RU2595835C2 - Method of producing analytical test system for multiplex identification and quantitative measurement of content of proteins of interest in biological sample by content of corresponding proteotipc marker peptides - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention relates to a method of producing an analytical test system (MRM-test) for multiplex identification and quantitative measurement of content of protein of interest in a biological sample based on content of corresponding proteotypic marker peptides, including detection of unique for protein proteotypic marker peptide sequences; extraction of at least two proteotypic marker peptide sequences of protein; prediction of peptide fragments; prediction of MRM-test in form of a list of marker peptides, their fragments and best detection parameters; synthetic peptide marker; determination of profile transitions of synthetic marker peptides; optimising MRM-test in accordance with obtained profiles; cleaning peptides; preparation of a biological sample; identification of protein in a biological sample with weir of synthetic peptides; determination of retention time of marker peptides with application of values determined in MRM tests; conducting multiplex calibration measurements; quantitative measurement of content of marker peptides in biological sample; and a judgment on content of protein of interest in a biological sample.

EFFECT: invention provides high sensitivity and reduced time for determining proteotypic peptides and target protein of interest.

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Description

FIELD OF THE INVENTION

The present invention relates to the field of biochemistry and, more specifically, proteomics. In particular, the invention relates to a method for producing an analytical test system for multiplex identification and quantitative measurement of the content of proteins of interest in a biological sample by the content of their corresponding proteotypic marker peptides.

Description of the Related Art of the Present Invention

The main task of proteomics - a relatively young area of modern biochemical research - is to conduct an inventory of proteins that are expressed in the cell, which implies their most accurate qualitative and quantitative determination.

The most modern approach to overcoming the lack of specificity and performance of protein analyzes based on the use of antibodies and their analogues is to use the unique capabilities of mass spectrometric technology for monitoring Multiple (Selected) Reaction Monitoring, MRM, SRM). This method consists in monitoring the fragmentation of peptide ions in a hydrolyzed sample of biological origin [Anderson L., Hunter C. L. Quantitative mass spectrometric multiple reaction monitoring assays for major plasma proteins. Mol Cell Proteomics. 2006; 5 (4): 573-588]. According to the MRM method, measurements are carried out on a mass spectrometer with a detector such as a triple quadrupole, which has high sensitivity and superior parameters to the competitive technologies in the range of measured concentrations. The MRM method is based on the registration of charged molecules resulting from the ionization and fragmentation of specific peptide molecules originating from the protein of interest and characterizing it exclusively. In other words, individual peptides serve as a “prototype” of the whole protein after its artificial hydrolysis. Such peptides are called proteotypic. For the purposes of the present invention, the terms “proteotypic”, “marker”, or a combination thereof are used interchangeably.

In contrast to traditional immunochemical methods of protein registration, using this approach, there is essentially no possibility of cross-reactions with other proteins, since the bioinformation choice of a proteotypic peptide is based, first of all, on the uniqueness of the sequence and mass spectrometric parameters of the test [Picotti P., Aebersold R. Selected reaction monitoring-based proteomics: workflows, potential, pitfalls and future directions. Nat Methods. 2012; 9 (6): 555-566].

A sufficient number of works on this topic is known from the prior art, however, the closest to the present invention similar solution to the authors is the invention according to international patent publication WO 2009/141141, which discloses a method for identifying and quantifying proteins and peptides using mass spectrometry using technology monitoring multiple reactions.

More specifically, said prototype relates to a method for forming an MRM test for detecting and / or quantifying one or more peptides and / or proteins, comprising the following steps:

(1) a protein / peptide of interest, a group of proteins / peptides of interest, a subproteome or a whole proteome is optionally cleaved, followed by optional purification / separation / enrichment, to obtain a mixture of peptides from which at least one peptide is essentially uniquely associated with the method of interest by mass spectrometry a protein or a target protein from a specified group of proteins of interest or from a specified whole proteome, and it is associated by differential quantitative behavior in various experimental conditions ditions;

(2) this at least one peptide is synthesized and / or generated essentially without subsequent purification;

(3) this at least one crude peptide is analyzed by the method of optionally coupled with liquid chromatography monitoring of multiple reactions;

(4) development, validation and / or optimization of an appropriate test of at least one peptide with determination of the MRM coordinates of the peptide / protein of interest and / or the regulator of interest.

However, this prototype is characterized by several disadvantages.

