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A method for protein identification using high-energy collision-induced ionization fragmentation technique

A protein and high-energy technology, applied in the field of bioinformatics, can solve the problems of limited modification types, optimize the library construction method and library search engine, and the HCD data modification type pre-screening method that has not yet been seen, so as to improve the identification success rate, Improving the sensitivity of peptide identification and the effect of improving identification sensitivity

Active Publication Date: 2015-12-16
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] (1) Conventional search engines such as MASCOT, SEQUEST, and X!Tandem have not designed corresponding theoretical maps and matching scoring methods for HCD, and the processing of HCD still uses the same theoretical spectrum and scoring methods as CID;
[0011] (2) Quality control methods for peptide identification do not make full use of HCD data characteristics
[0012] (3) The existing de novo sequencing algorithm for HCD maps considers limited modification types, and there is no pre-screening method for modification types for HCD data.
[0013] (4) In terms of spectral library search, there are no spectral library construction methods and spectral library search engines optimized for HCD data (Lam2011)

Method used

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  • A method for protein identification using high-energy collision-induced ionization fragmentation technique
  • A method for protein identification using high-energy collision-induced ionization fragmentation technique

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1. Using HCD tandem MS / MS data to analyze and identify peptide sequences

[0037] 1. Enzymatic hydrolysis to obtain peptides

[0038] Saccharomyces cerevisiae strain ATCC201388 (BY4741, MATahis3delta1leu2delta0met15delta0ura3delta) was purchased from American Type Culture Collection.

[0039] Strain culture: use YPD medium to cultivate yeast ATCC201388, cultivate it on a constant temperature shaker at 30°C until the OD600 is 1.5, collect the bacteria by centrifugation at 5000rpm for 5 minutes, pour out the supernatant, rinse the precipitate with 0.1% sodium azide phosphate buffer, After the supernatant was removed by centrifugation, the bacterial cells were collected and stored in a -80°C refrigerator.

[0040] Cell lysis: add urea lysate (8M urea, 50mM ammonium bicarbonate, 50mM iodoacetamide) to the yeast cell precipitation, then add glass beads equal to the cell volume, and place it on a vortex mixer at the maximum speed Vortex to lyse for 5 minutes, centri...

Embodiment 2

[0068] Example 2. Optimization of Peptide Sequence Analysis and Identification Using HCD Tandem MS / MS Spectrogram Data

[0069] The HCD secondary mass spectrograms of 16,479 peptides obtained in 2 of Example 1 were processed according to the basic method of Step 3 and Step 4 of Example 1 as follows:

[0070] C1 is the first isotope peak added, "onlyy" is the isotope peak with only y ion added. "onlyy"+C1 is the best condition, that is, only the first isotope peak of y ion is added. C0 means no isotopic peaks added.

[0071] A, "b&y"+C0 treatment group (for the control group in Example 1)

[0072] B. "b&y"+C1 treatment group

[0073] B.1) Format conversion: the method is the same as step 3 of embodiment 1;

[0074] B.2) Search, generate theoretical spectrum and perform matching scoring: it is basically the same as step 4 of Example 1, except that in the theoretical spectrum, the mass-to-charge ratio of the b and y ion monoisotope peaks of candidate peptides and the addition o...

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Abstract

The invention discloses a method for identification of a protein by utilizing high collision induced dissociation. The invention provides the method for identification of the unknown target protein by utilizing high collision induced dissociation. The method comprises the following main steps: adopting a search engine for candidate peptide fragment searching of obtained second-level HCD mass spectrograms, and generating a theoretical spectrum; and then matching an experimental spectrum with the theoretical spectrum, and outputting identification results. The generation of the theoretical spectrum comprises simultaneously generating theoretical b ion monoisotopic peak mass-to-charge ratios, generating theoretical y ion monoisotopic peak mass-to-charge ratios and increasing first isotopic peak ion mass-to-charge ratios of y ions. The method is simple and effective, and can significantly improve the peptide fragment identification sensitivity without changing a database search engine data structure and a matched grading algorithm.

Description

technical field [0001] The invention relates to the field of bioinformatics, in particular to a method for identifying proteins using high-energy collision-induced ionization fragmentation technology. Background technique [0002] With the development of mass spectrometry technology, high-energy collision fragmentation technology (HCD, high-energycollisioninduced dissociation) is widely used in the qualitative and quantitative identification of proteome expression profiles and modification profiles (Olsen, Macek et al. 2007; Nagaraj, D'Souza et al. 2010 ; Savitski, Mathiesone et al. 2010; deGraaf, Altelaar et al. 2011; Frese, Altelaar et al. 2011). The output sensitivity of the HCD secondary spectrum of the new generation of high-precision mass spectrometer LTQ-OrbiTrapVelos is close to or equivalent to that of CID (collision induced dissociation), both of which are higher than that of the electron transfer fragmentation (ETD, electron transfer dissociation) spectrum, but th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/86
Inventor 徐平李宁
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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