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

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.