EP3973296A1 - Method for preparing a peptide sample - Google Patents

Method for preparing a peptide sample

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Publication number
EP3973296A1
EP3973296A1 EP20728688.1A EP20728688A EP3973296A1 EP 3973296 A1 EP3973296 A1 EP 3973296A1 EP 20728688 A EP20728688 A EP 20728688A EP 3973296 A1 EP3973296 A1 EP 3973296A1
Authority
EP
European Patent Office
Prior art keywords
sample
protein
proteins
complement
alpha
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP20728688.1A
Other languages
German (de)
French (fr)
Inventor
Sylvain Lehmann
Christophe HIRTZ
Jérôme VIALARET
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Spot To Lab
Universite de Montpellier I
Centre Hospitalier Universitaire de Montpellier CHUM
Original Assignee
Spot To Lab
Universite de Montpellier I
Centre Hospitalier Universitaire de Montpellier CHUM
Universite de Montpellier
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Spot To Lab, Universite de Montpellier I, Centre Hospitalier Universitaire de Montpellier CHUM, Universite de Montpellier filed Critical Spot To Lab
Publication of EP3973296A1 publication Critical patent/EP3973296A1/en
Pending legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4044—Concentrating samples by chemical techniques; Digestion; Chemical decomposition
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/34—Purifying; Cleaning
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4005—Concentrating samples by transferring a selected component through a membrane
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry

Definitions

  • the present application relates to a method of preparing a peptide sample from a biological sample. It also relates to a method for detecting and quantifying proteins using said method for preparing the peptide sample. Finally, it relates to the use of these methods for detecting or monitoring a condition or a disease.
  • the technical field of the invention is that of the biochemical analysis of proteins obtained from a biological sample.
  • the detection of protein clinical markers in a complex biological sample requires, for a proteomic analysis or by mass spectrometry, a first step of preparing a peptide sample, the quality of which is essential to then allow a sensitive and reproducible analysis. Indeed, the analysis of these media is subject to interference due to the presence of very predominant proteins and in certain cases to the release of the intracellular content during the preparation of the sample. This is particularly the case with blood products in which hemoglobin, albumin or immunoglobulins are very abundant.
  • WO 2014/118474 describes a method for determining the presence of prolidase for the detection of colorectal cancer, this method comprises a step of enzymatic digestion followed by a step of cleaning the proteins on a HLB solid phase support from Waters.
  • Yakundi et al. (Journal of Pharmaceutical and Biomedical analysis, 56, 1057-1063, 2011) cites a method for quantifying ranitidine in a dried blood sample, for clinical application. This method comprises a step of cleaning the proteins prior to an analysis by mass spectrometry, and does not include an enzymatic digestion step.
  • Rosting et al. ⁇ American Chemical Society, 87, 7918-24, 2015) discloses a method for detecting a model protein in a dried blood sample, comprising an enzymatic digestion step followed by pre-concentration by solid phase extraction (Solid- Extraction phase, or SPE), before analysis by mass spectrometry.
  • SPE Solid- Extraction phase
  • the inventors have now developed a method for the in vitro preparation of a sample of peptides, this method comprising successively steps of denaturation, reduction / alkylation, cleaning of the proteins by chromatography on a solid support comprising at least one polystyrene polymer. -divinyl benzene, then enzymatic digestion.
  • a method according to the invention allows the reproducible detection of a large number of clinical marker proteins from a small volume sample, this method can be applied successfully to a solid or liquid sample, such as plasma, and is automatable.
  • Analyzes of peptide samples by mass spectrometry have the advantage of absolute specificity in the detection of proteins and are considered to be standard methods.
  • a preparation method according to the invention, followed by an analysis by a method such as mass spectrometry for example makes it possible to have results statistically comparable to clinically validated tests, such as for example immunoassays.
  • a method according to the invention makes it possible to eliminate the interference due to the release of the intracellular content and to the presence of majority proteins, thus making it possible to improve the sensitivity, the specificity. and the analytical performance of the analysis of compounds.
  • a method for preparing a peptide sample according to the invention is suitable for the analysis of any type of biological sample, in particular blood samples, and more particularly solid blood samples, for which no reproducible and clinically validated method exists. 'is available today.
  • the use of a solid support of the blotter type is a recognized method of blood collection because it is non-invasive (finger tip sampling), easily transportable (in particular by post) and easy to implement; it can be carried out by the subject himself or by an unqualified person and is not dangerous, because the sample is decontaminated by drying.
  • a method according to the invention implemented on a sample on a solid support allows analysis at a lower cost, in particular for the longitudinal monitoring of subjects for multiple markers. Such a This approach is promising for monitoring populations whose access to analytical laboratories is restricted and for future developments in telemedicine.
  • the present application relates to a method for the in vitro preparation of a sample of peptides 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 from step a), c) cleaning of the proteins from step b) by reverse phase chromatography on a polymeric solid support, said solid support comprising at least one polystyrene-divinyl benzene polymer, and d) digestion by a protease of the proteins obtained from step c).
  • a method according to the invention comprises at least one step of cleaning the denatured, reduced and alkylated proteins and a step of enzymatic digestion, said step of cleaning the proteins taking place prior to the step of enzymatic digestion of said proteins.
  • biological sample is meant a sample comprising tissues and cells derived from the body, human or animal, and their derivatives, and in particular the following products: blood, serum, plasma, urine, stools, saliva, sputum, biopsies, and all body fluids and secretions.
  • the biological sample is a blood product in liquid form, chosen from: whole blood, serum and plasma
  • the biological sample is a blood product in solid or dried form, chosen from whole blood, serum and plasma.
  • the biological sample consists of dried whole blood deposited on a solid support, also called Dried Blood Spot (DBS).
  • DBS Dried Blood Spot
  • taking such a sample consists of depositing a drop of blood with a volume of 30 to 50 DL on a suitable solid support followed by a drying step.
  • the solid support for the sample is then easily handled and can in particular be sent by simple courier.
  • suitable solid supports there may be mentioned: blotting or collection papers such as "Whatman 903 paper” or "Ahlstrom TFN / 226 paper", on which blood can be deposited.
  • polystyrene-divinylbenzene polymer is meant a polymer comprising polystyrene crosslinked with divinylbenzene.
  • Polystyrene, or poly (1-phenylethylene) is obtained by polymerization of styrene monomer.
  • Divinylbenzene, or DVB is an aromatic hydrocarbon of formula C10Hio used as a crosslinking agent.
  • a solid support for cleaning proteins comprises at least one polystyrene-divinyl benzene polymer optionally associated with another polymer or with any other suitable element.
  • the solid support of the reverse phase chromatography step is characterized by the presence of polystyrene-divinyl benzene polymer beads, the size of which is between 10 and 50 mm, preferably between 20 and 40 mm and preferably 30 m. , on the one hand, and pores whose size is between 500 and 10,000 Angstroms, and preferably between 2,000 and 4,000 Angstroms, on the other hand.
  • solid supports comprising at least one polystyrene-divinyl benzene polymer which can be used in a process according to the invention
  • solid supports comprising at least one polystyrene-divinyl benzene polymer which can be used in a process according to the invention
  • RP-W® cartridges sold by the company Agilent.
  • Said RP-W® cartridges are described for use in a method and an application very different from that of the method according to the invention. Indeed, the methods of the state of the art typically describe the use of these cartridges during the production of recombinant antibodies, for a “desalination” step, that is to say in particular to ensure the elimination of the 6M guanidine necessary for the denaturation of the antibodies, prior to the digestion of the latter with trypsin.
  • each of the steps of denaturation, reduction, alkylation and enzymatic digestion of proteins is carried out by means of any method well known to those skilled in the art, under conditions of buffer, concentration, temperature and duration. appropriate.
  • the denaturation is carried out thanks to the addition of a protein denaturing agent, for example 8M urea
  • the reduction is carried out thanks to the addition of an agent capable of reducing the disulfide bonds of proteins, for example DTT
  • the alkylation of the cysteines released during the reduction is carried out by the addition of an alkylating agent, for example iodoacetic acid (IAA) .
  • an alkylating agent for example iodoacetic acid (IAA)
  • the enzymatic digestion of the proteins is preferably carried out by the addition of at least one protease chosen from: trypsin, endoproteinase GluC and endoproteinase LysC. According to a particular aspect of a process according to the invention, the enzymatic digestion is carried out for a duration greater than 2 hours, preferably greater than or equal to 10 hours, preferably greater than or equal to 14 hours, preferably equal to 14 hours. .
  • the enzymatic digestion is carried out in the presence of a ratio between the quantity of enzyme present and the quantity of substrate protein of between 1/10 and 1/200 and preferably between 1 / 50 and 1/100.
  • a ratio between the quantity of enzyme present and the quantity of substrate protein of between 1/10 and 1/200 and preferably between 1 / 50 and 1/100.
  • the present application relates to a method for the in vitro preparation of a sample of peptides from a blood sample in solid or dried form, said method comprising a step of extracting proteins from said solid blood sample prior to the denaturation, reduction and alkylation, protein cleaning and protease digestion steps.
  • the extraction of the proteins is carried out by any extraction method known to a person skilled in the art, in particular by placing the sample in the presence of ammonium bicarbonate at room temperature, optionally in the presence of ovalbumin.
  • the present application relates to a method for detecting the presence of at least one protein considered as a clinical marker in a biological sample, said detection method comprising an in vitro method for preparing a peptide sample, according to the invention, followed by a step of detecting the presence of at least one protein in said sample.
  • the present application relates to a method for detecting and quantifying at least one protein in a biological sample, said method for detecting and quantifying comprising an in vitro process for preparing a peptide sample, according to the invention, followed by a step of detecting and quantifying at least one protein in said sample.
  • the detection and / or quantification of said at least one protein is carried out by any known technical means, preferably by mass spectrometry, more preferably by mass spectrometry coupled to liquid chromatography, or Liquid Chromatography - Mass Spectrometry (LC-MS ). Said detection and / or quantification step can be implemented for one, two or more proteins. Said mass spectrometry can be carried out in a multiplex form.
  • Detection and quantification of proteins in a complex biological sample by LC-MS typically includes preparation of a peptide sample, followed by liquid chromatographic separation of said peptide sample, followed by mass spectrometry analysis, with l identification followed optionally by protein quantification, then statistical analysis of the results.
  • the detection and / or quantification of proteins is carried out using at least one standard consisting of a labeled or unlabeled peptide, making it possible to detect the corresponding specific protein.
  • Said standards are well known to those skilled in the art who can easily choose them from the available commercial standards, depending on the nature of the protein (s) of interest.
  • the subject of the present invention is a method for detecting, optionally followed by quantification, of at least one protein chosen from the group consisting of the following proteins: afamine, alpha-1-antichymotrypsin, alpha- lB-glycoprotein (A1BG), alpha 1 acid glycoprotein, albumin (ALBU), alpha-2-HS-glycoprotein, alpha-2- antiplasmin, 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 Al), Apolipoprotein A2 (Apo A2), apolipoprotein A2 (Apeta A2), apolipoprotein 2-4 glycoprotein 1, beta-2
  • the subject of the present invention is a method of detection, optionally followed by quantification, of one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty , twenty-five, thirty, thirty-five, forty or more than forty proteins selected from the group defined above.
