JP4832168B2 - De novo sequence analysis method, analysis software, storage medium storing analysis software, reagent kit - Google Patents

Description

  The present invention relates to a tool capable of accurate amino acid sequence analysis in a de novo sequence for performing amino acid sequence analysis on proteins and peptides that do not have a database among proteomics in the biochemical field.

  For protein identification and amino acid sequence analysis, the target protein is enzymatically digested into peptide fragments of several to several tens of amino acid residues, and then the peptide obtained by a liquid chromatograph mass spectrometer equipped with collision-induced cleavage is used. This has been done on the basis of mathematical values that match from the database created based on the mass number information and gene information of the mass spectrum of the fragment. Biological species such as humans and mice, which are well studied genetically, have a well-developed database, but most other species have few data registrations and do not function as databases. Or it does not exist as a database at all. In the absence of a database, the mass spectrum obtained in a liquid chromatograph mass spectrometer equipped with collision-induced cleavage alone can be used for the b-series having an N-terminus and the y-series having a C-terminus after collision-induced cleavage of the peptide. Discrimination becomes difficult and the amino acid sequence cannot be analyzed accurately.

Protein Nucleic Acid Enzyme Vol29 No.1 (1984) p49-56

  In general, de novo sequence analysis is based on the mass number information of the mass spectrum obtained from a liquid chromatograph mass spectrometer equipped with collision-induced cleavage, and the values of the b series including the N terminus and the y series including the C terminus. The amino acid sequence is applied without distinction. For this reason, the possibility of an amino acid sequence increases, and an accurate amino acid sequence cannot be determined. In the present invention, when enzymatic digestion of a protein is performed, an enzyme selection and a reaction condition that give priority to the condensation reaction are set, and by introducing another functional group selectively on the C-terminal side, dehydration obtained by ordinary enzyme digestion is performed. Compared with the reaction, the difference in mass number becomes clear. By using this reaction, it is possible to clearly distinguish the y series and the b series, and this is a technique for easily performing a de novo sequence using programmed software.

  For enzymatic digestion of the protein to be measured, a known lysyl endopeptidase (Lys-C) enzyme with high condensation efficiency is selected, and the reaction conditions are an enzyme reaction in the presence of a high organic solvent (dimethylformamide). The optimum pH is slightly shifted to the acidic side so as to be advantageous for the condensation reaction, and the introduction reaction is carried out by 10 times or more of the substrate so that the condensation reaction is carried out during the enzymatic digestion. This introduces a functional group having a large mass number on the C-terminal side. After introduction, when measured with a mass spectrometer, the y series (with no functional group introduced) and the b series with a functional group introduced can be easily distinguished from the mass number of the mass spectrometer.

  According to the present invention, de novo sequence analysis independent of a database can be performed accurately and in a short time.

  An embodiment of the present invention will be described with reference to the drawings.

FIG. 1 is a configuration diagram of a de novo sequence analysis reaction procedure and a condensation reaction reagent kit. The reagent kit organic solvent (dimethylformamide) 1 is added in the range of 25-75% to the tube 5 containing the protein, and then the reagent kit Tris buffer 2 is added in the range of 75-25%. Next, the reagent kit introduction substance (phenylhydrazine) 3 is added about 10 to 100 times the amount of protein. After stirring well, 2 to 20 μg of reagent kit enzyme (Lys-C) 4 is added and maintained at 37 ° C., and the enzyme reaction is carried out for 4 to 16 hours. According to this reaction kit and reaction conditions, as shown in FIG. 2, the introduction group phenylhydrazine is introduced into the C-terminal side after cleaving the peptide bond (NC) instead of H ₃ O ⁺ (OH). The enzymatically digested peptide fragment is subjected to amino acid sequence analysis with a liquid chromatograph mass spectrometer equipped with collision-induced cleavage. FIG. 3 shows an example of a mass spectrum of a peptide fragment composed of 8 amino acid residues. When peptide fragments are analyzed by mass-induced collision-induced gas fragmentation, peptide bonds are randomly broken, and it is difficult to clearly distinguish the b-series having an N-terminus and the y-series having a C-terminus from the mass spectrum pattern. However, in the reaction in which the introduction group phenylhydrazine is introduced, a mass spectrum shifted by the mass number 106 of phenylhydrazine on the C-terminal side is confirmed. FIG. 4 is a mass number list obtained by changing the enzyme digestion method from the same measurement sample by a mass spectrometer. FIG. 5 is a flowchart of a method for de novo sequence analysis from the two mass number lists of FIG. Compared with the value of the mass number list of the de novo enzyme digestion from the first value of the mass number list of the enzyme digestion in FIG. The value extracted by performing it until the last value is a candidate for the y series. Similarly, b series candidates are found by extracting the same mass number from the two lists. A value that does not meet the above conditions can be classified as an ion derived from a solvent or an internal fragment. FIG. 6 shows a list extracted as a b-sequence candidate and a list extracted as a b-sequence candidate. The difference corresponds to an amino acid by subtracting a close small value from a large value. From this result, the amino acid sequence analysis of SFL (I) DSGYR becomes possible.

  In the above, the same effect can be obtained even if the enzyme having a condensation effect is at least one selected from lysyl endopeptidase, trypsin, chymotrypsin, and thermolysin.

  Further, the introduced substance may be a substance having at least one selected from amide, aniline, hydrazine, and phenylhydrazine.

  The organic solvent may be dimethylformamide, ethyl acetate, or acetonitrile.

De novo sequence analysis procedure and condensation reaction reagent kit. Chemical formula of the condensation reaction. Mass spectrum of peptide fragments. Mass number list obtained from mass spectrometer. Flow chart of de novo sequence analysis. Analysis result example.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 1 ... Organic solvent (dimethylformamide), 2 ... pH buffer solution (Tris buffer), 3 ... Introduction group solution (phenylhydrazine), 4 ... Enzyme (Lys-C), 5 ... Reaction tube.

Claims (7)

A de novo enzyme digestion step that simultaneously performs enzymatic digestion and condensation reaction using an enzyme having a condensing effect and adding an introduction substance more than 10 times the substrate in the presence of an organic solvent when the protein to be measured is enzymatically digested. When,
Obtaining a mass spectrum of the sample subjected to the de novo enzyme digestion using a mass spectrometer having a collision-induced cleavage function;
The mass spectrum obtained in this step is compared with the mass spectrum obtained using a mass spectrometer equipped with a collision-induced cleaving function for the sample subjected to normal enzyme digestion, and shifted by the mass number of the introduced substance. Extracting a mass spectrum that is being performed, and setting the extracted value as a candidate for a y-series;
A de novo sequence analysis method comprising: In claim 1,
The de novo sequence analysis method, wherein the enzyme having the condensation effect is at least one selected from lysyl endopeptidase, trypsin, chymotrypsin, and thermolysin. In claim 1 or 2,
The de novo sequence analysis method, wherein the introduced substance is a substance having at least one selected from amide, aniline, hydrazine, and phenylhydrazine. In any one of Claims 1-3,
The de novo sequence analysis method, wherein the organic solvent is dimethylformamide, ethyl acetate, or acetonitrile. A reagent kit for de novo sequencing, comprising at least the introduction substance for performing the de novo sequence analysis method according to any one of claims 1 to 4. A de novo sequence analysis software comprising a program for executing the de novo sequence analysis method according to claim 1.   A storage medium storing the de novo sequence analysis software according to claim 6.

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