RU2399627C2 - Method of producing isotope-modified peptides and proteins - Google Patents

Abstract

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 H₂ ¹⁸O 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.

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Description

The invention relates to methods for introducing an isotopic label into peptides and proteins, for their subsequent quantitative determination using mass spectrometry, a method which, in terms of sensitivity and universality of approaches for quantitative analysis of bioorganic compounds, is not equal, but the use of which for these purposes is possible only if standards chemically identical to the measured compounds, but differing in isotopic composition, by including stable isotopes ( ² N, ¹³ C, ¹⁵ N, ¹⁸ O).

It is especially important to obtain proteins and their peptide fragments, which include stable isotopes, as this opens up new possibilities for clinical proteomics and for the branch of biological science studying proteins expressed by the genome. It is known that the occurrence of pathological conditions in humans is due to a change in the concentrations of individual proteome components. It is the monitoring of these changes that opens the possibility, both for understanding pathological processes and for developing 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.

A known method of introducing stable isotopes into a bioorganic compound for quantitative mass spectrometry, which consists in using isotope-containing starting materials in their biosynthesis using isotope-labeled precursors. So, to obtain isotope-labeled proteins for biosynthesis, amino acids that already have stable isotopes are used [1]. The disadvantage of this method is that it is suitable only for peptides and proteins obtained in the process of microbiological biosynthesis only, and is not suitable for the production of mammalian proteins, including humans. As a source of isotope-containing components, commercial samples of isotope-containing amino acids are used, of which only a small fraction is used for inclusion in the target product, so the process is quite expensive. In addition, the method requires the development of special technologies for the isolation of each of the individual isotope-containing objects, which are used further as standards.

Thus, the known method is far from universal and not technologically advanced, and although it retains the primary structure of the sample, and the isotopic label obtained as a result of its use is stable, it is mainly used to study cell models.

There is also known a method [2], based on the introduction of isotopes into a substance by treating specific sections (functional groups) of the molecules of a substance with a reagent containing stable isotopic labels, often using modifications to SH groups or amino groups. With this method, a sufficiently complete or identical degree of modification must be provided, both in the sample and in the standard, to obtain reliable results. In addition, subsequent fractionation is necessary to separate the reagents from the target products, which greatly complicates the process of preparing samples for analysis. Thus, the method for producing isotopically modified compounds is complicated by the need to isolate the desired products and does not preserve the primary structure of the sample.

The closest known method for producing isotope-modified bioorganic compounds is the method adopted as a prototype [3], which consists in introducing the ¹⁸ O isotope during proteolysis of proteins or peptides in the presence of H ₂ ¹⁸ O and enzymes, the advantage of which is compared to the known methods additional stages in the process of sample preparation, however, this method has several significant drawbacks. In the process of labeling, the initial integrity of the object is violated, and not the original peptides and proteins, but their fragments are isotope-labeled.

Since the known method consists in the introduction of the ¹⁸ O isotope during the proteolysis of proteins or peptides in the presence of H ₂ ¹⁸ O and enzymes, the use and preservation of such a label requires the observance of special conditions: so at a certain stage it is necessary to inactivate the enzymes (for example, change the pH of the solution to values at which the enzyme is not active) before being transferred to a medium containing H ₂ ¹⁶ O.

The objective of the invention is to obtain resistant to destruction (decay) of isotope-modified labels suitable for quantitative mass spectrometric analysis while maintaining the original structure of the compounds while simplifying the method.

This problem is solved by introducing an isotopic label into the carboxyl groups of peptides and proteins, which are placed in a mixture of H ₂ ¹⁸ O with trifluoroacetic acid and kept in it at a temperature that does not cause damage to the primary structure. The acid is then removed by neutralization, or the solution is dried or passed through a chromatographic column.

The trifluoroacetic acid proposed here as a catalyst meets two necessary requirements at once: 1) it has high catalytic activity in the isotope exchange reaction 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 and alkali is destroyed [4].

Due to such a unique combination of properties, the method of introducing the ¹⁸ O isotope in the presence of trifluoroacetic acid proved to be suitable for obtaining previously inaccessible isotope standards of proteins and peptides required for quantitative analysis by mass spectrometry.

Labeling is carried out independently in each of the carboxyl groups of the compound; therefore, the degree of introduction of the stable ¹⁸ O isotope is proportional to the number of carboxyl groups present in the sample. The resulting compounds retain their label after removal of the strong acid and are thus suitable for use as standards.