  • the present application relates to a kit suitable for the in vitro preparation of a sample of peptides from a biological sample according to the invention, said kit comprising at least:
  • solid support comprising at least one polymer chosen from the family of polystyrene-divinyl benzene polymers.
  • Said reagents are chosen from existing reagents, well known to those skilled in the art.
  • the present application relates to the use of a method for the in vitro preparation of a sample of peptides from a biological sample according to the invention.
  • the present application relates to the use of a method for detecting and potentially quantifying at least one protein in a biological sample, according to the invention.
  • the present invention relates to the use of a method for the in vitro preparation of a sample of peptides from a biological sample, according to the invention, and / or the use of a detection and potentially quantification method according to the invention, for the detection and potentially quantification of at least one protein chosen from the group consisting of the following proteins: afamine, alpha-1-antichymotrypsin, alpha-1B- glycoprotein (A1BG), alpha 1 acid glycoprotein, albumin (ALBU), alpha-2-HS-glycoprotein, alpha-2-antiplasmin, 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 Al),
  • the present invention relates to the use of a method for the in vitro preparation of a sample of peptides from a biological sample, according to the invention, and / or the use of a detection and potentially quantification method according to the invention, for the detection and potentially of quantification of one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, twenty -five, thirty, thirty-five, forty or more than forty proteins selected from the group defined above.
  • the present application relates to the use of a method for the in vitro preparation of a sample of peptides from a biological sample according to the invention for the detection and / or monitoring of a condition. or a particular disease chosen from:
  • the detection and / or the monitoring of a disease or a particular condition can be carried out by the detection and / or the quantification of at least one protein considered as a clinical marker, possibly associated with the detection and / or the quantification at least one non-protein clinical marker.
  • the present application relates to the use of a method for the in vitro preparation of a sample of peptides from a biological sample, according to the invention, for the detection and / or monitoring.
  • the state of health, nutritional and frailty and of a subject in particular an elderly subject.
  • elderly subject is meant a subject of 75 years or more, more particularly a subject of 85 years or more.
  • a use according to the invention comprises the detection and / or the quantification of one or more proteins chosen from: albumin, alpha 1 acid glycoprotein, transthyretin and CRP (C Reactive Protein).
  • FIG. l schematically represents an embodiment of a method for preparing a peptide sample according to the invention and from a DBS sample (FIG. 1A) and an embodiment of a method for preparing a. peptide sample according to the state of the art from plasma (FIG. IB).
  • 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) method according to the invention applied to a dried blood sample (“DBS RPW” ), b) method according to the invention applied to a plasma sample (“plasma”), c) method for preparing DBS samples according to the state of the art (“DBS preparation standard”).
  • DBS preparation standard For each of the proteins, the values of the areas obtained during the analysis by mass spectrometry under the conditions "DBS RPW" and "plasma” are divided, respectively, by the corresponding values of the areas obtained with the standard method for preparing the protein. 'sample.
  • C reactive protein CRP
  • serotransferrin Fig. 3B
  • EXAMPLE 1 Preparation of a peptide sample from a dried blood sample (DBS) and comparative analysis by LC-MS.
  • the efficiency of the method according to the invention was evaluated by detecting and quantifying 26 proteins in LC / MS, by respectively comparing the analysis of a DBS sample treated by a method according to the invention (“DBS-RPW”) and the analysis of a plasma sample treated by a method according to the state of the art (“plasma”) to an analysis carried out directly from a DBS sample (“DBS standard”).
  • DBS-RPW analysis of a DBS sample treated by a method according to the invention
  • plasma plasma sample treated by a method according to the state of the art
  • FIG. I B The steps of the method according to the invention are shown diagrammatically in FIG. 1A, the steps of the method for preparing a sample from plasma and according to a method of the state of the art are diagrammed in FIG. I B.
  • the experiment was performed in duplicate and each LC / MS analysis was performed in duplicate.
  • the 26 proteins are as follows: A1 BG, A2MG, ANT3, Apo Al, Apo A2, Apo B100, Apo C2, Apo E, B2M, CERU, CF_X, C2, C3, C4B, CRP, CysC, FIBA, H PT, FIBB, IGFB3, PLMN, ORM, RET4, TRANSF, TTHY, ALBU.
  • DBS-RPW The preparation process from a DBS sample according to the invention “DBS-RPW” is carried out as follows.
  • a “DBS punch” is produced by punching the solid support (type 226 blotting paper) containing the sample. Said punch is transferred to a plate
  • the proteins are extracted by adding 200 m ⁇ - of 50 mM ammonium bicarbonate, then stirring for 30 minutes using a shaker. bench at 350 rpm on an Eppendorf® Thermomixer Compact device.
  • the sample is then denatured by adding 200 ml of 8M urea and stirring again for 10 minutes.
  • the disulfide bonds of the proteins in the sample are then reduced by adding 21 m ⁇ - of 200 mM DTT in 1 M tris pH 8.5, and 12 pL 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 liberated cysteines are then alkylated by adding 18 m ⁇ - of 1 M IAA, 6 ⁇ l of 1 M Tris pH 10, and further stirring for 30 minutes at 37 ° C.
  • the alkylation step is stopped by adding 20 m ⁇ - of 200 mM DTT.
  • the sample is then acidified by adding 10 m ⁇ - of formic acid before transferring the 210 m ⁇ - of the supernatant into two new wells.
  • the sample cleaning step is then carried out on RP-W® cartridges sold by the company Agilent.
  • the cartridges are washed and conditioned with 100 ⁇ L at 300 ⁇ L / min of a solution of acetonitrile (70%) / TFA (0.1%) / water (29.9%), and are equilibrated with 50 ⁇ L at 10 pL / min of a solution of 0.1% formic acid.
  • the sample is loaded onto an RP-W cartridge (Cat # G5496-60086) at 5 pL / min.
  • the RP-W phase contained in the cartridge is washed with 50 m ⁇ - at 10 m ⁇ / Gh ⁇ h of a 0.1% TFA solution and then the cleaned sample is eluted from the cartridge with 20 m ⁇ - at 5 pL / min of a solution of acetonitrile (70%) / formic acid (0.1%) / water (29.9%).
  • the elution is put to dryness (Speedvac TM).
  • the dry sample is resuspended in 37.4 ⁇ L of 20 mM Tris pH 8.5; 5.6 ⁇ g of trypsin / LysC are added to carry out the tryptic digestion. This reaction lasts 14 hours at 37 ° C.
  • the preparation of the “standard DBS” 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 spot of DBS. It is also possible to deposit with the use of a pipette 70 ⁇ L of venous whole blood collected in a tube on a DBS spot. After deposit, the cards are left to dry for 2 hours at room temperature. They are put in an individual plastic bag and can be stored according to use at room temperature, at 4 ° C or frozen (-20 ° C or -80 ° C). Before analysis, the cards are returned at room temperature. A 6 mm diameter punch is then taken for each spot and transferred into a 2 ml Eppendorf LoBind tube.
  • the preparation of the "plasma” samples is carried out as follows: the process is shown diagrammatically in FIG. IB, it represents a typical process known in the state of the art and comprises the following stages: starting from 2 ⁇ L of liquid or deposited plasma 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 the peptides obtained carried out on a ZORBAX Eclipse Plus C18 type column, followed by analysis by LC-MS.
  • the intensities in mass spectrometry found after treatment of the DBS sample by a method according to the invention are 19 times more intense than those obtained directly after standard DBS. These intensities correspond for the majority of proteins to those obtained on a sample of plasma prepared by a standard method.
  • the median of the overall CV obtained this time on the plasma collected from the patients at the same time as the DBS was 6.7% with a range CVs that ranged from 2 to 48%.
  • a correlation between the values obtained in the plasma (those which are therefore used for clinical purposes) and those obtained in the DBS was calculated. 35 peptides show a very strong correlation between “standard plasma” and “DBS-RPW” (correlation coefficient> 0.8), 32 an intermediate correlation (between 0.6 and 0.8) and 24 a weak correlation ( ⁇ 0.6 ).
  • the plasmas of 95 different patients, recruited in chronological order and without any particular pathology were analyzed, on the one hand, by mass spectrometry on samples prepared by a method according to the invention and in parallel by immunoassay, on a COBAS 6000 automaton. (Roche Diagnostic) on the Biochemistry site of the adjoin hospital. The results obtained are shown in figure 3, together with the analysis of CRP (figure 3A) and serotransferrin (figure 3B) respectively. The results obtained by the two methods were compared by software R. The comparison shows a statistically significant correlation for each of the two quantified proteins.

Abstract

The present application relates to a method for preparing a peptide sample from a biological sample, and to a method for detecting or quantifying proteins comprising said method for preparing a sample. The invention also relates to the use of these methods for the detection or monitoring of a condition or a disease.

Description

PROCÉDÉ DE PRÉPARATION D'UN ÉCHANTILLON PEPTIDIQUE PROCESS FOR PREPARING A PEPTIDIC SAMPLE
Domaine de l'invention Field of the invention
[La présente demande concerne un procédé de préparation d'un échantillon peptidique à partir d'un échantillon biologique. Elle concerne aussi un procédé de détection et de quantification de protéines mettant en œuvre ledit procédé de préparation de l'échantillon peptidique. Elle concerne enfin l'utilisation de ces procédés pour la détection ou le suivi d'une condition ou d'une maladie. [The present application relates to a method of preparing a peptide sample from a biological sample. It also relates to a method for detecting and quantifying proteins using said method for preparing the peptide sample. Finally, it relates to the use of these methods for detecting or monitoring a condition or a disease.
Le domaine technique de l'invention est celui de l'analyse biochimique des protéines issues d'un échantillon biologique. The technical field of the invention is that of the biochemical analysis of proteins obtained from a biological sample.
Etat de l'art State of the art
La détection et le suivi de l'évolution de maladies ou de conditions physiques particulières, telles que carences ou malnutrition, demandent la détection et la quantification de protéines spécifiques, dits « marqueurs cliniques protéiques», outils essentiels à la prévention, au diagnostic, au pronostic et aux choix thérapeutiques. The detection and monitoring of the evolution of diseases or particular physical conditions, such as deficiencies or malnutrition, require the detection and quantification of specific proteins, known as “protein clinical markers”, essential tools for prevention, diagnosis, treatment. prognosis and therapeutic choices.
La détection des marqueurs cliniques protéiques dans un échantillon biologique complexe requiert, pour une analyse protéomique ou par spectrométrie de masse, une première étape de préparation d'un échantillon peptidique dont la qualité est primordiale pour permettre ensuite une analyse sensible et reproductible. En effet, l'analyse de ces milieux est soumise à des interférences dues à la présence de protéines très majoritaires et dans certain cas à la libération du contenu intracellulaire lors de la préparation de l'échantillon. C'est le cas notamment des produits sanguins dans lesquels l'hémoglobine, l'albumine ou les immunoglobulines sont très abondantes. The detection of protein clinical markers in a complex biological sample requires, for a proteomic analysis or by mass spectrometry, a first step of preparing a peptide sample, the quality of which is essential to then allow a sensitive and reproducible analysis. Indeed, the analysis of these media is subject to interference due to the presence of very predominant proteins and in certain cases to the release of the intracellular content during the preparation of the sample. This is particularly the case with blood products in which hemoglobin, albumin or immunoglobulins are very abundant.
