Method of producing isotope-modified peptides and proteins

FIELD: chemistry.

SUBSTANCE: invention discloses a method of producing isotope-modified peptides and proteins, based on introduction of a stable 18O isotope (isotopic tag) into carboxyl groups of peptides and proteins by holding in a solution simultaneously containing H218O and trifluoroacetic acid.

EFFECT: method enables to obtain isotope-modified tags which are resistant to destruction and suitable for quantitative mass-spectrometric analysis with retention of the initial structure of the analysed compounds.

1 dwg, 3 ex

 

The invention relates to methods for introducing isotopic labels in peptides and proteins for subsequent quantification using mass spectrometry, which in terms of sensitivity and versatility of approaches for quantitative analysis of organic compounds has no equal, but the use of which for these purposes is possible only if there are standards, chemically identical to the measured compounds, but different isotopic composition, by incorporating stable isotopes (2H,13C,15N18O).

Especially important to obtain proteins and their peptide fragments that include stable isotopes, because it opens up new opportunities for clinical proteomics and for the section of biological Sciences expressed genome proteins. It is known that the occurrence of pathological conditions in humans caused by the changing concentrations of the individual components of the proteome. It is monitoring these changes opens the opportunity for understanding pathological processes and to develop new methods of treatment or diagnosis of various diseases.

Thus, the invention will find wide application in biotechnology, medicine, organic chemistry and other fields of science and technology.

The known method of introducing stable isotopes the Bioorganic connection for quantitative mass spectrometry, consisting in the use of stopstreaming starting materials in the processes of biosynthesis using isotope-labeled precursors. So to obtain the isotope-labeled proteins for the biosynthesis of use amino acids that already have stable isotopes [1]. The disadvantage of this method is that it is only suitable for peptides and proteins produced in the process of microbial biosynthesis, and are not suitable for protein mammals, including man. Source stopstreaming components used commercial samples stopstreaming amino acids, of which only a small fraction is used for inclusion in the target product, thus the process is quite expensive. Furthermore, the method requires the development of special technologies highlight each individual isotopomers objects, which are used later as standards.

Thus, the known method is not universal and not technological, and although he retains the primary structure of the sample, and the resulting isotope label is stable, is used primarily for the study of cellular models.

Also known method [2]based on the introduction of isotopes in substance by processing specific sites (functional groups) of molecules of washes the VA reagent, containing stable isotope labels, often using a modification of SH-groups or amino groups. This method must be provided sufficiently complete or the same degree of modification, as in the sample and the standard to obtain reliable results. In addition, the subsequent fractionation to separate the reactants from the target products, which significantly complicates the process of preparation of samples for analysis. Thus, the method of obtaining isotopically modified compounds is complicated by the necessity of selection of target products and does not retain the primary structure of the sample.

The closest known way of obtaining isotopically modified Bioorganic compounds is taken as a prototype [3] method, which consists in the introduction of isotope18About when proteolysis of proteins or peptides in the presence of N218Oh and enzymes, which in comparison with known methods is the absence of additional stages in the preparation of the sample, but this method has some significant drawbacks. In the process of introducing labels violated the original integrity of the object, and isotope-labeled are not the original peptides and proteins, and fragments thereof.

Since the known method consists in the introduction of isotope18At about ealize proteins or peptides in the presence of N 218Oh and enzymes, the use and preservation of such marks requires compliance with special conditions: so at some stage it is necessary to inactivate enzymes (for example, to change the pH of the solution to values at which the enzyme is not active) to transfer the medium containing H216O.

Object of the present invention to provide resistant to degradation (decay) isotope-modified labels, suitable for quantitative mass spectrometric analysis while maintaining the original structure of the connections and simplify the method.

This problem is solved by introducing isotopic label in the carboxyl groups of peptides and proteins, which are placed in a mixture of H218O triperoxonane acid and maintain it at a temperature which will not cause violations of the primary structure. Next, the acid is removed by neutralization, or the solution is dried or passed through the chromatographic column.

Offered here as a catalyst triperoxonane acid meets two requirements: 1) has a high catalytic activity in the reaction of isotopic exchange like strong mineral acids and caustic alkalis, and 2) practically does not violate the primary structure of peptides and proteins, which in the presence of strong mineral acids exelica destroyed [4].

Thanks to this unique combination of properties, the route of administration of the isotope18In the presence of triperoxonane acid was suitable for the production of previously inaccessible isotopic standards of proteins and peptides necessary for quantitative analysis by mass spectrometry.

The introduction of the label is carried out in an independent manner and in each of the carboxyl groups of the compounds, therefore, the degree of introduction of stable isotope18About proportional to the amount present in the sample carboxyl groups. The compounds retain the label after removal of the strong acid and thus suitable for use as standards.

