Electrophoresis Letters
Online ISSN : 2189-2636
Print ISSN : 2189-2628
ISSN-L : 2189-2636
A novel proteomics strategy based on dissolvable polyacrylamide gel electrophoresis
Nobuaki Takemori
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2021 Volume 65 Issue 2 Pages 63-68


SDS-polyacrylamide gel electrophoresis (SDS-PAGE) is a promising technique for the high-resolution separation of protein components extracted from crude biological samples and is popular for the use in sample prefractionation processing for mass spectrometry-based proteomics. In order to analyze SDS-PAGE-separated proteins via mass spectrometry, the proteins must first be enzymatically digested in the gel matrix and then recovered from the gel as peptide fragments. Such an in-gel digestion procedure often requires overnight reaction and is more prone to sample loss than in-solution digestion. Here, the focus is on dissolvable polyacrylamide gels prepared using N,N’-bis(acryloyl)cystamine (BAC) as a cross-linker to overcome these problems. The BAC cross-linked polyacrylamide gel is easily dissolved by reductive treatment, allowing lossless protein recovery into the surrounding solution. The suitability of recovered proteins for rapid trypsin digestion under high temperature conditions led to the establishment of a sample preparation workflow for protein mass spectrometry called BAC-DROP (BAC-Gel Dissolution to Digest PAGE-Resolved Objective Proteins). According to this workflow, the entire process from SDS-PAGE separation to the end of trypsin digestion can be completed in approximately 5 h. Combining reduced proteome complexity via high-resolution PAGE fractionation with rapid trypsin digestion provides a unique opportunity for conducting high-throughput analysis of in-depth proteome with a focus on specific molecular weight ranges.

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