the process for preparing a sample from plasma and according to a process of the state of the art are represented diagrammatically in FIG. 1B. The experiment was carried out in duplicate and each LC-MS analysis was carried out twice. The 26 proteins are the following: A1BG, A2MG, ANT3, Apo A1, Apo A2, Apo B100, Apo C2, Apo E, B2M, CERU, CF_X, C2, C3, C4B, CRP, CysC, FIBA, HPT, FIBB, IGFB3, PLMN, ORM, RET4, TRANSF, TTHY, ALBU.

[0062] The process of preparation according to the invention from a DBS sample according to the invention “DBS-RPW” is carried out as follows. To extract the proteins, a “DBS punch” is effected by punching the solid support (type 226 blotting paper) containing the sample. Said punch is transferred into a 96-well plate, then the proteins are extracted by adding 200 μL 50 mM ammonium bicarbonate, then by stirring for 30 minutes using a benchtop mixer at 350 rpm on an Eppendorf® Thermomixer Compact apparatus. The sample is then denatured by addition of 200 μL 8 M urea and stirring again for 10 minutes. The disulfide bonds of the proteins in the sample are then reduced by addition of 21 μL 200 mM DTT in 1 M Tris pH 8.5, and of 12 μL 1 M Tris pH 8.5, and stirring for one hour at 37° C. with stirring at 350 rpm on an Eppendorf® Thermomixer Compact. The released cysteines are then alkylated by addition of 18 μL 1 M IAA, 6 μL 1 M Tris pH 10, and stirring again for 30 minutes at 37° C. The alkylation step is stopped by addition of 20 μL 200 mM DTT. The sample is then acidified by the addition of 10 μL formic acid before transferring the 210 μL of supernatant into two new wells.

[0063] The step of cleaning of the sample is then carried out on RP-W® cartridges marketed by Agilent. The cartridges are washed and conditioned with 100 μL of an acetonitrile (70%)/TFA (0.1%)/water (29.9%) solution at 300 μL/min, and are brought into balance with 50 μL of a 0.1% formic acid solution at 10 μL/min. The sample is loaded onto an RP-W cartridge (Cat #G5496-60086) at 5 μL/min. The RP-W phase contained in the cartridge is washed with 50 μL of a 0.1% TFA solution at 10 μL/min and then the cleaned sample is eluted from the cartridge with 20 μL of an acetonitrile (70%)/formic acid (0.1%)/water (29.9%) solution at 5 μL/min. The elution is evaporated to dryness (Speedvac™). The dry sample is resuspended in 37.4 μL 20 mM Tris pH 8.5; 5.6 μg trypsin/LysC is added in order to carry out the trypsin digestion. This reaction lasts 14 hours at 37° C. with stirring (350 rpm on an Eppendorf® Thermomixer Compact). The digestion reaction is stopped by addition of 3 μL formic acid. The 96-well plate is sealed with film and the samples are ready to be injected into the LC-MS system.

[0064] The preparation of the “DBS standard” samples is carried out as follows: one or two drops of capillary blood, obtained after pricking the fingertip with a lancet, are deposited on each DBS. It is also possible to deposit 70 μL venous whole blood collected in a tube on a DBS using a pipette. After deposition, the cards are left to dry for 2 hours at ambient temperature. They are placed in an individual plastic bag and can be stored, depending on the use, at ambient temperature, at 4° C. or frozen (−20° C. or −80° C.). Before analysis, the cards are returned to ambient temperature. A punch with a diameter of 6 mm is then removed for each spot and transferred into a 2 ml Eppendorf LoBind tube.

[0065] The preparation of the “plasma” samples is carried out as follows: the process is represented diagrammatically in FIG. 1B, it represents a typical process known in the state of the art and comprises the following steps: from 2 μL plasma that is liquid or deposited on a “Whatman 903 paper” or “Ahlstrom FN/226 paper” support, the proteins are denatured and reduced, then undergo an alkylation, an enzymatic digestion (in the presence of trypsin/LysC for 14 h at 37° C.), then a step of cleaning of the obtained peptides carried out on a column of the ZORBAX Eclipse Plus C18 type, followed by analysis by LC-MS.

[0066] The results are illustrated in FIG. 2. A positive value is observed for the majority of the proteins. This indicates a larger detectable quantity (better sensitivity) in comparison with the standard process, and a good correspondence between standard plasma and DBS RPW values is also noted.

[0067] As a whole, the mass spectrometry intensities found after treating the DBS sample using a process according to the invention (DBS RPW) are 19 times more intense than those obtained directly after standard DBS. These intensities correspond, for the majority of the proteins, to those obtained on a plasma sample prepared using a standard process.

[0068] To validate the process for preparing a peptide sample according to the invention and to define its performance, the following study was conducted: ten independent samples taken from patients on DBS (DBS-RPW process according to the invention) were used for the study. For each patient, two DBS punches were analysed independently and in duplicate. The coefficients of variation (CV) of 91 peptides, corresponding to 76 proteins, thus measured twice independently and in duplicate, were calculated. This makes it possible to estimate the variability of the whole process for analysing the DBSs (pre-analytical and analytical). The median of the CVs obtained over all of the peptides is 9.3%. The CVs range from 2 to 52%. By comparison, during the same study, the median of all of the CVs obtained, this time on the plasma taken from the patients at the same time as the DBS, was 6.7% with the CVs ranging from 2 to 48%. To validate the clinical relevance of the measures, a correlation between the values obtained in the plasma (those which are thus used for clinical medicine) and those obtained in the DBSs was calculated. 35 peptides show a very strong correlation between “standard plasma” and “DBS-RPW” (coefficient of correlation >0.8), 32 show an intermediate correlation (between 0.6 and 0.8) and 24 show a weak correlation (<0.6).

[0069] The plasmas of 95 different patients, recruited in chronological order and without a particular pathology, were analysed, on the one hand, by mass spectrometry on samples prepared using a process according to the invention and in parallel by immunoassay, on a COBAS 6000 machine (Roche Diagnostic) in the Biochemistry department of Montpellier hospital. The results obtained are presented in FIG. 3, with analysis of CRP (FIG. 3A) and of serotransferrin (FIG. 3B) respectively. The results obtained by the two methods were compared using the R software. The comparison shows a statistically significant correlation for each of the two proteins quantified.

[0070] The results thus obtained therefore make it possible to validate the clinical relevance of the analysis process comprising a process for preparing a peptide sample according to the invention.