Peptide fragment-based targeted proteome accurate quantification method

Abstract

The invention discloses a peptide fragment-based targeted proteome accurate quantification method. A series of polypeptide sequences which comprise affinity tags, have similar physicochemical properties and have the same amino acid length are designed, and the polypeptide sequences comprise affinity tag sequences, characteristic amino acid sequences of labeled proteins and enzyme cutting sites of protease. Nucleotide sequences for coding the sequences are added to 5' ends or 3' ends of open reading frames of different genes to be expressed and then serve as optimized peptide fragments for mass spectrometric detection for protein quantification. all sequences contain the same affinity tag, so that the peptide fragments can be enriched by using the same antibody, the signal difference caused by the difference of the enrichment efficiency of the antibody is avoided, the detection of the low-concentration peptide fragments can be realized, and the accurate quantification of the protein in the targeted proteomics is realized.

Description

technical field [0001] The invention relates to the field of targeted proteomics, in particular to a peptide-based quantitative method for targeted proteomics. Background technique [0002] Targeted proteomics, that is, only select signals related to the detection of the target protein, and ignore other irrelevant signals, so as to achieve high specificity and high accuracy in the quantitative identification of the target protein. At present, targeted proteomics technology mainly includes two methods: SRM / MRM (Selected / Multiple reaction monitoring, selection / multiple reaction monitoring) and PRM (Parallel reaction monitoring, parallel reaction monitoring). Among them, PRM combines the high selectivity of quadrupole and the high resolution and high precision of Orbitrap, which can independently identify the secondary spectrum, and the method process is more convenient. Compared with SRM / MRM, it has better anti-interference ability and detection sensitivity in more complex ba...

AI Technical Summary

Problems solved by technology

[0006] (1) For proteins without suitable enzyme cleavage sites or proteins with small molecular weight, suitable peptides cannot be screened;

[0007] (2) For proteins with low copy number, it may not be possible to make the mass spectrometry detection signal within the linear range of the peptide without enrichment;

[0008] (3) Even if suitable peptides are screened for proteins and antibodies are obtained, there may be problems with inconsistent enrichment efficiencies of peptide antibodies for different proteins, which need to be optimized one by one;

[0009] (4) At present, it is costly to realize the optimal peptide screening of each protein for the proteome. It is necessary to achieve quantitative comparison between proteins through later data analysis, and the dominant peptides to be searched must maintain similar physical and chemical properties.

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