1DE-MS Profiling for Proteoform-Correlated Proteomic Analysis, by Combining SDS-PAGE, Whole-Gel Slicing, Quantitative LC–MS/MS, and Reconstruction of Gel Distributions of Several Thousands of Proteins

SDS-PAGE has often been used in proteomic analysis, but
generally
for sample prefractionation although the technique separates proteins
by molecular masses (Mws) and the information
would contribute to proteoform-level analysis. Here, we report a method
that combines SDS-PAGE, whole-gel slicing, and quantitative LC–MS/MS
for establishing gel distributions of several thousand proteins in
a proteome. A previously obtained data set on rat cerebral cortex
with cerebral ischemia-reperfusion injury1 was analyzed,
and the gel distributions of 5906 proteins were reconstructed. These
distributions, referred to as 1DE-MS profiles, revealed that about
30% of the proteins had more than one proteoform detected in the gels.
The profiles were categorized into six types by distribution (narrow,
dispersed, or broad) and relative deviations between the abundance-peak
apparent Mws and calculated Mws. Only 56% of the proteins showed narrow distributions
and matched Mws, while the others had
rather complex profiles. Bioinformatic analysis on example profiles
showed the resolved proteoforms involved alternative splicing, proteolytic
processing, glycosylation and ubiquitination, fragmentation, and probably
transmembrane structures. Profile-based differential analysis revealed
that many of the disease-caused changes were proteoform dependent.
This work provided a proteome-scale view of protein distributions
in SDS-PAGE gels, and the method would be useful to obtain proteoform-correlated
information for in-depth proteomics.