Detection and quantification method for post-translational modification proteomics

Abstract

The invention relates to the technical field of comparative proteomics, and in particular to a detection and quantification method for post-translational modification proteomics. The method is characterized in that equiponderous tandem mass tag labeling is carried out on a to-be-detected protein sample and an internal standard substance, and tandem mass spectral analysis is carried out on a labeled mixture, wherein the internal standard substance is a mixture which is rich in to-be-detected post-translational modification peptide fragments. According to the method, a signal containing the to-be-detected post-translational modification peptide fragments can be amplified, and therefore under the circumstances that the sensitivity of the mass spectrum is not changed and the post-translationalmodification peptide fragments do not need to be enriched, the probability that the post-translational modification peptide fragments are subjected to mass spectrometry detection and MS / MS analysis can be improved.

Description

technical field [0001] The invention relates to the technical field of comparative proteomics, in particular to a method for detecting and quantifying post-translational modification proteomics. Background technique [0002] Protein post-translational modification is an important mechanism to regulate gene expression and protein function, and plays a vital regulatory role in many biological processes. The abnormality of protein post-translational modification is closely related to the occurrence and development of many diseases. In the past few decades, a variety of new types of protein post-translational modifications have been discovered and identified, providing an important basis for studying protein post-translational modifications through omics methods, and discovering and understanding the functions of protein post-translational modifications. [0003] Due to the extremely low abundance of protein post-translational modifications, it has brought great difficulties to...

AI Technical Summary

Problems solved by technology

However, there are still many shortcomings and deficiencies in the current strategies for studying the modified proteome: (1) a large number of biological samples need to be used

Antibodies and enrichment reagents are very expensive, and it is very difficult to obtain a specific enrichment reagent, which increases the difficulty of post-translational modification research

(3) The throughput of qualitative and quantitative detection is very low

The current strategy is to identify the enriched peptides by mass spectrometry, but because the peptides where the post-translational modifications are located can affect the binding of pan-antibodies and modifications, many peptides containing post-translational modifications cannot be enriched, which makes the detection throughput very low

(4) Poor repeatability and reproducibility

The enrichment of protein post-translational modifications is a very difficult experiment, and the difference in the technical level of operators has caused great deviations in the experimental results

In addition, the detection of mass spectrometry itself will also cause certain deviations to the results.

(5) Using the current research strategy, only one post-translational modification can be studied at a time. If multiple post-translational modifications in the same sample are to be studied, the amount of sample, antibody, and analysis time need to be doubled increase, which ultimately substantially increases the cost of the overall study

These methods are not very versatile. SILAC labeling quantification is usually only suitable for the post-translational modification omics study of cultured cells; isobaric tandem mass labeling reagent labeling quantification is not suitable for the study of peptide modification, because a large number of biological samples are subjected to isostatic Retandem mass tag labeling reagents are expensive to label, and after labeling, the efficiency of enrichment of modified peptides is greatly affected; non-standard quantification is currently a promising strategy, but its quantitative accuracy is very high. low, poor reproducibility

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