Method used for large-scale screening of protein biomarkers

A biomarker and protein technology, applied in the field of molecular biology, can solve the problems of high cost, small dynamic range, and poor consistency, and achieve the effect of low cost, large number of screenings, and small total amount of samples

Active Publication Date: 2018-12-07
湖北普罗金科技有限公司
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  • Abstract
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Problems solved by technology

[0011] When the above method is used for biomolecular marker screening, there

Method used

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  • Method used for large-scale screening of protein biomarkers

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Embodiment 1

[0026] Take 4 g of mouse liver tissue control samples and 4 g of pathological samples, cut into pieces, and transfer them to a homogenizer. Add PMSF / isopropanol stock solution (2 mL) to pre-cooled lysis buffer. Quickly add 2mL of pre-cooled lysis buffer into the homogenizer, and grind thoroughly under ice bath conditions. Transfer the tissue grinding solution to a 1.5mL centrifuge tube and centrifuge at 4°C (14000rpm, 10min). After centrifugation, draw the supernatant and transfer it to a new 1.5mL centrifuge tube, which is the crude tissue protein extract. The obtained protein extract can be stored at -20°C for subsequent experiments.

[0027] The above protein extracts were sampled with a spotting instrument and fixed in 96-well plates for 1 hour of reaction. Block the plate with a solution containing skim milk to prevent non-affinity substances from sticking to the chip. Take out the material in the orifice plate, use a glass rod to crush the gel, dissolve it with buffe...

Embodiment 2

[0054] Take 4g of mouse spleen tissue control sample and 4g of pathological tissue and put them in 4ml of protein lysate A (10mmol / LMgCl2, 1mmol / LKCl, 1mmol / L pyrenetetraylsulfonyl fluoride, 1mmol / L dithiothreitol), and mix well. Slurry until the particles are invisible to the naked eye, then add a few drops of 3mmol / L sucrose and mix well, centrifuge at 10000r / min at 4°C for 30min, and discard the supernatant. Dissolve the precipitate in 3-5 times protein lysate (0.5mol / L KCl) and mix well, centrifuge at 10000r / min at 4°C for 30min, take the supernatant and store it at -80°C for future use.

[0055] The above protein extracts were sampled with a spotting instrument and fixed in 96-well plates for 1 hour of reaction. Block the plate with a solution containing skim milk to prevent non-affinity substances from sticking to the chip. Take out the material in the orifice plate, use a glass rod to crush the gel, dissolve it with buffer solution (0.3% bromophenol blue solution: 0.3%...

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Abstract

The invention discloses a method used for large-scale screening of protein biomarkers. The method comprises following steps: 1, a biological tissue check sample and a reference sample (a pathologicalsample) are subjected to homogenization and digestion; 2, an obtained lysate is subjected to enzymatic hydrolysis and desalting; 3, LC-MS/MS liquid chromatography-mass spectrometry is adopted for protein identification; 4, mass spectrometry frequency or mass spectrum peak intensity are compared for analysis of sample protein abundance change, MS1 is taken as a base, the integration of the signal intensity of each peptide segment on the LC-MS is calculated, MS2 identification results are taken as quantification base, and large scale correction is carried out; and 5, analysis results of corrected data are adopted for screening of obviously up-regulated or down-regulated protein biomarkers. In operation, no expensive marking reagent is needed; the cost is relatively low; screening amount of each time is large; and screening results are accurate.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a method for large-scale screening of protein biomarkers using proteomics quantitative technology. Background technique [0002] Biomarkers refer to biochemical indicators that can mark changes or possible changes in system, organ, tissue, cell and subcellular structure or function, and have a very wide range of uses. Biomarkers can be used for disease diagnosis, judgment of disease stage, or to evaluate the safety and effectiveness of new drugs or new treatments in the target population. [0003] For disease research, biomarkers generally refer to certain characteristic biochemical indicators in a common physiological or pathological or therapeutic process that can be objectively measured and evaluated. Through its measurement, the current biological process of the body can be known. in the process. Examining a disease-specific biomarker may help in disease identif...

Claims

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Application Information

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IPC IPC(8): G01N30/02
CPCG01N33/6848
Inventor 葛峰杨明坤张珈熊倩洪斌李俊峰刘光猛
Owner 湖北普罗金科技有限公司
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