So, from the current level of technology it follows that the use of a data-independent mode always precedes a data-dependent (information-dependent, targeted) mode of recording mass spectra. This is reflected in a large number of publications where, before developing an MRM test, it is proposed to analyze mass spectral databases obtained in a data-independent mode [Picotti P, Aebersold R. Selected reaction monitoring-based proteomics: workflows, potential, pitfalls and future directions . Nat Methods. 2012; 9 (6): 555-566; Gallien S, Duriez E, Demeure K, Domon B. Selectivity of LC-MS / MS analysis: implication for proteomics experiments. J Proteomics. 2013; 81: 148-158]. According to international patent publication WO 2009/141141, a mass spectrometric analysis step of a biological material is also provided for prior to the development of an MRM test. In the example shown in the patent, as well as in the application of the invention set forth by the patent authors in the publication [Picotti P, Rinner O, Stallmach R, Dautel F, Farrah T, Domon B, Wenschuh H, Aebersold R. High-throughput generation of selected reaction-monitoring assays for proteins and proteomes. Nat Methods. 2010 Jan; 7 (1): 43-6], it is indicated that peptide fragmentation independent of the MS / MS data is used as the primary mass spectrometric analysis, while subsequent test development is performed using information-dependent ion monitoring.

As already mentioned, the main task of proteomics is the accurate qualitative and quantitative determination of the proteins expressed in the cell, and it is natural that the higher the sensitivity of the determination method, the more reliable and valuable results can be obtained by the researcher. Thus, in the prior art there remains a need for a method for identifying and quantifying proteins and peptides using mass spectrometry, which would be characterized by greater sensitivity.

In addition, the implementation of the method according to international patent publication WO 2009/141141 is very time-consuming and other resources, and in the prior art there remains a need for a faster and cheaper method for the identification and quantification of proteins and peptides using mass spectrometry.

Disclosure of the present invention

The fundamental difference of the present invention is that already at the preliminary stage, information-dependent monitoring of ions at a triple quadrupole type detector is also used. The results obtained allow us to say that the “targeted”, targeted determination of the content of peptides at all stages significantly increases the sensitivity of the method. Therefore, the present invention provides an increase in sensitivity with respect to conventional methods through the use of information-dependent mode in the first stage of the formation of the MRM test.

Reducing the time spent on obtaining the result is also achieved due to the fact that all measurements are performed in an information-dependent mode. Since modern devices are limited in the number of measured transitions in a certain period of time (up to 100-150), the use of an information-dependent mode provides an optimal time for loading the device. First, the device measures all transitions related to one or more peptides. That is, 15-20 transitions are measured for each peptide, which, due to technical limitations, allows obtaining data for no more than 5-10 peptides in 5-20 minutes. Further, the most intense transitions (3-5 per peptide) are selected from the measurement results and combined into one measurement protocol, which, with the restriction of, for example, 100 transitions, can now theoretically account for up to 100/5 = 20, and in real conditions up to 40- 50 peptides. Measurements for this number of peptides are carried out in an increased gradient of 20-40 minutes, which allows us to determine the values of the exit time from the chromatographic column, which actually correlate with the values in a complex bio-sample. Thus, according to the present invention, the formation and use of the MRM test is disclosed, during the implementation of which, between operations, the device is reconfigured with a change in the number of measured transitions, which is carried out simultaneously with an increase in the number of peptides corresponding to these transitions.

In accordance with the present invention, a method is developed that overcomes the disadvantages of the prototype by taking measurements in an information-dependent mode at all stages of the method involving the use of mass spectrometry.

So, the authors of the present invention have developed an improved, more efficient way to obtain an analytical test system for multiplex identification and quantitative measurement of the content of proteins of interest in a biological sample by the content of their corresponding proteotypic marker peptides. This method involves performing a number of stages:

1) bioinformational identification of protein-unique proteotypic marker peptide sequences;

2) the selection of at least two marker proteotypic peptide sequences of the protein, the most suitable for research by monitoring multiple reactions;

3) prediction of fragments of peptides;

4) prediction of the MRM test in the form of a list of marker peptides, their fragments and the best detection parameters by monitoring multiple reactions;

5) the synthesis of one or more marker peptides;

6) determining the transition profile of one or more synthetic marker peptides by monitoring multiple reactions under conditions of an accelerated chromatographic gradient;

7) optimization of the MRM test in accordance with the obtained profiles, removal from the set of peptide fragments characterized by the lowest intensity values in the mass spectrum;

8) purification of the peptides synthesized in stage 5);

9) preparation of a biological sample, involving the removal of major proteins;

10) identification of the protein in a biological sample with the introduction of synthetic peptides by monitoring multiple reactions under normal chromatographic gradient based on the profiles obtained in stage 6);

11) determination of retention times of marker peptides with the introduction of established values in MRM tests;

12) conducting multiplex calibration measurements under conditions of a normal chromatographic gradient in a buffer solution of purified synthetic peptides against a background of a mixture of proteins and / or peptides not related to them; and

13) a quantitative measurement of the content of marker peptides in a biological sample under conditions of a normal chromatographic gradient with the introduction of synthetic peptides;

14) a judgment on the content of proteins of interest in a biological sample by the content of their corresponding proteotypic marker peptides, quantitatively determined at stage 13).

