Protein isotope dilution tandem mass spectrometry method based on chemical labeling technology

Abstract

The invention discloses a protein isotope dilution tandem mass spectrometry method based on a chemical labeling technology. The method comprises (1) labeling a sample to be detected by a chemical label A to obtain a labeled sample, (2) labeling an internal standard substance with a chemical label iso-A with an isotope label to obtain a labeled internal standard substance, (3) uniformly mixing the labeled sample and the labeled internal standard substance according to a preset ratio to obtain an initial sample, (4) pretreating the initial sample to obtain a fed sample, and (5) carrying out mass spectrometry on the fed sample. The sample and internal standard substance are respectively labeled, then are mixed, then added into a reaction system and then are treated so that the sample and internal standard substance are subjected to the same matrix effect and have the same operation error in the same system and thus the error can be corrected.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a protein isotope dilution tandem mass spectrometry detection method based on chemical labeling technology. Background technique [0002] The quantitative analysis of protein is often involved in the analysis of biochemistry and other life sciences. In biochemical experiments, accurate and reliable quantitative analysis of proteins in samples is a very important work that is often carried out. However, since protein is a very important biological macromolecule, it has many types, uneven structure, large molecular weight difference and different functions, which brings great advantages to the establishment of an ideal and general protein quantitative analysis method. There are many specific reasons for the difficulty. [0003] Existing protein quantification methods are mainly based on liquid chromatography-mass spectrometry. In order to control and reduc...

AI Technical Summary

Problems solved by technology

There are many deficiencies in the existing labeling methods based on the polypeptide level. For example, first, the labeling method at the polypeptide level makes the two samples mixed after enzymatic hydrolysis. Before enzymatic hydrolysis (extraction, purification, reduction, alkylation) All errors cannot be corrected, reducing the accuracy of the test results

Second, although the iTRAQ method, the TMT method, and the ICAT method have commercialized reagents, the commercialized labeling reagents are about 1000-3000 yuan per reaction, which is expensive

However, SILAC technology has the following disadvantages: (1) SILAC protein synthesis is expensive. According to the valuation of SILAC synthesis companies, about 10 mg of SILAC protein needs about 200,000 euros, which can meet the detection requirements of hundreds to a thousand samples, equivalent to each time The detection cost is about 200-400 euros; (2) The production of SILAC proteins is based on transgene expression technology, and due to the limitations of existing technologies, not all proteins can be successfully expressed through this technology; (3) After the expression of normally expressed proteins is completed, Post-expression modification and protein folding will be carried out, but the protein expressed by the transgene will not carry out these two steps, so the protein expressed by the transgene is significantly different from the normal expression protein in terms of spatial structure

[0006] From the above, it can be seen that the existing protein isotope labeling technology still has many deficiencies such as high price, cumbersome operation, and certain errors in the operation process.

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