Les méthodes conventionnelles d'analyses de produits sanguins s'appliquent à des produits sous forme liquide ou solide, telle que par exemple du sang séché sur un buvard, cette dernière forme présentant en effet de nombreux avantages pratiques, analytiques, cliniques et financiers. A partir des échantillons solides, des méthodes d'extraction de petites molécules non protéiques (acides aminés, hormones stéroïdiennes, vitamines et médicaments) sont déjà connues et validées. Au contraire, l'analyse des marqueurs cliniques protéiques rencontre de nombreux problèmes analytiques du fait des interférences dues aux autres protéines très majoritaires, de la faible efficacité et reproductibilité des méthodes de préparation ou de digestion des protéines et de la présence possible d'impuretés dans le support solide. The conventional methods of analyzing blood products apply to products in liquid or solid form, such as, for example, blood dried on a blotter, the latter form in fact exhibiting numerous practical, analytical, clinical and financial advantages. From solid samples, methods of extracting small non-protein molecules (amino acids, steroid hormones, vitamins and drugs) are already known and validated. On the contrary, the analysis of Protein clinical markers encounter many analytical problems due to interference due to the other very predominant proteins, the low efficiency and reproducibility of the methods of preparation or digestion of proteins and the possible presence of impurities in the solid support.
Des progrès récents dans la spectrométrie de masse permettent une alternative intéressante à des procédés classique d'analyses cliniques déjà validés (immunodétection...). La différence de composition protéique entre un échantillon de sang solide et un échantillon de sérum ou de plasma, ainsi que la complexité de ces matrices rend actuellement l'analyse par spectrométrie de masse peu reproductible, peu sensible et peu compatible avec une utilisation clinique acceptable. Recent progress in mass spectrometry allows an interesting alternative to conventional methods of clinical analyzes already validated (immunodetection ...). The difference in protein composition between a solid blood sample and a serum or plasma sample, as well as the complexity of these matrices currently makes the analysis by mass spectrometry not very reproducible, not very sensitive and not very compatible with acceptable clinical use.
Il existe donc un besoin important de disposer d'un procédé reproductible, sensible, rapide et validé cliniquement de préparation d'échantillons peptidiques préalables à la détection et au suivi analytique de marqueurs cliniques protéiques, notamment par spectrométrie de masse. There is therefore a significant need to have a reproducible, sensitive, rapid and clinically validated method for preparing peptide samples prior to the detection and analytical monitoring of protein clinical markers, in particular by mass spectrometry.
Des procédés de préparation d'échantillons peptidiques préalables à une analyse sont décrits dans l'art antérieur. Ces procédés comprennent des étapes de dénaturation, élution, alkylation et digestion trypsique des protéines. Methods of preparing peptide samples prior to analysis are described in the prior art. These methods include denaturation, elution, alkylation and tryptic digestion of proteins.
WO 2014/118474 décrit un procédé de détermination de la présence de la prolidase pour la détection du cancer colorectal, ce procédé comprend une étape de digestion enzymatique suivie d'une étape de nettoyage des protéines sur un support en phase solide HLB de Waters. WO 2014/118474 describes a method for determining the presence of prolidase for the detection of colorectal cancer, this method comprises a step of enzymatic digestion followed by a step of cleaning the proteins on a HLB solid phase support from Waters.
Yakundi et al. ( Journal of Pharmaceutical and Biomédical analysis, 56, 1057- 1063, 2011) cite un procédé destiné à quantifier la ranitidine dans un échantillon de sang séché, pour une application clinique. Ce procédé comprend une étape de nettoyage des protéines préalablement à une analyse par spectrométrie de masse, et ne comprend pas d'étape de digestion enzymatique. Yakundi et al. (Journal of Pharmaceutical and Biomedical analysis, 56, 1057-1063, 2011) cites a method for quantifying ranitidine in a dried blood sample, for clinical application. This method comprises a step of cleaning the proteins prior to an analysis by mass spectrometry, and does not include an enzymatic digestion step.
Rosting et al. Ç American Chemical Society, 87, 7918-24, 2015) divulgue un procédé de détection d'une protéine modèle dans un échantillon de sang séché, comprenant une étape de digestion enzymatique suivie d'une pré concentration par extraction en phase solide (Solid-Phase Extraction, ou SPE), avant analyse par spectrométrie de masse. Description de l'invention Rosting et al. Ç American Chemical Society, 87, 7918-24, 2015) discloses a method for detecting a model protein in a dried blood sample, comprising an enzymatic digestion step followed by pre-concentration by solid phase extraction (Solid- Extraction phase, or SPE), before analysis by mass spectrometry. Description of the invention
Les inventeurs ont maintenant mis au point un procédé pour la préparation in vitro d'un échantillon de peptides, ce procédé comprenant successivement des étapes de dénaturation, réduction/alkylation, nettoyage des protéines par chromatographie sur un support solide comprenant au moins un polymère de polystyrène-divinyl benzène, puis digestion enzymatique. The inventors have now developed a method for the in vitro preparation of a sample of peptides, this method comprising successively steps of denaturation, reduction / alkylation, cleaning of the proteins by chromatography on a solid support comprising at least one polystyrene polymer. -divinyl benzene, then enzymatic digestion.
Un procédé selon l'invention permet la détection reproductible d'un grand nombre de protéines marqueurs cliniques à partir d'un échantillon de faible volume, ce procédé peut être appliqué avec succès à un échantillon solide ou liquide, tel que du plasma, et est automatisable. Les analyses d'échantillons peptidiques par spectrométrie de masse présentent l'avantage d'une spécificité absolue dans la détection de protéines et sont considérées comme des méthodes de référence. Un procédé de préparation selon l'invention, suivi d'une analyse par un procédé comme la spéctrométrie de masse par exemple, permet de disposer de résultats statistiquement comparables aux tests validés cliniquement, comme par exemple les immunoessais. A method according to the invention allows the reproducible detection of a large number of clinical marker proteins from a small volume sample, this method can be applied successfully to a solid or liquid sample, such as plasma, and is automatable. Analyzes of peptide samples by mass spectrometry have the advantage of absolute specificity in the detection of proteins and are considered to be standard methods. A preparation method according to the invention, followed by an analysis by a method such as mass spectrometry for example, makes it possible to have results statistically comparable to clinically validated tests, such as for example immunoassays.
Par rapport aux procédés de l'état de l'art, un procédé selon l'invention permet d'éliminer les interférences dues à la libération du contenu intracellulaire et à la présence de protéines majoritaires, permettant ainsi d'améliorer la sensibilité, la spécificité et les performances analytiques de l'analyse des composés. Compared to the methods of the state of the art, a method according to the invention makes it possible to eliminate the interference due to the release of the intracellular content and to the presence of majority proteins, thus making it possible to improve the sensitivity, the specificity. and the analytical performance of the analysis of compounds.
Un procédé de préparation d'un échantillon peptidique selon l'invention est adapté pour l'analyse de tout type d'échantillon biologique, en particulier les échantillons sanguins, et plus particulièrement les échantillons sanguins solides, pour lesquels aucun procédé reproductible et validé cliniquement n'est disponible à ce jour. L'utilisation d'un support solide de type buvard est une méthode reconnue de collection du sang car non invasive (prélèvement au bout du doigt), facilement transportable (notamment par courrier postal) et facile à mettre en œuvre ; elle peut être réalisée par le sujet lui-même ou par une personne non qualifiée et est non dangereuse, car le prélèvement est décontaminé par le séchage. Un procédé selon l'invention mis en œuvre sur échantillon sur support solide permet une analyse à moindre coût, notamment pour le suivi longitudinal de sujets pour de multiples marqueurs. Une telle approche est prometteuse pour le suivi de populations dont l'accès aux laboratoires d'analyse est contraint et pour des développements futurs de télémédecine. A method for preparing a peptide sample according to the invention is suitable for the analysis of any type of biological sample, in particular blood samples, and more particularly solid blood samples, for which no reproducible and clinically validated method exists. 'is available today. The use of a solid support of the blotter type is a recognized method of blood collection because it is non-invasive (finger tip sampling), easily transportable (in particular by post) and easy to implement; it can be carried out by the subject himself or by an unqualified person and is not dangerous, because the sample is decontaminated by drying. A method according to the invention implemented on a sample on a solid support allows analysis at a lower cost, in particular for the longitudinal monitoring of subjects for multiple markers. Such a This approach is promising for monitoring populations whose access to analytical laboratories is restricted and for future developments in telemedicine.
Selon un premier objet, la présente demande concerne un procédé pour la préparation in vitro d'un échantillon de peptides à partir d'un échantillon biologique, comprenant les étapes successives suivantes : a) dénaturation des protéines présentes dans ledit échantillon, b) réduction et alkylation des protéines issues de l'étape a), c) nettoyage des protéines issues de l'étape b) par chromatographie en phase inverse sur support solide polymérique, ledit support solide comprenant au moins un polymère polystyrène-divinyl benzène, et d) digestion par une protéase des protéines issues de l'étape c). According to a first object, the present application relates to a method for the in vitro preparation of a sample of peptides 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 from step a), c) cleaning of the proteins from step b) by reverse phase chromatography on a polymeric solid support, said solid support comprising at least one polystyrene-divinyl benzene polymer, and d) digestion by a protease of the proteins obtained from step c).
Un procédé selon l'invention comprend au moins une étape de nettoyage des protéines dénaturées, réduites et alkylées et une étape de digestion enzymatique, ladite étape de nettoyage des protéines ayant lieu préalablement à l'étape de digestion enzymatique desdites protéines. A method according to the invention comprises at least one step of cleaning the denatured, reduced and alkylated proteins and a step of enzymatic digestion, said step of cleaning the proteins taking place prior to the step of enzymatic digestion of said proteins.
Par « échantillon biologique » on entend un échantillon comprenant des tissus et cellules issus du corps, humain ou animal, et leurs dérivés, et en particulier les produits suivants : sang, sérum, plasma, urines, selles, salive, crachats, biopsies, et toutes les sécrétions et liquides corporels. By “biological sample” is meant a sample comprising tissues and cells derived from the body, human or animal, and their derivatives, and in particular the following products: blood, serum, plasma, urine, stools, saliva, sputum, biopsies, and all body fluids and secretions.
Selon un premier aspect particulier du procédé selon l'invention, l'échantillon biologique est un produit sanguin sous forme liquide, choisi parmi : le sang total, le sérum et le plasma According to a first particular aspect of the method according to the invention, the biological sample is a blood product in liquid form, chosen from: whole blood, serum and plasma
Selon un deuxième aspect particulier du procédé selon l'invention, l'échantillon biologique est un produit sanguin sous forme solide, ou desséchée, choisi parmi le sang total, le sérum et le plasma. Selon un aspect encore plus particulier, l'échantillon biologique est constitué par du sang total desséché déposé sur un support solide, aussi appelé Dried Blood Spot (DBS). According to a second particular aspect of the method according to the invention, the biological sample is a blood product in solid or dried form, chosen from whole blood, serum and plasma. According to an even more particular aspect, the biological sample consists of dried whole blood deposited on a solid support, also called Dried Blood Spot (DBS).
