3 years ago

Top-down/Bottom-up Mass Spectrometry Workflow Using Dissolvable Polyacrylamide Gels

Top-down/Bottom-up Mass Spectrometry Workflow Using Dissolvable Polyacrylamide Gels
Ayako Takemori, Nobuaki Takemori, Frederik Lermyte, Joseph A. Loo, Rachel R. Ogorzalek Loo, Michael Nshanian, Piriya Wongkongkathep
Biologists’ preeminent toolbox for separating, analyzing, and visualizing proteins is SDS-PAGE, yet recovering the proteins embedded in these polyacrylamide media as intact species is a long-standing challenge for mass spectrometry. In conventional workflows, protein mixtures from crude biological samples are electrophoretically separated at high-resolution within N,N′-methylene-bis-acrylamide cross-linked polyacrylamide gels to reduce sample complexity and facilitate sensitive characterization. However, low protein recoveries, especially for high molecular weight proteins, often hinder characterization by mass spectrometry. We describe a workflow for top-down/bottom-up mass spectrometric analyses of proteins in polyacrylamide slab gels using dissolvable, bis-acryloylcystamine-cross-linked polyacrylamide, enabling high-resolution protein separations while recovering intact proteins over a broad size range efficiently. The inferior electrophoretic resolution long associated with reducible gels has been overcome, as demonstrated by SDS-PAGE of crude tissue extracts. This workflow elutes intact proteins efficiently, supporting MS and MS/MS from proteins resolved on biologists’ preferred separation platform.

Publisher URL: http://dx.doi.org/10.1021/acs.analchem.7b00357

DOI: 10.1021/acs.analchem.7b00357

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