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

Abstract

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 injury 1 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.

Mass Spectrometry-Based Proteomic Technology and Its Application to Study Skeletal Muscle Cell Biology

“…High-resolution native MS techniques have been developed to study the dynamic nature of protein complexes [147][148][149]. Very large protein species that can often not be properly separated by conventional 2D-GE can be analyzed by combining 1D-GE, liquid chromatography and tandem MS (GeLC-MS/MS) methodology [139][140][141]. The stabilization of sensitive protein-protein interactions in skeletal muscle tissues can be supported by chemical cross-linking [142], a method that is becoming increasingly used to study dynamic protein conformations [80,81,84].…”

Extracellular Matrix Proteomics: The mdx-4cv Mouse Diaphragm as a Surrogate for Studying Myofibrosis in Dystrophinopathy

“…Although the majority of mass spectrometric surveys are carried out with peptides and proteins that are isolated by gel-free systems, top-down proteomics routinely employs one-dimensional or two-dimensional gel electrophoretic techniques, or biochemical isolation methods such as affinity chromatography, for the preparation of intact proteoforms. As summarized in Figure 2, gel-based top-down proteomics can be based on GeLC-MS/MS using one-dimensional gradient gels in combination with liquid chromatography [97][98][99] or classical two-dimensional gel electrophoresis using isoelectric focusing in the first dimension and slab gel electrophoresis in the second dimension [100][101][102]. A gel-based method that has overcome the technical limitation of gel-to-gel variations is represented by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) [103][104][105].…”