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Electroblotting through a tryptic membrane for LC-MS/MS analysis of proteins separated in electrophoretic gels.

ABSTRACT: Digestion of proteins separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) remains a popular method for protein identification using mass-spectrometry based proteomics. Although robust and routine, the in-gel digestion procedure is laborious and time-consuming. Electroblotting to a capture membrane prior to digestion reduces preparation steps but requires on-membrane digestion that yields fewer peptides than in-gel digestion. This paper develops direct electroblotting through a trypsin-containing membrane to a capture membrane to simplify extraction and digestion of proteins separated by SDS-PAGE. Subsequent liquid chromatography-tandem mass spectrometry (LC-MS/MS) identifies the extracted peptides. Analysis of peptides from different capture membrane pieces shows that electrodigestion does not greatly disturb the spatial resolution of a standard protein mixture separated by SDS-PAGE. Electrodigestion of an Escherichia coli (E. coli) cell lysate requires four hours of total sample preparation and results in only 13% fewer protein identifications than in-gel digestion, which can take 24 h. Compared to simple electroblotting and protein digestion on a poly(vinylidene difluoride) (PVDF) capture membrane, adding a trypsin membrane to the electroblot increases the number of protein identifications by 22%. Additionally, electrodigestion experiments using capture membranes coated with polyelectrolyte layers identify a higher fraction of small proteolytic peptides than capture on PVDF or in-gel digestion.

SUBMITTER: Bickner AN

PROVIDER: S-EPMC7704035 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Electroblotting through a tryptic membrane for LC-MS/MS analysis of proteins separated in electrophoretic gels

Project description:Electroblotting proteins separated by SDS-PAGE through a trypsin-containing membrane and onto a capture membrane simplifies extraction and digestion of proteins. Subsequent liquid chromatography-tandem mass spectrometry (LC-MS/MS) identifies the extracted peptides.

2020-08-25 | MSV000086010 | MassIVE

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