Thus, the contribution of the authors of the present invention to the prior art is determined primarily by the fact that they have created a method that provides for the measurement in an information-dependent mode at all stages of the method involving the use of mass spectrometry, and thereby provides an increase in sensitivity and reduction of temporary breakfasts on the determination of proteotypic peptides and, accordingly, target proteins of interest, the content of which in the initial biological sample determines the content Table of contents proteotipicheskih peptides.

Examples of the method according to the present invention are given below.

Brief Description of the Figures

Figure 1 illustrates the selection of proteotypic peptides and fragments thereof for proteins O60543 and O95427 using Skyline software.

Figure 2 shows the chromatogram of the target peptides obtained in isocratic mode.

Figure 3 shows a fragment of a table of intensities of peaks of fragments of peptides. Fading out fragments giving the most intense signal.

Figure 4 illustrates the selection of the most intense transitions of target peptides to create a multiplex method. The graphs on the abscissa axis show the time of exit from the column in minutes, and the number of pulses on the ordinate axis.

Figure 5 shows the chromatogram of the target peptides obtained in the gradient elution mode to determine the retention time.

Figure 6 shows the calibration curve of the peptide GVMASSLQELISK.

Figure 7 shows the calibration curve of the VTFDLYR peptide.

On Fig shows the calibration curve of the peptide AESMFTNAVQILEQFK.

Figure 9 shows the calibration curve of the EVTLPFLFTPFK peptide.

Examples of the implementation of the present invention

Example 1. Prediction of the amino acid sequence of proteotypic peptides

The prediction was made using the free Skyline software based on the amino acid sequences of the O60543 and O95427 proteins from the Uniprot database. Two proteotypic peptides were selected taking into account physicochemical properties. Peptides GVMASSLQELISK and VTFDLYR were selected for protein O60543, and AESMFTNAVQILEQFK and EVTLPFLFTPFK for protein O95427. For each peptide, a list of 6–10 b- and y-ions with charge states of +1, +2, and +3 formed during the decay of the precursor peptide ion was compiled. The selection of proteotypic peptides and their fragments is illustrated in figure 1.

Example 2. Synthesis of proteotypic peptides

Peptides were prepared according to the solid-phase peptide synthesis protocol on an Overture automated peptide synthesizer (Protein Technologies, Inc.) using protected 9-fluorenylmethyloxycarbonyl (Fmoc) at the α-amino groups of amino acid derivatives. As the solid phase, we used resins with a low loading (0.35 mmol / g of active groups) produced by Novabiochem with the attached first amino acid lysine Fmoc-Lys (Boc) -Wangresin LL or arginine Fmoc-Arg (Pbf) -Wangresin LL. The following (α-N-Fmoc-L-) amino acids (Novabiochem) were used for synthesis:

1) alanine (Fmoc-Ala-OH);

2) methionine (Fmoc-Met-OH);

3) asparagine (Fmoc-Asn (Trt) -OH);

4) phenylalanine (Fmoc-Phe-OH);

5) aspartic acid (Fmoc-Asp (OtBu) -OH);

6) proline (Fmoc-Pro-OH);

7) glutamine (Fmoc-Gln (Trt) -OH);

8) serine (Fmoc-Ser (tBu) -OH);

9) glutamic acid (Fmoc-Glu (OtBu) -OH);

10) threonine (Fmoc-Thr (tBu) -OH);

11) glycine (Fmoc-L-Gly-OH)

12) tyrosine (Fmoc-Tyr (tBu) -OH);

13) isoleucine (Fmoc-Ile-OH);

14) leucine (Fmoc-Leu-OH);

15) valine (Fmoc-Val-OH).

