A comparative analysis of human plasma and serum proteins by combining native PAGE, whole‐gel slicing and quantitative LC‐MS/MS: Utilizing native MS‐electropherograms in proteomic analysis for discovering structure and interaction‐correlated differences

Abstract

MS identification has long been used for PAGE-separated protein bands, but global and systematic quantitation utilizing MS after PAGE has remained rare and not been reported for native PAGE. Here we reported on a new method combining native PAGE, whole-gel slicing and quantitative LC-MS/MS, aiming at comparative analysis on not only abundance, but also structures and interactions of proteins. A pair of human plasma and serum samples were used as test samples and separated on a native PAGE gel. Six lanes of each sample were cut, each lane was further sliced into thirty-five 1.1 mm × 1.1 mm squares and all the squares were subjected to standardized procedures of in-gel digestion and quantitative LC-MS/MS. The results comprised 958 data rows that each contained abundance values of a protein detected in one square in eleven gel lanes (one plasma lane excluded). The data were evaluated to have satisfactory reproducibility of assignment and quantitation. Totally 315 proteins were assigned, with each protein assigned in 1-28 squares. The abundance distributions in the plasma and serum gel lanes were reconstructed for each protein, named as "native MS-electropherograms". Comparison of the electropherograms revealed significant plasma-versus-serum differences on 33 proteins in 87 squares (fold difference > 2 or < 0.5, p < 0.05). Many of the differences matched with accumulated knowledge on protein interactions and proteolysis involved in blood coagulation, complement and wound healing processes. We expect this method would be useful to provide more comprehensive information in comparative proteomic analysis, on both quantities and structures/interactions.

Proteomic Analysis of Rat Cerebral Cortex in the Subacute to Long-Term Phases of Focal Cerebral Ischemia-Reperfusion Injury

“…Slab SDS-PAGE gels (38 mm wide × 43 mm high × 1 mm thick, 4–17.85% T linear pore-size gradient and 5% C, 1% w/v SDS) were prepared in 16-gel batches with a PC-controlled gradient gel former . Plexiglas acrylic combs were inserted from the top of the gel molds before polymerization to form six 4 mm wide wells in each gel . On the day of electrophoresis, the protein samples were thawed at 4 °C and centrifuged at 15 000 g. The supernatants were taken out and supplemented with SDS, DTT, and sucrose solutions with carefully calculated ratios to ensure all of the samples had the same final concentrations of 2 μg/μL total protein, 2% w/v SDS, 100 mM DTT, and 12% w/v sucrose in 0.01 M Tris-0.02 M glycine buffer.…”

“…The stained patterns were visually examined on band distribution, resolution and densities to make sure gels with high separation quality and constant sample loading quantity were obtained for the next step. Gel slicing was done as we previously reported . A set of laboratory-made cutters comprising multiple blades were used to simultaneously cut two 1.1 mm-wide gel strips from each lane and then each strip into thirty-five 1.1 mm × 1.1 mm square pieces as shown in Figure B.…”

“…Gel slicing was done as we previously reported. 33 A set of laboratory-made cutters comprising multiple blades were used to simultaneously cut two 1.1 mm-wide gel strips from each lane and then each strip into thirty-five 1.1 mm × 1.1 mm square pieces as shown in Figure 1B. The gel pieces were kept at 4 °C in 50 μL 7% v/v acetic acid.…”

“…All of the (35 × 24 rat samples = ) 840 square gel pieces were subjected to standardized procedures of destaining, reduction, and alkylation of cysteine residues, in-gel trypsin digestion, and peptide extraction as we previously reported. 33 The same numbered gel pieces of the 24 samples, e.g., all squares 1 of the 24 samples, were processed in one batch with immediate LC-MS/MS measurement. In a few cases that the gel pieces were lost during processing or the LC-MS/MS measurement was interrupted, the gel pieces in the backup sets were used.…”

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

“…Previously, we have reported on MS-based profiling of proteins on nondenaturing 1DE or 2DE gels. By combining lane slicing or grid cutting and quantitative LC–MS/MS, gel distributions could be reconstructed for all the identified proteins, and the analysis based on pattern correlation and bioinformatics provided abundant information on complex formation and subunit structures. − Compared with nondenaturing gel electrophoresis, SDS-PAGE provides better analysis for insoluble proteins, and also the dissociated polypeptides could be directly correlated with gene products and their proteoforms. Therefore, in this work, we aimed to examine if a method coupling SDS-PAGE with systematic LC–MS/MS could provide extra information that correlates with proteoforms.…”