Typiquement, le prélèvement d'un tel échantillon consiste en un dépôt d'une goutte de sang d'un volume de 30 à 50 DL sur un support solide approprié suivi d'une étape de séchage. Le support solide de l'échantillon est ensuite facilement manipulable et peut notamment être envoyé par simple courier. Parmi les supports solides appropriés, on peut citer : les papiers buvard ou de recueil tels que le « papier Whatman 903 » ou le « papier Ahlstrom TFN/226 », sur lesquels du sang peut être déposé. Typically, taking such a sample consists of depositing a drop of blood with a volume of 30 to 50 DL on a suitable solid support followed by a drying step. The solid support for the sample is then easily handled and can in particular be sent by simple courier. Among the suitable solid supports, there may be mentioned: blotting or collection papers such as "Whatman 903 paper" or "Ahlstrom TFN / 226 paper", on which blood can be deposited.
Par « polymère de polystyrène-divinyl benzène » on entend un polymère comprenant du polystyrène réticulé par le divinylbenzène. Le polystyrène, ou poly(l-phényléthylène), est obtenu par polymérisation du monomère styrène. Le divinylbenzène, ou DVB est un hydrocarbure aromatique de formule CioHio utilisé comme agent de réticulation. By “polystyrene-divinylbenzene polymer” is meant a polymer comprising polystyrene crosslinked with divinylbenzene. Polystyrene, or poly (1-phenylethylene), is obtained by polymerization of styrene monomer. Divinylbenzene, or DVB is an aromatic hydrocarbon of formula C10Hio used as a crosslinking agent.
Dans un procédé selon l'invention, un support solide pour le nettoyage de protéines comprend au moins un polymère de polystyrène-divinyl benzène associé éventuellement à un autre polymère ou à tout autre élément approprié In a method according to the invention, a solid support for cleaning proteins comprises at least one polystyrene-divinyl benzene polymer optionally associated with another polymer or with any other suitable element.
Le support solide de l'étape de chromatographie en phase inverse se caractérise par la présence de billes de polymère de polystyrène-divinyl benzène dont la taille est comprise entre 10 et 50 mm, de préférence entre 20 et 40 mm et de préférence de 30 m, d'une part, et des pores dont la taille est comprise entre 500 et 10.000 Ângstrom, et de préférence entre 2.000 et 4.000 Ângstrom, d'autre part. The solid support of the reverse phase chromatography step is characterized by the presence of polystyrene-divinyl benzene polymer beads, the size of which is between 10 and 50 mm, preferably between 20 and 40 mm and preferably 30 m. , on the one hand, and pores whose size is between 500 and 10,000 Angstroms, and preferably between 2,000 and 4,000 Angstroms, on the other hand.
Parmi les supports solides comprenant au moins un polymère de polystyrène- divinyl benzène utilisables dans un procédé selon l'invention, on peut citer notamment les cartouches RP-W® commercialisées par la société Agilent. Among the solid supports comprising at least one polystyrene-divinyl benzene polymer which can be used in a process according to the invention, there may be mentioned in particular the RP-W® cartridges sold by the company Agilent.
Lesdites cartouches RP-W® sont décrites pour une utilisation dans un procédé et une application très différente de celle du procédé selon l'invention. En effet, les procédés de l'état de l'art décrivent typiquement l'util isation de ces cartouches lors de la production d'anticorps recombinants, pour une étape de « dessalement », c'est-à-dire en particulier pour assurer l'élimination de la guanidine 6M nécessaire à la dénaturation des anticorps, préalablement à la digestion de ceux-ci par la trypsine. Said RP-W® cartridges are described for use in a method and an application very different from that of the method according to the invention. Indeed, the methods of the state of the art typically describe the use of these cartridges during the production of recombinant antibodies, for a “desalination” step, that is to say in particular to ensure the elimination of the 6M guanidine necessary for the denaturation of the antibodies, prior to the digestion of the latter with trypsin.
Dans un procédé selon l'invention, chacune des étapes de dénaturation, réduction, alkylation et digestion enzymatique des protéines est réalisée au moyen de tout procédé bien connu de l'homme du métier, dans les conditions de tampon, de concentration, température et durée appropriées. De préférence, la dénaturation est réalisée grâce à l'addition d'un agent dénaturant les protéines, par exemple l'urée 8M, la réduction est réalisée grâce à l'addition d'un agent capable de réduire les ponts disulfures des protéines, par exemple le DTT, l'alkylation des cystéines libérées lors de la réduction est réalisée grâce à l'addition d'un agent alkylant, par exemple l'acide iodoacétique (IAA). In a method according to the invention, each of the steps of denaturation, reduction, alkylation and enzymatic digestion of proteins is carried out by means of any method well known to those skilled in the art, under conditions of buffer, concentration, temperature and duration. appropriate. Preferably, the denaturation is carried out thanks to the addition of a protein denaturing agent, for example 8M urea, the reduction is carried out thanks to the addition of an agent capable of reducing the disulfide bonds of proteins, for example DTT, the alkylation of the cysteines released during the reduction is carried out by the addition of an alkylating agent, for example iodoacetic acid (IAA) .
La digestion enzymatique des protéines est réalisée de préférence par l'addition d'au moins une protéase choisie parmi : la trypsine, l'endoprotéinase GluC et l'endoprotéinase LysC. Selon un aspect particulier d'un procédé selon l'invention, la digestion enzymatique est réalisée pendant une durée supérieure à 2 heures, de préférence supérieure ou égale à 10 heures, de préférence supérieure ou égale à 14 heures, de préférence égale à 14 heures. The enzymatic digestion of the proteins is preferably carried out by the addition of at least one protease chosen from: trypsin, endoproteinase GluC and endoproteinase LysC. According to a particular aspect of a process according to the invention, the enzymatic digestion is carried out for a duration greater than 2 hours, preferably greater than or equal to 10 hours, preferably greater than or equal to 14 hours, preferably equal to 14 hours. .
Plus particulièrement, dans un procédé selon l'invention, la digestion enzymatique est réalisée en présence d'un ratio entre la quantité d'enzyme présente et la quantité de protéine substrat compris entre 1/10 et 1/200 et de préférence compris entre 1/50 et 1/100. L'homme du métier spécialiste du domaine est capable d'identifier et calculer précisément ledit ratio. More particularly, in a process according to the invention, the enzymatic digestion is carried out in the presence of a ratio between the quantity of enzyme present and the quantity of substrate protein of between 1/10 and 1/200 and preferably between 1 / 50 and 1/100. Those skilled in the art who specialize in the field are able to identify and calculate precisely said ratio.
Selon un autre aspect particulier dudit premier objet, la présente demande concerne un procédé pour la préparation in vitro d'un échantillon de peptides à partir d'un échantillon sanguin sous forme solide ou desséchée, ledit procédé comprenant une étape d'extraction des protéines dudit échantillon sanguin solide préalablement aux étapes de dénaturation, réduction et alkylation, nettoyage des protéines et digestion par une protease. According to another particular aspect of said first object, the present application relates to a method for the in vitro preparation of a sample of peptides from a blood sample in solid or dried form, said method comprising a step of extracting proteins from said solid blood sample prior to the denaturation, reduction and alkylation, protein cleaning and protease digestion steps.
L'extraction des protéines est réalisée par tout procédé d'extraction connu d'un homme du métier, en particulier par la mise en présence de l'échantillon avec du bicarbonate d'ammonium à température ambiante, éventuellement en présence d'ovalbumine. The extraction of the proteins is carried out by any extraction method known to a person skilled in the art, in particular by placing the sample in the presence of ammonium bicarbonate at room temperature, optionally in the presence of ovalbumin.
Selon un deuxième objet, la présente demande concerne un procédé de détection de la présence d'au moins une protéine considérée comme un marqueur clinique dans un échantillon biologique, ledit procédé de détection comprenant un procédé in vitro de préparation d'un échantillon peptidique, selon l'invention, suivi d'une étape de détection de la présence d'au moins une protéine dans ledit échantillon. According to a second object, the present application relates to a method for detecting the presence of at least one protein considered as a clinical marker in a biological sample, said detection method comprising an in vitro method for preparing a peptide sample, according to the invention, followed by a step of detecting the presence of at least one protein in said sample.
Selon un mode de réalisation particulier, la présente demande concerne un procédé de détection et de quantification d'au moins une protéine dans un échantillon biologique, ledit procédé de détection et de quantification comprenant un procédé in vitro de préparation d'un échantillon peptidique, selon l'invention, suivi d'une étape de détection et de quantification d'au moins une protéine dans ledit échantillon. According to a particular embodiment, the present application relates to a method for detecting and quantifying at least one protein in a biological sample, said method for detecting and quantifying comprising an in vitro process for preparing a peptide sample, according to the invention, followed by a step of detecting and quantifying at least one protein in said sample.
La détection et/ou la quantification de ladite au moins une protéine est réalisée par tout moyen technique connu, de préférence par spectrométrie de masse, plus préférentiellement par spectrométrie de masse couplée à la chromatographie liquide, ou Liquid Chromatography - Mass Spectrometry (LC- MS). Ladite étape de détection et/ou de quantification peut être mise en œuvre pour une, deux ou plusieurs protéines. Ladite spectrométrie de masse peut être réalisée sous une forme multiplexe. The detection and / or quantification of said at least one protein is carried out by any known technical means, preferably by mass spectrometry, more preferably by mass spectrometry coupled to liquid chromatography, or Liquid Chromatography - Mass Spectrometry (LC-MS ). Said detection and / or quantification step can be implemented for one, two or more proteins. Said mass spectrometry can be carried out in a multiplex form.
La détection et la quantification de protéines dans un échantillon biologique complexe par LC-MS comprend typiquement la préparation d'un échantillon peptidique, puis la séparation par chromatographie en phase liquide dudit échantillon peptidique, suivie par l'analyse par spectrométrie de masse, avec l'identification suivie éventuellement de la quantification de protéines, puis l'analyse statistique des résultats. Detection and quantification of proteins in a complex biological sample by LC-MS typically includes preparation of a peptide sample, followed by liquid chromatographic separation of said peptide sample, followed by mass spectrometry analysis, with l identification followed optionally by protein quantification, then statistical analysis of the results.
La détection et/ou la quantification de protéines est réalisée en utilisant au moins un standard consistant en un peptide marqué ou non marqué, permettant de détecter la protéine spécifique correspondante. Lesdits standards sont bien connus de l'homme du métier qui peut aisément les choisir parmi les standards commerciaux disponibles, selon la nature de la ou des protéines d'intérêt. The detection and / or quantification of proteins is carried out using at least one standard consisting of a labeled or unlabeled peptide, making it possible to detect the corresponding specific protein. Said standards are well known to those skilled in the art who can easily choose them from the available commercial standards, depending on the nature of the protein (s) of interest.
