PEPPI-MS: Polyacrylamide-Gel-Based Prefractionation for Analysis of Intact Proteoforms and Protein Complexes by Mass Spectrometry

Prefractionation
of complex mixtures of proteins derived
from biological samples is indispensable for proteome analysis via
top-down mass spectrometry (MS). Polyacrylamide gel electrophoresis
(PAGE), which enables high-resolution protein separation based on
molecular size, is a widely used technique in biochemical experiments
and has the potential to be useful in sample fractionation for top-down
MS analysis. However, the lack of a means to efficiently recover the
separated proteins in-gel has always been a barrier to its use in
sample prefractionation. In this study, we present a novel experimental
workflow, called Passively Eluting Proteins from Polyacrylamide gels
as Intact species for MS (“PEPPI-MS”), which allows
top-down MS of PAGE-separated proteins. The optimization of Coomassie
brilliant blue staining followed by the passive extraction step in
the PEPPI-MS workflow enabled the efficient recovery of proteins,
separated on commercial precast gels, from a wide range of molecular
weight regions in under 10 min. Two-dimensional separation combining
offline PEPPI-MS with online reversed-phase liquid chromatographic
separation resulted in identification of over 1000 proteoforms recovered
from the target region of the gel (≤50 kDa). Given the widespread
availability and relatively low cost of traditional sodium dodecyl
sulfate (SDS)-PAGE equipment, the PEPPI-MS workflow will be a powerful
prefractionation strategy for top-down proteomics.