A method for enriching 12 medium-abundance and low-abundance proteins in plasma or serum

Abstract

The invention discloses a method for enriching 12 kinds of medium-abundance and low-abundance protein in plasma or serums. Phosphoric acid cellulose is used as a column material; a chromatographic column running buffer solution comprises 20 mmol / L tris(hydroxymethyl)methyl aminomethane, 100 mmol / L sodium chloride and 1 mmol / L ethylenediamine tetraacetic acid; elution fluid comprises 20 mmol / L tris(hydroxymethyl)methyl aminomethane, 1 mmol / L sodium chloride and 1 mmol / L ethylenediamine tetraacetic acid, and 12 kinds of medium-abundance and low-abundance proteins are enriched simultaneously. Enriched samples are detected through the combination of a high-performance liquid chromatography and a high-resolution mass spectrometry, and the 12 kinds of medium-abundance and low-abundance protein can be identified and quantitatively analyzed. Without using expensive equipment or consumables, the method for enriching the 12 kinds of medium-abundance and low-abundance protein in the plasma or the serums is remarkable in enrichment effect, simple, low in cost and convenient to popularize and apply; without using antibodies, the detection cost can be greatly reduced, and the uncertainty caused by the stability problems of the quality of the antibodies can be avoided.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for enriching middle and low-abundance proteins in blood plasma or serum. Background technique [0002] Proteomics can conduct high-throughput research on proteins and can be used to discover diagnostic markers and drug targets for various diseases. The International Human Proteomic Organization (HUPO) launched the Human Plasma Proteome Project (HPPP) as early as 2002. Its scientific goals include comprehensive analysis of human plasma and serum protein components, and identification of different human The degree of variability among various plasma proteins and the determination of individual differences in different physiological and pathological states. However, there is a huge difference in the abundance of plasma proteins, 21 main high-abundance and medium-abundance proteins in plasma, such as albumin, IgG, ɑ1 antitrypsin, ɑ2 macroglobulin, transferrin, etc....

AI Technical Summary

Problems solved by technology

In clinical practice, these medium- and low-abundance proteins are difficult to detect. How to effectively detect and analyze medium- and low-abundance proteins has always been a challenging problem in the field of analytical science

At present, the quantitative analysis of these medium- and low-abundance proteins is mainly realized by antibody-dependent techniques such as ELISA. The accuracy of these methods is mainly determined by the quality of the antibody, and the quality of the antibody is easily affected by many factors, which will causing instability in the measurement results

In addition, these antibody-based methods are relatively expensive, and the cost will further increase when multiple antibodies need to be detected simultaneously

[0017] Mass spectrometry has made great progress in recent years and can quantify many kinds of proteins at the same time. However, there are still some shortcomings in the direct analysis of complex biological samples such as plasma or serum by mass spectrometry, which affects the application of this technology in clinical testing.

Difficulties mainly lie in two aspects: On the one hand, there are huge differences in the contents of high, medium and low abundance proteins in plasma or serum proteins. The signals of high-abundance proteins are easily covered by high-abundance proteins, resulting in the inability to detect medium and low-abundance target proteins, and the high rate of missed detection of medium- and low-abundance proteins; on the other hand, multidimensional liquid chromatography and electrophoresis pretreatment Separation and other methods, as well as pretreatment methods for eliminating high-abundance proteins through immunoaffinity techniques, combined with mass spectrometry can improve the detection probability of medium and low-abundance proteins, but these methods have strict requirements on equipment and operating techniques. Time-consuming and labor-intensive or expensive consumables are required, and it is not easy to promote in practical applications

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