Selon un mode de réalisation particulier, la présente invention a pour objet un procédé de détection, suivie éventuellement de la quantification, d'au moins une protéine choisie dans le groupe constitué par les protéines suivantes : afamine, alpha-l-antichymotrypsine, alpha-lB-glycoprotéine (A1BG), alpha 1 glycoprotéine acide, albumine (ALBU), alpha-2-HS-glycoprotéine, alpha-2- antiplasmine, alpha-2-macroglobuline (A2MG), antithrombine-3 (ANT3), apolipoprotéine B100 (Apo B100), apolipoprotéine C2 (Apo C2), apolipoprotéine D, apolipoprotéine E (Apo E), apolipoprotéine M, apolipoprotéine (a), apolipoprotéine al (Apo Al), Apolipoprotéine A2 (Apo A2), apolipoprotéine a4, bêta-2-glycoprotéine 1, bêta-2-microglobuline (B2M), bêta-Ala-his-dipeptidase, protéine liant la C4b (chaîne alpha), CD5 antigène- like, CDNA-FU53327, céruloplasmine (CERU), cholinestérase, clustérine, facteur de coagulation X (CF-X), facteur de coagulation XI, facteur de coagulation XII, complément Clq sous-unité de sous-composante B, sous- unité C du complément Clq, complément Clr sous-composant, complément C1S sous-composant, complément C2 (C2), complément C3 (C3), complément C4-B (C4B), complément C5, composant de complément C8 (chaîne bêta), complément composante C9, facteur de complément B, facteur de complément D, facteur de complément I, Cystatine C (CysC), globuline liant les corticoïdes, protéine C réactive (CRP), fibrinogène (chaîne alpha) (FIBA), fibrinogène (chaîne beta) (FIBB), fibronectine, fibuline-1, gelsoline, haptoglobine, sous- unité d'hémoglobine alpha, hémopexine, héparine cofacteur 2, facteur de croissance analogue à l'insuline liant la sous-unité acide labile, facteur de croissance analogue à l'insuline liant la protéine 3, Haptoglobuline (HPT), inhibiteur de l'inter-alpha-trypsine chaîne lourde Hl, inhibiteur de l'inter-alpha- trypsine chaîne lourde H2, Insulin-like growth factor binding protein 3 (IGFB3), protéine liant les lipopolysaccharides, lumican, Neuropiline-2, orosomucoïde (ORM), PEDF, Plasminogène (PLMN), Protéine_AMBP, prothrombine, protéine 4 liant le rétinal, serotransferrine (TRANSF), protéine sérum amyloïde A-4, globuline liant la thyroxine, transthyrétine (pré-albumine)(TTHY), vasorine, protéine liant la vitamine D, protéine C dépendante de la vitamine K, protéine dépendante de la vitamine K S, protéine 4 liant le rétinol (Retinol binding protein 4, RET4). According to a particular embodiment, the subject of the present invention is a method for detecting, optionally followed by quantification, of at least one protein chosen from the group consisting of the following proteins: afamine, alpha-1-antichymotrypsin, alpha- lB-glycoprotein (A1BG), alpha 1 acid glycoprotein, albumin (ALBU), alpha-2-HS-glycoprotein, alpha-2- antiplasmin, 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 Al), Apolipoprotein A2 (Apo A2), apolipoprotein A2 (Apeta A2), apolipoprotein 2-4 glycoprotein 1, beta-2-microglobulin (B2M), beta-Ala-his-dipeptidase, C4b binding protein (alpha chain), CD5 antigen- like, CDNA-FU53327, ceruloplasmin (CERU), cholinesterase, clusterin, coagulation factor X (CF-X), coagulation factor XI, coagulation factor XII, complement Clq subunit of sub-component B, subunit C complement Clq, complement Clr subcomponent, complement C1S subcomponent, complement C2 (C2), complement C3 (C3), complement C4-B (C4B), complement C5, complement component C8 (beta chain), complement component C9, complement factor B, complement factor D, complement factor I, Cystatin C (CysC), corticosteroid-binding globulin, C reactive protein (CRP), fibrinogen (alpha chain) (FIBA), fibrinogen (beta chain) ( FIBB), fibronectin, fibulin-1, gelsolin, haptoglobin, hemoglobin alpha subunit, hemopexin, heparin cofactor 2, insulin-like growth factor binding labile acid subunit, growth factor analogous to insulin binding protein 3, Haptoglobulin (HPT), inter-alpha-trypsin inhibitor l chain urde Hl, inter-alpha-trypsin heavy chain inhibitor H2, Insulin-like growth factor binding protein 3 (IGFB3), lipopolysaccharide binding protein, lumican, Neuropilin-2, orosomucoid (ORM), PEDF, Plasminogen (PLMN) , Protein_AMBP, prothrombin, retinal binding protein 4, serotransferrin (TRANSF), serum amyloid A-4 protein, thyroxine binding globulin, transthyretin (pre-albumin) (TTHY), vasorin, vitamin D binding protein, protein C dependent vitamin K, a protein dependent on vitamin KS, retinol binding protein 4 (Retinol binding protein 4, RET4).
Selon un mode de réalisation plus particulier, la présente invention a pour objet un procédé de détection, suivie éventuellement de la quantification, d'une, deux, trois, quatre, cinq, six, sept, huit, neuf, dix, quinze, vingt, vingt-cinq, trente, trente-cinq, quarante ou plus de quarante protéines choisies dans le groupe défini ci-dessus. According to a more particular embodiment, the subject of the present invention is a method of detection, optionally followed by quantification, of one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty , twenty-five, thirty, thirty-five, forty or more than forty proteins selected from the group defined above.
Selon un troisième objet, la présente demande concerne un kit convenant pour la préparation in vitro d'un échantillon de peptides à partir d'un échantillon biologique selon l'invention, ledit kit comprenant au moins : According to a third subject, the present application relates to a kit suitable for the in vitro preparation of a sample of peptides from a biological sample according to the invention, said kit comprising at least:
- des réactifs convenant pour la dénaturation des protéines, - reagents suitable for the denaturation of proteins,
- des réactifs convenant pour la réduction des ponts disulfure et éventuellement des agents alkylants, - des réactifs convenant pour la digestion enzymatique des protéines, et- reagents suitable for the reduction of disulfide bridges and optionally alkylating agents, - reagents suitable for the enzymatic digestion of proteins, and
- des réactifs convenant pour la chromatographie en phase inverse sur un support solide, ledit support solide comprenant au moins un polymère choisi dans la famille des polymères de polystyrène-divinyl benzene. - reagents suitable for reverse phase chromatography on a solid support, said solid support comprising at least one polymer chosen from the family of polystyrene-divinyl benzene polymers.
Lesdits réactifs sont choisis parmi les réactifs existants, bien connus de l'homme du métier. Said reagents are chosen from existing reagents, well known to those skilled in the art.
Selon un quatrième objet, la présente demande concerne l'utilisation d'un procédé pour la préparation in vitro d'un échantillon de peptides à partir d'un échantillon biologique selon l'invention. Selon un mode de réalisation particulier, la présente demande concerne l'utilisation d'un procédé de détection et potentiellement de quantification d'au moins une protéine dans un échantillon biologique, selon l'invention. According to a fourth object, the present application relates to the use of a method for the in vitro preparation of a sample of peptides from a biological sample according to the invention. According to a particular embodiment, the present application relates to the use of a method for detecting and potentially quantifying at least one protein in a biological sample, according to the invention.
Selon un mode de réalisation particulier, la présente invention a pour objet l'utilisation d'un procédé pour la préparation in vitro d'un échantillon de peptides à partir d'un échantillon biologique, selon l'invention, et/ou l'utilisation d'un procédé de détection et potentiellement de quantification selon l'invention, pour la détection et potentiellement de quantification d'au moins une protéine choisie parmi le groupe constitué par les protéines suivantes : afamine, alpha- 1-antichymotrypsine, alpha-lB-glycoprotéine (A1BG), alpha 1 glycoprotéine acide, albumine (ALBU), alpha-2-HS-glycoprotéine, alpha-2-antiplasmine, alpha-2-macroglobuline (A2MG), antithrombine-3 (ANT3), apolipoprotéine B100 (Apo B100), apolipoprotéine C2 (Apo C2), apolipoprotéine D, apolipoprotéine E (Apo E), apolipoprotéine M, apolipoprotéine (a), apolipoprotéine al (Apo Al), Apolipoprotéine A2 (Apo A2), apolipoprotéine a4, bêta-2-glycoprotéine 1, bêta-2-microglobuline (B2M), bêta-Ala-his- dipeptidase, protéine liant la C4b (chaîne alpha), CD5 antigène-like, cDNA- FU53327, céruloplasmine (CERU), cholinestérase, clustérine, facteur de coagulation X (CF-X), facteur de coagulation XI, facteur de coagulation XII, complément Clq sous-unité de sous-composante B, sous-unité C du complément Clq, complément Clr sous-composant, complément C1S sous- composant, complément C2 (C2), complément C3 (C3), complément C4-B (C4B), complément C5, composant de complément C8 (chaîne bêta), complément composante C9, facteur de complément B, facteur de complément D, facteur de complément I, Cystatine C (CysC), globuline liant les corticoïdes, protéine C réactive (CRP), fibrinogène (chaîne alpha) (FIBA), fibrinogène (chaîne beta) (FIBB), fibronectine, fibuline-1, gelsoline, haptoglobine, sous- unité d'hémoglobine alpha, hémopexine, héparine cofacteur 2, facteur de croissance analogue à l'insuline liant la sous-unité acide labile, facteur de croissance analogue à l'insuline liant la protéine 3, Haptoglobuline (HPT), inhibiteur de l'inter-alpha-trypsine chaîne lourde Hl, inhibiteur de l'inter-alpha- trypsine chaîne lourde H2, Insulin-like growth factor binding protein 3 (IGFB3), protéine liant les lipopolysaccharides, lumican, Neuropiline-2, orosomucoïde (ORM), PEDF, Plasminogène (PLMN), Protéine_AMBP, prothrombine, protéine 4 liant le rétinal, serotransferrine (TRANSF), protéine sérum amyloïde A-4, globuline liant la thyroxine, transthyrétine (pré-albumine)(TTHY), vasorine, protéine liant la vitamine D, protéine C dépendante de la vitamine K, protéine dépendante de la vitamine K S, protéine 4 liant le rétinol (Retinol binding protein 4, RET4). According to a particular embodiment, the present invention relates to the use of a method for the in vitro preparation of a sample of peptides from a biological sample, according to the invention, and / or the use of a detection and potentially quantification method according to the invention, for the detection and potentially quantification of at least one protein chosen from the group consisting of the following proteins: afamine, alpha-1-antichymotrypsin, alpha-1B- glycoprotein (A1BG), alpha 1 acid glycoprotein, albumin (ALBU), alpha-2-HS-glycoprotein, alpha-2-antiplasmin, 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 Al), Apolipoprotein A2 (Apo A2), apolipoprotein 2-a4, glycoprotein 2-a4 , beta-2-microglobulin (B2M), beta-Ala-his-dipeptidase, lian protein t C4b (alpha chain), antigen-like CD5, cDNA- FU53327, ceruloplasmin (CERU), cholinesterase, clusterin, coagulation factor X (CF-X), coagulation factor XI, coagulation factor XII, complement Clq sub- sub-component unit B, C sub-unit of complement Clq, complement Clr sub-component, complement C1S sub-component, complement C2 (C2), complement C3 (C3), complement C4-B (C4B), complement C5, complement component C8 (beta chain), complement component C9, complement factor B, complement factor D, complement factor I, Cystatin C (CysC), corticosteroid binding globulin, C reactive protein (CRP), fibrinogen (alpha chain) (FIBA), fibrinogen (beta chain) (FIBB), fibronectin, fibulin-1, gelsolin , haptoglobin, hemoglobin alpha subunit, hemopexin, heparin cofactor 2, insulin-like growth factor binding labile acid subunit, insulin-like growth factor binding protein 3, Haptoglobulin (HPT) , H1 heavy chain inter-alpha-trypsin inhibitor, H2 heavy chain inter-alpha-trypsin inhibitor, Insulin-like growth factor binding protein 3 (IGFB3), lipopolysaccharide binding protein, lumican, Neuropilin-2, orosomucoid (ORM), PEDF, Plasminogen (PLMN), Protein_AMBP, prothrombin, retinal binding protein 4, serotransferrin (TRANSF), serum amyloid protein A-4, thyroxine binding globulin, transthyretin (pre-albumin) (TTHY), vasorTHY , vitamin D binding protein, vitamin K dependent protein C, pro vitamin KS dependent tein, retinol binding protein 4 (Retinol binding protein 4, RET4).
