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 (M(w)s) 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 M(w)s and calculated M(w)s. Only 56% of the proteins showed narrow distributions and matched M(w)s, 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.

Automated summary

This study introduces a method that combines SDS-PAGE, whole-gel slicing, and quantitative LC-MS/MS to analyze protein distributions in a proteome, providing proteoform-correlated information for in-depth proteomics. The research revealed that about 30% of proteins have multiple proteoforms in gels, with complex profiles categorized into six types based on distribution and deviations between abundance-peak apparent M(w)s and calculated M(w)s.