A method for enriching 5 low-abundance proteins in plasma or serum

Abstract

A method for enriching five low-abundance proteins in blood plasma or serum is disclosed. Cellulose phosphate is utilized as a column material. A chromatographic column operating buffer liquid includes 20 mmol/L of tris(hydroxymethyl)aminomethane, 300 mmol/L of sodium chloride, and 1 mmol/L of ethylenediaminetetraacetic acid. An eluant includes 20 mmol/L of tris(hydroxymethyl)aminomethane, 1 mol/L of sodium chloride, and 1 mmol/L of ethylenediaminetetraacetic acid. Simultaneous enrichment of the five low-abundance proteins is achieved. Samples after enrichment can be detected by adopting high performance liquid chromatography combined with high resolution mass spectrum so that the five low-abundance proteins can be identified and subjected to quantitative analysis. The enriching effects of the method are obvious. The method is simple and convenient, does not need expensive equipment or consumables and is low in cost, thus facilitating popularization and application. No antibody is required, thus greatly reducing the detection cost and avoiding nondeterminacy brought by an antibody quality stability issue.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for enriching low-abundance proteins in 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. More than 99%, ...

AI Technical Summary

Problems solved by technology

[0009] In clinical practice, these low-abundance proteins are difficult to detect, how to effectively detect and analyze low-abundance proteins has always been a challenging problem in the field of analytical science

[0010] At present, the quantitative analysis of these low-abundance proteins is mainly realized by antibody-dependent techniques such as ELISA, etc., 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 cause Instability of 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

[0011] 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.

The difficulty mainly lies in two aspects: on the one hand, there is a huge difference in the content of high-abundance proteins and low-abundance proteins in plasma or serum proteins. It is easily covered by high-abundance proteins, causing low-abundance target proteins to be undetectable, and low-abundance proteins have a high rate of missed detection; Although the pretreatment method for removing high-abundance proteins combined with mass spectrometry can improve the detection probability of low-abundance proteins, these methods have strict requirements on equipment and operating techniques, and are time-consuming and labor-intensive or require expensive consumables. Difficult to promote in practical applications

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