Selon un mode de réalisation plus particulier, la présente invention a pour objet l'utilisation d'un procédé pour la préparation in vitro d'un échantillon de peptides à partir d'un échantillon biologique, selon l'invention, et/ou l'utilisation d'un procédé de détection et potentiellement de quantification selon l'invention, pour la détection et potentiellement de quantification d'une, deux, trois, quatre, cinq, six, sept, huit, neuf, dix, quinze, vingt, vingt-cinq, trente, trente-cinq, quarante ou plus de quarante protéines choisies dans le groupe défini ci- dessus. According to a more particular embodiment, the present invention relates to the use of a method for the in vitro preparation of a sample of peptides from a biological sample, according to the invention, and / or the use of a detection and potentially quantification method according to the invention, for the detection and potentially of quantification of one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, twenty -five, thirty, thirty-five, forty or more than forty proteins selected from the group defined above.
Selon un cinquième objet, la présente demande concerne l'utilisation d'un procédé pour la préparation in vitro d'un échantillon de peptides à partir d'un échantillon biologique selon l'invention pour la détection et/ou le suivi d'une condition ou d'une maladie particulière choisie parmi : According to a fifth object, the present application relates to the use of a method for the in vitro preparation of a sample of peptides from a biological sample according to the invention for the detection and / or monitoring of a condition. or a particular disease chosen from:
- une neuro-dégénérescence, notamment la maladie d'Alzheimer, - neurodegeneration, in particular Alzheimer's disease,
- une maladie neurologique ou psychiatrique, et notamment la sclérose en plaques et l'autisme, - a neurological or psychiatric disease, and in particular multiple sclerosis and autism,
- un état de carence, infectieux, d'inflammation ou de malnutrition, - a state of deficiency, infection, inflammation or malnutrition,
- une maladie chronique ou évolutive, notamment un cancer, une hépatite ou une maladie métabolique. La détection et/ou le suivi d'une maladie ou d'une condition particulière peuvent être réalisés par la détection et/ou la quantification d'au moins une protéine considérée comme un marqueur clinique, éventuellement associée à la détection et/ou la quantification d'au moins un marqueur clinique non protéique. - a chronic or progressive disease, in particular cancer, hepatitis or metabolic disease. The detection and / or the monitoring of a disease or a particular condition can be carried out by the detection and / or the quantification of at least one protein considered as a clinical marker, possibly associated with the detection and / or the quantification at least one non-protein clinical marker.
Selon un aspect particulier, la présente demande a pour objet l'utilisation d'un procédé pour la préparation in vitro d'un échantillon de peptides à partir d'un échantillon biologique, selon l'invention, pour la détection et/ou le suivi de l'état de santé, nutritionnel et de fragilité et d'un sujet, en particulier un sujet âgé. Par sujet âgé on entend un sujet de 75 ans ou plus, plus particulièrement un sujet de 85 ans ou plus. Plus particulièrement, une utilisation selon l'invention comprend la détection et/ou la quantification d'une ou plusieurs protéines choisies parmi : l'albumine, l'alpha 1 glycoprotéine acide, la transthyrétine et la CRP (Protéine C Réactive). According to one particular aspect, the present application relates to the use of a method for the in vitro preparation of a sample of peptides from a biological sample, according to the invention, for the detection and / or monitoring. the state of health, nutritional and frailty and of a subject, in particular an elderly subject. By elderly subject is meant a subject of 75 years or more, more particularly a subject of 85 years or more. More particularly, a use according to the invention comprises the detection and / or the quantification of one or more proteins chosen from: albumin, alpha 1 acid glycoprotein, transthyretin and CRP (C Reactive Protein).
D'autres avantages et caractéristiques de l'invention apparaîtront à l'examen de la description détaillée d'un mode de mise en œuvre nullement limitatif, et des dessins annexes. Other advantages and characteristics of the invention will become apparent on examination of the detailed description of a non-limiting embodiment, and of the accompanying drawings.
[Fig. l] représente schématiquement un mode de réalisation d'un procédé de préparation d'un échantillon peptidique selon l'invention et à partir d'un échantillon DBS (Fig. IA) et un mode de réalisation d'un procédé de préparation d'un échantillon peptidique selon l'état de l'art à partir de plasma (Fig. IB). [Fig. l] schematically represents an embodiment of a method for preparing a peptide sample according to the invention and from a DBS sample (FIG. 1A) and an embodiment of a method for preparing a. peptide sample according to the state of the art from plasma (FIG. IB).
[Fig. 2] représente un histogramme montrant les résultats comparés de l'analyse LC-MS de 26 protéines différentes, réalisées sur des échantillons préparés selon trois protocoles différents : a) procédé selon l'invention appliqué à un échantillon de sang séché (« DBS RPW »), b) procédé selon l'invention appliqué à un échantillon de plasma (« plasma »), c) procédé de préparation des échantillons DBS selon l'état de l'art (« standard DBS préparation »). Pour chacune des protéines, les valeurs des aires obtenues lors de l'analyse par spectrométrie de masse dans les conditions « DBS RPW » et « plasma » sont divisées, respectivement, par les valeurs correspondantes des aires obtenues avec le procédé standard de préparation de l'échantillon. En ordonnées, LOG (aire/aire après préparation DBS standard) est représenté, en abscisse, les résultats pour chacune des 26 protéines sont représentés, avec « DBS RPW » en histogramme clair et « plasma » en histogramme foncé.[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) method according to the invention applied to a dried blood sample ("DBS RPW" ), b) method according to the invention applied to a plasma sample (“plasma”), c) method for preparing DBS samples according to the state of the art (“DBS preparation standard”). For each of the proteins, the values of the areas obtained during the analysis by mass spectrometry under the conditions "DBS RPW" and "plasma" are divided, respectively, by the corresponding values of the areas obtained with the standard method for preparing the protein. 'sample. On the ordinate, LOG (area / area after standard DBS preparation) is represented, on abscissa, the results for each of the 26 proteins are shown, with “DBS RPW” in light histogram and “plasma” in dark histogram.
[Fig.3] représente schématiquement la comparaison de la quantification, dans[Fig.3] schematically represents the comparison of the quantification, in
95 échantillons de plasma, de deux protéines sériques : la protéine C réactive (CRP) (Fig. 3A) et la serotransferrine (TRANSF) (Fig. 3B). Pour chacune des protéines, le résultat de la quantification par immunoessai validé cliniquement est exprimé sur l'axe des abscisses (en mg/L pour CRP et g/L pour TRANSF) et le résultat de la quantification par spectrométrie de masse sur des échantillons préparés par un procédé selon l'invention est exprimé sur l'axe des ordonnées (unités arbitraires). 95 plasma samples, of two serum proteins: C reactive protein (CRP) (Fig. 3A) and serotransferrin (TRANSF) (Fig. 3B). For each protein, the result of 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 quantification by mass spectrometry on prepared samples by a method according to the invention is expressed on the y-axis (arbitrary units).
La présente invention se comprendra mieux à la lecture des exemples suivants qui sont donnés pour illustrer l'invention et non pour limiter sa portée. 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.
Exemple Example
EXEMPLE 1 : Préparation d'un échantillon peptidique à partir d'un échantillon de sang séché (DBS) et analyse comparative par LC-MS. EXAMPLE 1 Preparation of a peptide sample from a dried blood sample (DBS) and comparative analysis by LC-MS.
L'efficacité du procédé selon l'invention a été évaluée en détectant et quantifiant 26 protéines en LC/MS, en comparant respectivement l'analyse d'un échantillon DBS traité par un procédé selon l'invention (« DBS-RPW ») et l'analyse d'un échantillon de plasma traité par un procédé selon l'état de l'art (« plasma ») à une analyse effectuée directement à partir d'un échantillon DBS (« standard DBS »). Les étapes du procédé selon l'invention sont schématisées en Figure IA, les étapes du procédé de préparation d'un échantillon à partir de plasma et selon un procédé de l'état de l'art sont schématisées en Figure I B. L'expérience a été réalisée en duplicat et chaque analyse LC/MS a été réalisée en double. Les 26 protéines sont les suivantes : A1 BG, A2MG, ANT3, Apo Al, Apo A2, Apo B100, Apo C2, Apo E, B2M, CERU, CF_X, C2, C3, C4B, CRP, CysC, FIBA, H PT, FIBB, IGFB3, PLMN, ORM, RET4, TRANSF, TTHY, ALBU. The efficiency of the method according to the invention was evaluated by detecting and quantifying 26 proteins in LC / MS, by respectively comparing the analysis of a DBS sample treated by a method according to the invention (“DBS-RPW”) and the analysis of a plasma sample treated by a method according to the state of the art (“plasma”) to an analysis carried out directly from a DBS sample (“DBS standard”). The steps of the method according to the invention are shown diagrammatically in FIG. 1A, the steps of the method for preparing a sample from plasma and according to a method of the state of the art are diagrammed in FIG. I B. The experiment was performed in duplicate and each LC / MS analysis was performed in duplicate. The 26 proteins are as follows: A1 BG, A2MG, ANT3, Apo Al, Apo A2, Apo B100, Apo C2, Apo E, B2M, CERU, CF_X, C2, C3, C4B, CRP, CysC, FIBA, H PT, FIBB, IGFB3, PLMN, ORM, RET4, TRANSF, TTHY, ALBU.
