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


ABSTRACT: 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.

SUBMITTER: Takemori N 

PROVIDER: S-EPMC5590889 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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

Takemori Nobuaki N   Takemori Ayako A   Wongkongkathep Piriya P   Nshanian Michael M   Loo Rachel R Ogorzalek RRO   Lermyte Frederik F   Loo Joseph A JA  

Analytical chemistry 20170802 16


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.  ...[more]

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