Amino acids were selected with the following protective groups of the side functional groups, taking into account their final release by trifluoroacetic acid: aspartic acid (D), glutamic acid (E) - tert-butoxy (OtBu '); glutamine (Q), asparagine (N); arginine (R) - 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf); serine (S), threonine (T) and tyrosine (Y) - tert-butyl (tBu). To condense Fmoc amino acids, diisopropylcarbodiimide (DIC) was used in the presence of 1-hydroxybenzotriazole (HOBt) and (1-cyano-2-ethoxy-2-oxoethyldeaminooxy) diethylaminomorpholinocarbenium (COMU) in the presence of 2,4,6-trimethylpyridine. As a solvent in peptide synthesis, N, N-dimethylformamide (os.h) (DMF) manufactured by Spectrum-Chemical, Russia was used.

Example 3. Determination of the transition profile of synthetic marker peptides by MRM method under accelerated chromatographic gradient

In the course of one analysis, four peptides were examined, separated by experimental analytical time segments, where one peptide was analyzed in each segment for a short time interval (not more than 180 seconds). The analysis was performed using an Agilent 6490 TripleQuadrupole LC / MS system, a quadrupole mass spectrometer in isocratic mode, allowing the use of only a single-channel pump, an automatic sample injector, and a column. The segment duration depended on the set parameters, such as the sampling rate, the injection speed of the sample, the duration of washing the needle, the dead volume of the capillary system, the volume of the saddle, and the flow rate of the isocratic pump. During the analysis, the values of the intensity of the signal received from the device during registration of each fragment were recorded. The resulting chromatogram is shown in figure 2.

Example 4. The selection of peptide fragments with optimal intensity values

Based on the data obtained, for each peptide, 3-5 fragments with the highest intensity were selected and one multiplex measurement test was compiled to program the operation of a triple quadrupole mass spectrometer. The selection is illustrated in FIGS. 3 and 4.

Example 5. Determination of retention times of marker peptides

Conducted a combined analysis of all peptides in one analysis. The analysis was performed using an Agilent 6490 TripleQuadrupole LC / MS system, a quadrupole mass spectrometer configured to register 3-5 fragments for each peptide, in gradient elution mode (within 47 minutes). Retention time was set for each peptide. The determination of the retention times is illustrated in FIG.

Example 6. Conducting multiplex calibration measurements

A measurement method was created for detecting and / or quantifying the four indicated peptides using the transition profile obtained in step 6) and the exit time from the column set in step 11). Carrying out multiplex calibration measurements is illustrated in Fig.6.

Example 7. Measurement of the concentration of 4 studied peptides and calculation of the concentration of proteins containing them in a sample of human blood plasma and in a sample of cells of the standard HepG2 line

A measuring method for the four indicated peptides was used to measure their concentration in biological samples of blood plasma and immortalized cells of the standard line of HepG2 hepatocellular carcinoma.

Human blood was collected in vacuum tubes with EDTA. To obtain plasma, the tubes were centrifuged for 15 min at 2500 g. Plasma samples were centrifuged at 10,000 g for 10 minutes. The supernatant was filtered through 0.22 μm acetate filters (Whatman, USA) and stored at -80 ° C. The concentration of total protein was determined using protein analysis using bicinchononic acid Micro BCA (Thermo Scientific, USA). The predominant 14 proteins were removed from the obtained plasma samples using MARS (multi-affinity removal system) Hu-14 columns (10 × 100 mm) (Agilent, USA) according to the manufacturer's protocol. The collected fractions containing non-column related proteins were desalted using 5K MWCO cellulose acetate centrifuge columns (Agilent, USA) and concentrated to a final volume of 50 μl (from the initial volume of 0.5 μl of plasma). Protein concentration in the final aliquot was measured using Micro BCA protein analysis and was 24.9 g / L. HepG2 cell culture was grown on Dulbecco's modified Eagle’s medium supplemented with 10% fetal bovine serum and gentamicin at a concentration of 30 μg / ml and penicillin at a concentration of 25 μg / ml (Invitrogen, USA). The cell pellet, separated by centrifugation at 300 g for 5 minutes, was frozen in liquid nitrogen and stored at -80 ° C. Cells were washed in phosphate-buffered saline (PBS) at pH 7.4 three times and lysed in buffer containing 10 mM PBS, 5 mM EDTA, 10 μM E-64, 50 mM PMSF, 2% SDS and 1% NP-40 . Lysates were sonicated using a Branson type apparatus and then clarified by centrifugation at 10,000 g for 20 min at 4 ° C. The concentration of the total protein fraction was 15.7 g / l.