Le procédé de préparation à partir d'un échantillon DBS selon l'invention « DBS-RPW » est réalisé comme suit. Pour l'extraction des protéines, un « punch DBS » est réalisé par poinçonnage du support solide (papier buvard type 226) contenant l'échantillon. Ledit punch est transféré dans une plaque The preparation process from a DBS sample according to the invention “DBS-RPW” is carried out as follows. For the extraction of proteins, a “DBS punch” is produced by punching the solid support (type 226 blotting paper) containing the sample. Said punch is transferred to a plate
96 puits, puis les protéines sont extraites en ajoutant 200 m\- d'ammonium bicarbonate à 50 mM, puis en agitant 30 minutes à l'aide d'un agitateur de paillasse à 350 rpm sur un appareil Eppendorf® Thermomixer Compact. L'échantillon est ensuite dénaturé par ajout de 200 m\- d'urée 8M et une nouvelle agitation pendant 10 minutes. Les ponts disulfures des protéines de l'échantillon sont ensuite réduits par ajout de 21 m\- de DTT à 200 mM dans du tris 1 M pH 8.5, et de 12 pL tris 1 M pH 8.5, et agitation une heure à 37°C sous agitation à 350 rpm sur un Eppendorf® Thermomixer Compact. Les cystéines libérées sont ensuite alkylées par ajout de 18 m\- d'IAA à 1 M, 6 pL de Tris 1 M pH 10, et nouvelle agitation 30 minutes à 37°C. L'étape d'alkylation est stoppée par ajout de 20 m\- de DTT à 200 mM. L'échantillon est ensuite acidifié par l'ajout de 10 m\- d'acide formique avant transfert des 210 m\- du surnageant dans deux nouveaux puits. 96 wells, then the proteins are extracted by adding 200 m \ - of 50 mM ammonium bicarbonate, then stirring for 30 minutes using a shaker. bench at 350 rpm on an Eppendorf® Thermomixer Compact device. The sample is then denatured by adding 200 ml of 8M urea and stirring again for 10 minutes. The disulfide bonds of the proteins in the sample are then reduced by adding 21 m \ - of 200 mM DTT in 1 M tris pH 8.5, and 12 pL 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 liberated cysteines are then alkylated by adding 18 m \ - of 1 M IAA, 6 μl of 1 M Tris pH 10, and further stirring for 30 minutes at 37 ° C. The alkylation step is stopped by adding 20 m \ - of 200 mM DTT. The sample is then acidified by adding 10 m \ - of formic acid before transferring the 210 m \ - of the supernatant into two new wells.
L'étape de nettoyage de l'échantillon est ensuite réalisée sur des cartouches RP-W® commercialisées par la société Agilent. Les cartouches sont lavées et conditionnées avec 100 pL à 300 pL/min d'une solution d'acétonitrile (70%)/TFA (0,l%)/eau (29,9%), et sont équilibrées avec 50 pL à 10 pL/min d'une solution de 0,1% acide formique. L'échantillon est chargé sur cartouche RP-W (Cat# G5496-60086) à 5 pL/min. La phase RP-W contenue dans la cartouche est lavée avec 50 m\- à 10 mΰ/Ghίh d'une solution de 0,1% TFA puis l'échantillon nettoyé est élué de la cartouche avec 20 m\- à 5 pL/min d'une solution d'acétonitrile (70%)/acide formique (0,l%)/eau (29,9%). L'élution est mise à sec (Speedvac™). L'échantillon sec est resuspendu dans 37,4 pL de Tris 20 mM pH 8,5 ; 5,6 pg de trypsine/LysC sont ajoutés pour réaliser la digestion trypsique. Cette réaction dure 14 heures à 37°C sous agitation (350 rpm sur un Eppendorf® Thermomixer Compact). La réaction de digestion est arrêtée par ajout de 3 m\- d'acide formique. La plaque de 96 puits est filmée et les échantillons sont prêts pour injection sur le système LC/MS. The sample cleaning step is then carried out on RP-W® cartridges sold by the company Agilent. The cartridges are washed and conditioned with 100 µL at 300 µL / min of a solution of acetonitrile (70%) / TFA (0.1%) / water (29.9%), and are equilibrated with 50 µL at 10 pL / min of a solution of 0.1% formic acid. The sample is loaded onto an RP-W cartridge (Cat # G5496-60086) at 5 pL / min. The RP-W phase contained in the cartridge is washed with 50 m \ - at 10 mΰ / Ghίh of a 0.1% TFA solution and then the cleaned sample is eluted from the cartridge with 20 m \ - at 5 pL / min of a solution of acetonitrile (70%) / formic acid (0.1%) / water (29.9%). The elution is put to dryness (Speedvac ™). The dry sample is resuspended in 37.4 µL of 20 mM Tris pH 8.5; 5.6 µg of trypsin / LysC are added to carry out the tryptic digestion. This reaction lasts 14 hours at 37 ° C. with stirring (350 rpm on an Eppendorf® Thermomixer Compact). The digestion reaction is stopped by adding 3 m \ - of formic acid. The 96-well plate is filmed and the samples are ready for injection into the LC / MS system.
La préparation des échantillons "DBS standard" est réalisée comme suit : une ou deux gouttes de sang capillaire, obtenues après piqûre au bout du doigt par une lancette, sont déposées sur chaque spot de DBS. Il est possible également de déposer avec l'usage d'une pipette 70 pL de sang total veineux collecté dans un tube sur un spot DBS. Après dépôt, les cartes sont mises à sécher pendant 2 heures à température ambiante. Elles sont mises dans un sachet plastique individuel et peuvent être conservées selon les usages à température ambiante, à 4°C ou congelées (-20°C ou -80°C). Avant analyse, les cartes sont remises à température ambiante. Un poinçon (« punch ») de 6 mm de diamètre est ensuite prélevé pour chaque spot et transféré dans un tube Eppendorf LoBind de 2 ml. The preparation of the “standard DBS” 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 spot of DBS. It is also possible to deposit with the use of a pipette 70 μL of venous whole blood collected in a tube on a DBS spot. After deposit, the cards are left to dry for 2 hours at room temperature. They are put in an individual plastic bag and can be stored according to use at room temperature, at 4 ° C or frozen (-20 ° C or -80 ° C). Before analysis, the cards are returned at room temperature. A 6 mm diameter punch is then taken for each spot and transferred into a 2 ml Eppendorf LoBind tube.
La préparation des échantillons "plasma" est réalisée comme suit : le procédé est schématisé en Figure IB, il représente un procédé typique connu dans l'état de l'art et comprend les étapes suivantes : à partir de 2 pL de plasma liquide ou déposé sur un support « papier Whatman 903 » ou le « papier Ahlstrom FN/226 », les protéines sont dénaturées et réduites, puis subissent une alkylation, une digestion enzymatique (en présence de trypsine/LysC pendant 14h à 37°C), puis une étape de nettoyage des peptides obtenus réalisée sur une colonne type ZORBAX Eclipse Plus C18, suivie de l'analyse par LC-MS. The preparation of the "plasma" samples is carried out as follows: the process is shown diagrammatically in FIG. IB, it represents a typical process known in the state of the art and comprises the following stages: starting from 2 μL of liquid or deposited plasma 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 the peptides obtained carried out on a ZORBAX Eclipse Plus C18 type column, followed by analysis by LC-MS.
Les résultats sont illustrés dans la Figure 2. On remarque pour la plupart des protéines une valeur positive. Ceci indique une plus grande quantité détectable (meilleure sensibilité) par rapport au procédé standard et on constate également une bonne correspondance entre valeurs plasma standard et DBS RPW. The results are shown in Figure 2. Most proteins show a positive value. This indicates a larger detectable amount (better sensitivity) compared to the standard method and there is also a good match between standard plasma values and DBS RPW.
Globalement, les intensités en spectrométrie de masse retrouvées après un traitement de l'échantillon DBS par un procédé selon l'invention (DBS RPW) sont 19 fois plus intenses que celles obtenues directement après DBS standard. Ces intensités correspondent pour la majorité des protéines à celles obtenues sur un échantillon de plasma préparé par un procédé standard. Overall, the intensities in mass spectrometry found after treatment of the DBS sample by a method 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 proteins to those obtained on a sample of plasma prepared by a standard method.
Pour valider le procédé de préparation d'un échantillon peptidique selon l'invention et définir ses performances, l'étude suivante a été faite : Dix prélèvements indépendants prélevés chez des patients sur DBS (procédé DBS- RPW selon l'invention) ont été utilisés pour l'étude. Pour chaque patient, deux punchs DBS ont été analysés indépendamment et en duplicat. Les coefficients de variation (CV) de 91 peptides, correspondant à 76 protéines, mesurés donc 2 fois indépendamment et en duplicat ont été calculés. Ceci permet d'estimer la variabilité de tout le processus d'analyse des DBS (pré-analytique et analytique). La médiane des CVs obtenus sur tous les peptides est de 9,3%. La gamme des CVs s'étend de 2 à 52%. Par comparaison, lors de la même étude, la médiane des CV globaux obtenus cette fois ci sur le plasma prélevé chez les patients en même temps que le DBS était de 6,7% avec une gamme des CVs qui s'étendait de 2 à 48%. Pour valider l'intérêt clinique des mesures une corrélation entre les valeurs obtenues dans le plasma (celles qui sont donc utilisées pour la clinique) et celles obtenues dans les DBS a été calculées. 35 peptides présentent une corrélation très forte entre « plasma standard » et « DBS-RPW » (coefficient de corrélation >0,8), 32 une corrélation intermédiaire (entre 0,6 et 0,8) et 24 une faible corrélation (<0.6). To validate the process for preparing a peptide sample according to the invention and define its performance, the following study was carried out: Ten independent samples taken from patients on DBS (DBS-RPW process according to the invention) were used for study. For each patient, two DBS punches were analyzed independently and in duplicate. The coefficients of variation (CV) of 91 peptides, corresponding to 76 proteins, therefore measured twice independently and in duplicate, were calculated. This makes it possible to estimate the variability of the whole DBS analysis process (pre-analytical and analytical). The median of the CVs obtained on all the peptides is 9.3%. The range of CVs extends from 2 to 52%. By comparison, in the same study, the median of the overall CV obtained this time on the plasma collected from the patients at the same time as the DBS was 6.7% with a range CVs that ranged from 2 to 48%. To validate the clinical interest of the measurements, a correlation between the values obtained in the plasma (those which are therefore used for clinical purposes) and those obtained in the DBS was calculated. 35 peptides show a very strong correlation between “standard plasma” and “DBS-RPW” (correlation coefficient> 0.8), 32 an intermediate correlation (between 0.6 and 0.8) and 24 a weak correlation (<0.6 ).
Les plasmas de 95 patients différents, recrutés par ordre chronologique et sans pathologie particulière ont été analysés, d'une part, par spectrométrie de masse sur des échantillons préparés par un procédé selon l'invention et en parallèle par immunoessai, sur un automate COBAS 6000 (Roche Diagnostic) sur le site de Biochimie de l'hôpital de Montpellier. Les résultats obtenus sont présentés sur la figure 3, avec l'analyse de la CRP (figure 3A) et de la serotransferrine (figure 3B) respectivement. Les résultats obtenus par les deux méthodes ont été comparés par le logiciel R. La comparaison montre une corrélation statistiquement significative pour chacune des deux protéines quantifiées. The plasmas of 95 different patients, recruited in chronological order and without any particular pathology were analyzed, on the one hand, by mass spectrometry on samples prepared by a method according to the invention and in parallel by immunoassay, on a COBAS 6000 automaton. (Roche Diagnostic) on the Biochemistry site of the Montpellier hospital. The results obtained are shown in figure 3, together with the analysis of CRP (figure 3A) and serotransferrin (figure 3B) respectively. The results obtained by the two methods were compared by software R. The comparison shows a statistically significant correlation for each of the two quantified proteins.
