PEPPI-MS: Strategies to Enhance the Extraction of Electrophoretically Separated Proteins from Polyacrylamide Gels and Their Application to Top-Down/native Mass Spectrometry

01 October 2019, Version 1


Polyacrylamide gel electrophoresis (PAGE) is a powerful technique for separating proteins from complex biological samples. However, the difficulty in recovering proteins with high yields from polyacrylamide matrices often precludes further analyses of intact proteins. Here, we propose a novel experimental workflow named Passively Eluting Proteins from Polyacrylamide gels as Intact species for MS (‘PEPPI-MS’), which allows intact mass spectrometry (MS) of PAGE separated proteins. We discovered that staining proteins with certain Coomassie brilliant blue formulations immediately after PAGE improves the efficiency of extraction in a medium with pH 7–11. Post-staining, proteins spanning a broad range of molecular weights were recovered efficiently in a 10-minute procedure. High recovery yields were also obtained from dried and archived gels. This workflow is effective for top-down proteomics analysis of the target molecular region in the gel. An alternative procedure was developed for the extraction of protein complexes exceeding 400 kDa, which were separated using native PAGE, from unstained gels. Non-covalent hemoglobin tetramer, purified from cell lysate with two-dimensional native PAGE and extracted with the mild detergent octyl-β-Dglucopyranoside, was amenable for native MS analysis. We anticipate that the established workflow will facilitate the purification, storage, and transport of proteins destined for detailed characterization by MS.


Top-down proteomics
Top-down mass spectrometry
Native mass spectrometry
Polyacrylamide gel electrophoresis
Native PAGE
Coomassie Brilliant Blue

Supplementary materials

PEPPI MS ChemRxiv Table S2


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