Note that almost all proteins are most widely represented carboxyl-containing amino acids and peptides, as a rule, have a C-terminal carboxyl group, also suitable for introduction of isotope labels introduced by isotopic exchange in carboxyl groups in a very acidic in the presence of water (H218O.

The proposed method is as follows.

The peptide or protein is maintained in the presence of H218O strong acid with PKα<2. The exchange is carried out in all carboxyl groups (for both lateral and terminal amino acids). Because of the carboxyl-containing amino the slots are widely represented in proteins, the method is suitable practically for most of these compounds, the degree of introduction of the18O is proportional to the number of available groups, and the aging time of the sample in the solution and the temperature of the solution are chosen in such a way that there was no breach of the primary structure. Further, the acid is neutralized or removed from the solution passing through the chromatographic column or drying. The samples containing organic compound in which the carboxy groups16About exchanged for18Oh, ready to use as a test object or standard.

Figure 1 presents fragments of mass spectra showing the isotopic distribution in the bradykinin before (a) and after an exchange of (B). From the comparison of the spectra shows that the exchange has occurred and is turning from one to two atoms18O.

Examples of the implementation of the proposed method.

1. Getting bradykinin exchanged with atoms16O18O.

Dry 0.3 mg of bradykinin was dissolved in 93 μl of a solution containing 40 µl (90%) H218Oh, 50 μl of acetonitrile and 3 ál triperoxonane acid. The peptide in the solution was incubated at 50°C for 5 hours, then dried in a vacuum centrifuge. Further, the peptide pererestorani in normal water and was used as with the of andarta for quantitative mass spectrometric analysis.

2. Getting trypsinogen (Tgn) exchanged with atoms16O18O. Dry 0.1 mg Tgn was dissolved in 50 μl of a solution containing 90% N218Oh and 3 ál triperoxonane acid. Protein in the solution was incubated at 50°C for 3 hours, then dried in a vacuum centrifuge. Further protein pererestorani in normal water and was used as a standard for quantitative mass spectrometric analysis.

3. Receiving nicotinic acid exchanged with atoms16O18O. Dry 0.3 mg of nicotinic acid was dissolved in 50 μl of a solution containing 90% N218About 5 μl triperoxonane acid. The acid in this solution was incubated at 50°C for 5 hours, then dried in a vacuum centrifuge. Next, nicotinic acid, pererestorani in normal water and was used as a standard for quantitative mass spectrometric analysis.

In conclusion, we point out the advantages of the proposed method to obtain isotopically modified peptides and proteins:

- The proposed method does not require chemical modification of the analyzed sample and does not contaminate the sample adulteration. The introduction of isotopes does not affect the chromatographic and electrophoretic mobility of the investigated compounds.

- Get the label stable in water is estorach with a wide range of pH (3 to 9).

- The proposed method can be used to study the kinetics of metabolism and enzymatic activity in vivo, because the resulting label is fairly stable in biological tissues.

- The proposed method is easy to use, runs fast enough (within a few hours), and the degree of introduction of the label can be controlled mass spectrometrically.

- Carboxyl group, which is currency16O18Oh, substantially distributed in nature, which significantly extends the application of the proposed method in comparison with others.

- The proposed method can be used for analysis of compounds in biological media without prior fractionation.

- The proposed method is applicable in medicine for quantitative measurements even if individual standards, in particular in the analysis of bioassay. In this case, the labeled standard can be successfully prepared from assay taken from control (or healthy) group, and the calculated quantitative data will represent the relative values between norm and pathology.

Sources of information

1. Aebersold, R. and Mann, M. Mass spectrometry-based proteomics // Nature. 2003. V.422. P.198-207.

2. Smolka M.B., Zhou H., Purkayastha S. and Aebersold R. Optimization of the isotope-coded affinity tag-labeling procedure for quantitative proteome analysis // Anal. Biochem. 2001. V.297. P.25-31.

3. United States atent 6864089. Labeling of proteomoc samples during proteolysis for quantitation and sample multiplexing (prototype).

4. Dippy, J.F.J.; Hughes, S.R.C.; Rozanski, A. The dissociation constants of some symmetrically disubstituted succinic acids. J. Chem. Soc. 1959, 2492.

The method of obtaining isotopically modified peptides and proteins, based on the introduction of stable isotope18On (isotope labels) in the carboxyl groups of peptides and proteins, characterized in that the isotope18O introducing carboxyl groups by incubation in a solution containing both H218Oh and triperoxonane acid, after which both reagents or only the acid is removed, both the reagent is removed by drying the solution, and the acid is removed by neutralization or passing through the chromatographic column.



 

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