A method for the extraction and detection of proteins in paraffin-embedded samples

Abstract

The invention specifically relates to a method for extracting, detecting and analyzing protein in a paraffin-embedded sample. According to the invention, an acetone precipitation method is adopted, cold acetone and 30mM of DTT are added into the fully cracked protein solution, the mixture is evenly mixed by vortex and precipitated at a temperature of -20 DEG C, the supernatant is removed during centrifugation, the protein is resuspended by adopting a solution with more compatible mass spectrum, surfactants such as SDS can be combined with the protein, and acetone precipitates while precipitating the protein, so that only part of SDS can be removed by acetone precipitation once. Cold acetone and 10 mM of DTT are added again after carrying out reductive alkylation on the protein to be resuspended, the supernatant is removed by centrifugation, the protein is resuspended by adopting the solution with more compatible mass spectrum once again, and the influence of SDS on subsequence experiments is basically eliminated through two times of acetone precipitation and lysate resuspension displacement. Each sample only needs about 3 ml of acetone, and the cost is about 0.1 yuan, so that morecost is saved compared with an ultrafiltration tube.

Description

technical field [0001] The invention relates to the technical field of protein extraction and analysis, in particular to a method for extracting, detecting and analyzing proteins in paraffin-embedded samples. Background technique [0002] Proteins form dimers, trimers and even polymers in only 20 minutes in formaldehyde, and the formation of methylene bridges after cross-linking causes some active cleavage sites to be buried, preventing trypsin or other endopeptidases from reaching their activity The cleavage site leads to incomplete enzymatic digestion, and cross-linked peptides still exist, affecting mass spectrometry detection. [0003] The method for performing proteomics quantitative analysis on paraffin-embedded samples mainly includes the following steps: removing paraffin with a dewaxing agent; adding a suitable protein lysate to extract protein; heating and incubating in a water bath to crack the covalent cross-linking of formaldehyde; FASP (Filter-aided sample pre...

AI Technical Summary

Problems solved by technology

However, because SDS will interfere with the enzymatic digestion of trypsin, the final concentration of SDS should be diluted to 0.15 before trypsin digestion; in addition, SDS inhibits the ionization of the analyte and interferes with the downstream reversed-phase separation, so in mass spectrometry Proper removal of SDS from the sample solution is essential prior to analysis

Using the FASP method to remove SDS from the sample requires ultrafiltration and centrifugation at least ten times, each time for 15 minutes. The operation is very cumbersome, and the cost of the ultrafiltration tube is relatively high. An ultrafiltration tube costs 30 yuan.

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