Then, SH-groups of proteins were reduced and alkylated, and protein was completely cleaved with modified pork trypsin (Promega, USA), as described previously [Zgoda VG, Moshkovskii SA, Ponomarenko EA, Andreewski TV, Kopylov AT, Tikhonova OV, Melnik SA, Lisitsa AV , Archakov AI. Proteomics of mouse liver microsomes: performance of different protein separation workflows for LC-MS / MS. Proteomics. 2009; 9 (16): 4102-4105]. The hydrolysates were analyzed on a triple quadrupole type mass spectrometer using the settings used in the examples above.

Table 1 The concentration of the four studied peptides UniProt Protein ID Peptide sequence The concentration in human blood plasma (mol / l) HepG2 cell concentration (mol / L) O60543 Gvmasslqelisk 6.4 × 10 ^-12 2.5 × 10 ^-8 O60543 VTFDLYR 8.5 × 10 ^-13 4.6 × 10 ^-8 O95427 EVTLPFLFTPFK 1,1 × 10 ^-12 2,3 × 10 ^-10 O95427 AESMFTNAVQILYQFK 1.0 × 10 ^-11 5.4 × 10 ^-9

To illustrate the invention, 2 peptides from the cell death activator proteins CIDE-A (accession number Uniprot O60543) and GPI-ethanolamine phosphate transferase-1 (accession number Uniprot O95427) were used. It is generally accepted in the art that the protein in the original sample and the peptides resulting from its cleavage in the hydrolyzate are in equimolar concentrations. Therefore, to determine the concentration of the protein, the measured concentrations of the peptides related to it should be averaged. Thus, the calculated concentration of the cell death activator CIDE-A (Uniprot O60543) according to Table 1 is 3.6 × 10 ^-12 mol / L in blood plasma, and 3.6 × 10 ^-8 mol / L in HepG2 cells. For GPI-ethanolamine phosphate transferase-1, these concentrations are 5.6 × 10 ⁻¹² and 2.8 × 10 ⁻⁹ mol / L, respectively.

Although the present invention is illustrated by specific examples of its implementation, it will be obvious to a person skilled in the art that many of the features of the disclosed method (carriers, columns, solvent systems, elution and detection modes, etc.) can be changed, modified, replaced by equivalent without departing from the scope of the present invention. It is intended that all such alterations, modifications and substitutions fall within the scope of the present invention.

Claims (4)

1. A method of obtaining an analytical test system (MRM test) for multiplex identification and quantitative measurement of the content of proteins of interest in a biological sample by the content of their corresponding proteotypic marker peptides, comprising the following steps:
1) the identification of protein-unique proteotypic marker peptide sequences;
2) the selection of at least two marker proteotypic peptide sequences of the protein, the most suitable for research by monitoring multiple reactions;
3) prediction of fragments of peptides;
4) prediction of the MRM test in the form of a list of marker peptides, their fragments and the best detection parameters by monitoring multiple reactions;
5) the synthesis of one or more marker peptides;
6) determination of the transition profile of one or several synthetic marker peptides by the method of monitoring multiple reactions under conditions of an accelerated chromatographic gradient;
7) optimization of the MRM test in accordance with the obtained profiles, removal from the set of peptide fragments characterized by the lowest intensity values in the mass spectrum;
8) purification of the peptides synthesized in stage 5);
9) preparation of a biological sample, involving the removal of major proteins;
10) identification of a protein in a biological sample with the sacrifice of synthetic peptides by monitoring multiple reactions under normal chromatographic gradient based on the profiles obtained in stage 6);
11) determination of retention times of marker peptides with the introduction of established values in MRM tests;
12) conducting multiplex calibration measurements under conditions of normal chromatographic gradient in a buffer solution of purified synthetic peptides against a background of a mixture of proteins and / or peptides not related to them;
13) a quantitative measurement of the content of marker peptides in a biological sample under conditions of a normal chromatographic gradient with the introduction of synthetic peptides; and
14) a judgment on the content of proteins of interest in a biological sample by the content of their corresponding proteotypic marker peptides, quantitatively determined at stage 13). 2. The method according to p. 1, in which the identification of protein-unique proteotypic marker peptide sequences and the selection of marker proteotypic peptide sequences of a protein that are most suitable for research by monitoring multiple reactions are carried out using literature data, data from repositories and / or software. 3. The method according to claim 1, in which the prediction of fragments of peptides and the creation of MRM tests in the form of lists of marker peptides, their fragments and the best detection parameters by monitoring multiple reactions are performed using software. 4. The method according to p. 1, in which the multiplex calibration measurements under normal chromatographic gradient in a buffer solution of purified synthetic peptides is performed against a background of an E. coli matrix or any other matrix.

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