Note that in almost all proteins, carboxyl-containing amino acids are most widely represented, and in peptides, as a rule, a C-terminal carboxyl group is present, which is also suitable for introducing an isotopic label introduced by isotopic exchange in carboxyl groups in a strongly acidic medium in the presence of water H ₂ ¹⁸ O.

The proposed method is as follows.

The peptide or protein is aged in the presence of H ₂ ¹⁸ O in a strong acid with pK _α <2. Metabolism is carried out in all carboxyl groups (both for side and terminal amino acids). Since carboxyl-containing amino acids are widely represented in proteins, the method is suitable for almost the majority of such compounds, the degree of ¹⁸ O introduction being proportional to the number of groups present, and the sample holding time in solution and the solution temperature are selected so that the primary structure is not disturbed. The acid is then neutralized or removed from the solution by passing through a chromatographic column or by drying. The samples obtained contain an organic compound in which ¹⁶ O in the carboxyl groups is exchanged for ¹⁸ O, ready for use as an analyzed object or standard.

Figure 1 presents fragments of mass spectra showing the isotopic distribution in bradykinin before (A) and after exchange (B). From a comparison of the spectra, it can be seen that the exchange occurred and the inclusion of one to two ¹⁸ O atoms occurs.

Examples of the proposed method.

1. Obtaining bradykinin with exchanged ¹⁶ O atoms at ¹⁸ O.

Dry 0.3 mg of bradykinin was dissolved in 93 μl of a solution containing 40 μl (90%) of H ₂ ¹⁸ O, 50 μl of acetonitrile and 3 μl of trifluoroacetic acid. The peptide in this solution was incubated at 50 ° C for 5 hours, then dried in a vacuum centrifuge. The peptide was then redissolved in ordinary water and was used as a standard for quantitative mass spectrometric analysis.

2. Preparation of trypsinogen (Tgn) with exchanged ¹⁶ O atoms to ¹⁸ O. Dry 0.1 mg Tgn was dissolved in 50 μl of a solution containing 90% H ₂ ¹⁸ O and 3 μl of trifluoroacetic acid. The protein in this solution was incubated at 50 ° C for 3 hours, then dried in a vacuum centrifuge. Then the protein was redissolved in ordinary water and was used as a standard for quantitative mass spectrometric analysis.

3. Preparation of nicotinic acid with exchanged ¹⁶ O atoms to ¹⁸ O. Dry 0.3 mg of nicotinic acid was dissolved in 50 μl of a solution containing 90% H ₂ ¹⁸ O and 5 μl of trifluoroacetic acid. The acid in this solution was incubated at 50 ° C for 5 hours, then dried in a vacuum centrifuge. Then, nicotinic acid was redissolved in ordinary water and was used as a standard for quantitative mass spectrometric analysis.

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

- The proposed method does not require chemical modification of the analyzed sample and does not contaminate the sample with extraneous impurities. The introduction of isotopes does not affect the chromatographic and electrophoretic mobility of the test compound.

- The resulting label is stable in aqueous solutions over a wide pH range (from 3 to 9).

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

- The proposed method is simple to use, proceeds quickly enough (within a few hours), and the degree of labeling can be controlled by mass spectrometry.

- Carboxyl groups, which exchange ¹⁶ O for ¹⁸ O, are significantly common in nature, which significantly expands the scope of the proposed method compared to others.

- The proposed method can be used to analyze compounds in biological media without prior fractionation.

- The proposed method is applicable in medicine for quantitative measurements even when individual standards are unavailable, in particular when analyzing bioassays. In this case, the labeled standard can be successfully prepared from a biological sample taken from a control (or healthy) group, and the calculated quantitative data will represent the relative values between the norm and the pathology.

Information sources

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 Patent 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.

Claims (1)

A method of producing isotope-modified peptides and proteins based on the introduction of a stable ¹⁸ O isotope (isotopic label) into the carboxyl groups of peptides and proteins, characterized in that the ¹⁸ O isotope is introduced into the carboxyl groups by incubation in a solution containing simultaneously H ₂ ¹⁸ O and trifluoroacetic acid, after which both reagents or only acid are removed, both reagents being removed by drying the solution, and the acid is removed by neutralization or passing through a chromatographic column.

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