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 crosslinked 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-crosslinked 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.

Proteomes Are of Proteoforms: Embracing the Complexity

“…The second issue concerns the capacity to recover intact protein species embedded in the polyacrylamide matrix; this has been a long-standing challenge in terms of subsequent MS analyses. As the Bis (N,N’-methylene-bis-acrylamide) cross-linked polyacrylamide matrix is insoluble, it is difficult, if not impossible, to quantitatively recover the embedded intact proteoforms; recovery via electroelution is possible but of variable-to-low efficiency, especially for high MW species, leading to a reduced depth of quantitative proteome analysis [ 138 ]. Alternatively, the peptides of resolved proteoforms are more routinely released via digestion, most commonly tryptic.…”

Detecting protein and post-translational modifications in single cells with iDentification and qUantification sEparaTion (DUET)

“…4). To achieve high-efficiency recovery, polyacrylamide gel with a reversible crosslinker was used 12 (see "Methods" section).…”

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

“…This recovery was superior to the previously reported recovery of BSA (59.7%) by dissolution of BAC gel. 56 Even when gels were dehydrated for 1 hour with a gel dryer and stored at 20 °C for 1 week, a recovery efficiency of 44 ± 6% was achieved. Finally, extraction using 0.1% SDS/100 mM ammonium bicarbonate yielded almost the same recovery (75± 2%) as that obtained before gel drying (72 ± 3%).…”

“…We have previously reported the recovery workflow of PAGE-separated proteins, employing a dissolvable polyacrylamide gel made with BAC-crosslinker with a disulfide bond. 56 That method recovers proteins in 30 minutes by completely dissolving the BAC gel. However, passive extraction post-CBB staining recovers BSA from gels in only 10 minutes, clearly faster than gel dissolution.…”