Les résultats ainsi obtenus permettent donc de valider l'intérêt clinique du procédé d'analyse comprenant un procédé de préparation d'un échantillon peptidique selon l'invention. The results thus obtained therefore make it possible to validate the clinical interest of the analysis method comprising a method for preparing a peptide sample according to the invention.

Claims

Revendications Claims
1. [Procédé pour la préparation in vitro d'un échantillon de peptides à partir d'un échantillon biologique, comprenant les étapes successives suivantes : a) dénaturation des protéines présentes dans ledit échantillon, 1. [Process for the in vitro preparation of a sample of peptides from a biological sample, comprising the following successive steps: a) denaturation of the proteins present in said sample,
b) réduction et alkylation des protéines issues de l'étape a), c) nettoyage des protéines issues de l'étape b) par chromatographie en phase inverse sur support solide polymérique, ledit support solide comprenant au moins un polymère de polystyrène-divinyl benzène, et b) reduction and alkylation of the proteins from step a), c) cleaning of the proteins from step b) by reverse phase chromatography on a polymeric solid support, said solid support comprising at least one polystyrene-divinyl benzene polymer , and
d) digestion par une protéase des protéines issues de l'étape c). d) digestion with a protease of the proteins resulting from step c).
2. Procédé selon la revendication 1, caractérisé en ce que ledit échantillon biologique est un échantillon sanguin sous forme liquide choisi dans le groupe constitué par : le sang total, le sérum et le plasma. 2. Method according to claim 1, characterized in that said biological sample is a blood sample in liquid form chosen from the group consisting of: whole blood, serum and plasma.
3. Procédé selon la revendication 1, caractérisé en ce que ledit échantillon biologique est un échantillon sanguin choisi dans le groupe constitué par : le sang total, le sérum et le plasma, que ledit échantillon est sous forme solide ou desséchée, et en ce que ledit procédé comprend, préalablement à l'étape de dénaturation des protéines, une étape d'extraction desdites protéines à partir dudit échantillon sous forme solide ou desséchée. 3. Method according to claim 1, characterized in that said biological sample is a blood sample chosen from the group consisting of: whole blood, serum and plasma, that said sample is in solid or dried form, and in that said method comprises, prior to the step of denaturing the proteins, a step of extracting said proteins from said sample in solid or dried form.
4. Procédé selon la revendication 3, caractérisé en ce que ledit échantillon sanguin sous forme solide ou desséchée est un échantillon de type DBS (Dried Blood Spot) déposé sur un papier de recueil, de préférence un papier de type buvard. 4. Method according to claim 3, characterized in that said blood sample in solid or dried form is a sample of DBS (Dried Blood Spot) type deposited on a collection paper, preferably a blotting paper.
5. Procédé selon l'une quelconque des revendications précédentes, caractérisé en ce que l'étape de digestion par une protéase des protéines est réalisée en présence de Trypsine / Endoprotéinase Lys- C avec un ratio quantité d'enzyme / quantité de protéine substrat, compris entre 1/10 et 1/200, et de préférence compris entre 1/50 et 1/100, pendant une durée supérieure ou égale à 2 heures, de préférence supérieure ou égale à 10 heures, de préférence supérieure ou égale à 14 heures, de préférence égale à 14 heures. 5. Method according to any one of the preceding claims, characterized in that the step of digestion with a protease of the proteins is carried out in the presence of trypsin / Lys-C endoproteinase with a ratio of quantity of enzyme / quantity of substrate protein, between 1/10 and 1/200, and preferably between 1/50 and 1/100, for a period greater than or equal to 2 hours, preferably greater than or equal to 10 hours, preferably greater than or equal to 14 hours , preferably equal to 14 hours.
6. Procédé selon l'une quelconque des revendications précédentes, dans lequel l'échantillon de type DBS est traité lors de l'étape d) en présence de Trypsine / Endoprotéinase Lys-C avec un ratio quantité d'enzyme / quantité de protéine substrat compris entre 1/50 et 1/100, pendant une durée supérieure ou égale à 2 heures. 6. Method according to any one of the preceding claims, in which the sample of DBS type is treated during step d) in the presence of Trypsin / Endoproteinase Lys-C with a ratio of quantity of enzyme / quantity of substrate protein. between 1/50 and 1/100, for a period greater than or equal to 2 hours.
7. Procédé de détection ou de quantification de protéines dans un échantillon biologique, comprenant un procédé pour la préparation in vitro d'un échantillon de peptides, selon l'une quelconque des revendications précédentes, suivi d'une étape de détection ou de quantification, d'au moins une protéine au moyen d'une technique de d'analyse, de préférence au moyen de la spectrométrie de masse, de préférence la technique de Chromatographie Liquide - Spectrométrie de Masse (LC-MS). 7. A method of detecting or quantifying proteins in a biological sample, comprising a method for the in vitro preparation of a sample of peptides, according to any one of the preceding claims, followed by a detection or quantification step, of at least one protein by means of an analysis technique, preferably by means of mass spectrometry, preferably the technique of Liquid Chromatography - Mass Spectrometry (LC-MS).
8. Procédé selon la revendication 7, caractérisé en ce que ladite au moins une protéine est choisie parmi les protéines suivantes : afamine, alpha-l-antichymotrypsine, alpha-lB-glycoprotéine (A1BG), alpha 1 glycoprotéine acide, albumine (ALBU), alpha-2-HS- glycoprotéine, alpha-2-macroglobuline (A2MG), antithrombine-3 (ANT3), apolipoprotéine B100 (Apo B100), apolipoprotéine C2 (Apo C2), apolipoprotéine D, apolipoprotéine E (Apo E), apolipoprotéine M, apolipoprotéine (a), apolipoprotéine al (Apo Al), Apolipoprotéine A2 (Apo A2), apolipoprotéine a4, bêta-2-glycoprotéine 1, bêta-2- microglobuline (B2M), bêta-Ala-his-dipeptidase, protéine liant la C4b (chaîne alpha), CD5 antigène-like, CDNA-FLJ53327, céruloplasmine (CERU), cholinestérase, clustérine, facteur de coagulation X (CF-X), facteur de coagulation XI, facteur de coagulation XII, complément Clq sous-unité de sous-composante B, sous-unité C du complément Clq, complément Clr sous-composant, complément C1S sous- composant, complément C2 (C2), complément C3 (C3), complément C4-B (C4B), complément C5, composant de complément C8 (chaîne bêta), complément composante C9, facteur de complément B, facteur de complément D, facteur de complément I, Cystatine C (CysC), globuline liant les corticoïdes, protéine C réactive (CRP), fibrinogène (chaîne alpha) (FIBA), fibrinogène (chaîne beta) (FIBB), fibronectine, fibuline- 1, gelsoline, haptoglobine, sous-unité d'hémoglobine alpha, hémopexine, héparine cofacteur 2, facteur de croissance analogue à l'insuline liant la sous-unité acide labile, facteur de croissance analogue à l'insuline liant la protéine 3, Haptoglobuline (HPT), inhibiteur de l'inter-alpha-trypsine chaîne lourde Hl, inhibiteur de l'inter-alpha-trypsine chaîne lourde H2, Insulin-like growth factor binding protein 3 (IGFB3), protéine liant les lipopolysaccharides, lumican, Neuropiline-2, orosomucoïde (ORM), PEDF, Plasminogène (PLMN), Protéine_AMBP, prothrombine, protéine 4 liant le rétinal, serotransferrine (TRANSF), protéine sérum amyloïde A-4, globuline liant la thyroxine, transthyrétine (pré- albumine)(TTHY), vasorine, protéine liant la vitamine D, protéine C dépendante de la vitamine K, protéine dépendante de la vitamine K S, protéine 4 liant le rétinol (Retinol binding protein 4, RET4). 8. Method according to claim 7, characterized in that said at least one protein is chosen from the following proteins: afamine, alpha-l-antichymotrypsin, alpha-lB-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 Eoprotein), apolipoprotein E (Apo Eoprotin) M, apolipoprotein (a), apolipoprotein al (Apo Al), Apolipoprotein A2 (Apo A2), apolipoprotein a4, beta-2-glycoprotein 1, beta-2-microglobulin (B2M), beta-Ala-his-dipeptidase, binding protein C4b (alpha chain), antigen-like CD5, CDNA-FLJ53327, ceruloplasmin (CERU), cholinesterase, clusterin, coagulation factor X (CF-X), coagulation factor XI, coagulation factor XII, complement Clq subunit of sub-component B, sub-unit C of the complement Clq, complement Clr subcomponent, complement C1S subcomponent, complement C2 (C2), complement C3 (C3), complement C4-B (C4B), complement C5, complement component C8 (beta chain), complement component C9, complement factor B, complement factor D, complement factor I, Cystatin C (CysC), corticosteroid-binding globulin, C reactive protein (CRP), fibrinogen (alpha chain) (FIBA), fibrinogen (beta chain) (FIBB) , fibronectin, fibulin-1, gelsolin, haptoglobin, hemoglobin alpha subunit, hemopexin, heparin cofactor 2, insulin-like growth factor binding labile acid subunit, insulin-like growth factor binding protein 3, Haptoglobulin (HPT), H1 heavy chain inter-alpha-trypsin inhibitor, H2 heavy chain inter-alpha-trypsin inhibitor, Insulin-like growth factor binding protein 3 (IGFB3), protein binding lipopolysaccharides, lumican, Neuropilin-2, orosomucoid (ORM), PEDF, Plasminogen (PLMN), Prot ein_AMBP, prothrombin, retinal binding protein 4, serotransferrin (TRANSF), serum amyloid protein A-4, thyroxine binding globulin, transthyretin (pre-albumin) (TTHY), vasorin, vitamin D binding protein, protein C dependent on vitamin K, vitamin KS dependent protein, retinol binding protein 4 (Retinol binding protein 4, RET4).
9. Utilisation d'un procédé selon l'une quelconque des revendications 1 à 8 pour la préparation d'un échantillon peptidique pour la détection ou le suivi de l'évolution d'une condition choisie parmi : 9. Use of a method according to any one of claims 1 to 8 for the preparation of a peptide sample for the detection or monitoring of the evolution of a condition chosen from:
- une neuro-dégénérescence, notamment la maladie d'Alzheimer, - neurodegeneration, in particular Alzheimer's disease,
- une maladie neurologique ou psychiatrique, et notamment la sclérose en plaques et l'autisme, - a neurological or psychiatric disease, and in particular multiple sclerosis and autism,
- un état de carence, infectieux, d'inflammation ou de malnutrition, - a state of deficiency, infection, inflammation or malnutrition,
- une maladie chronique ou évolutive, notamment un cancer, une hépatite ou une maladie métabolique. ] - a chronic or progressive disease, in particular cancer, hepatitis or metabolic disease. ]
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