Beneficial effects of Coomassie staining on proteomic analysis employing PAGE separation followed with whole-gel slicing, in-gel digestion and quantitative LC-MS/MS

Jin, Ya; Wen, Meiling; Qi, Yuan; Zhang, Jun; Tan, Wen

doi:10.1016/j.jchromb.2019.01.031

Cited by 8 publications

(3 citation statements)

References 33 publications

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“…The influence of the Coomassie‐staining on the identification of proteins in the mass range from 10 kDa to 600 kDa by GeLC‐MS was investigated earlier. [ 24 ] It was shown that significantly more proteins below 40 kDa were identified after staining compared to only fixing the gel. This observation was explained by the binding of Coomassie to the proteins, resulting in a lower diffusion due to the larger volume of the Coomassie‐protein‐complex.…”

Section: Resultsmentioning

confidence: 99%

Complementarity of Different SDS‐PAGE Gel Staining Methods for the Identification of Short Open Reading Frame‐Encoded Peptides

et al. 2020

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Short open reading frame-encoded peptides (SEP) have been identified across all domains of life and are predicted to be involved in many biochemical processes, however, for the vast majority of SEP their biological function is still unknown. Optimized methodologies have to be used for the mass spectrometric analysis of SEP, because traditional methods of bottom-up proteomics show a bias against small proteins. Here, different staining methods for SDS-PAGE gels prior in-gel digestion following LC-MS/MS analysis for the identification of SEP in the archaeon Methanosarcina mazei are investigated. In total, 45 SEP with at least one high confidence (FDR <1%) unique peptide and five consecutive b-or y-ions in the MS2 spectrum are identified. The staining methods provide complementary data. The highest number of SEP are identified in the samples stained with Coomassie brilliant blue. However, the highest quality of the identified SEP is achieved in the samples without staining. These comprehensive data sets demonstrate that in-gel digestion is well suited for the identification of SEP.

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Section: Resultsmentioning

confidence: 99%

Complementarity of Different SDS‐PAGE Gel Staining Methods for the Identification of Short Open Reading Frame‐Encoded Peptides

et al. 2020

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“…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].…”

Section: Sample Handling and Protein Extraction For Gel-based And Top...mentioning

confidence: 99%

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

et al. 2023

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The progressive degeneration of the skeletal musculature in Duchenne muscular dystrophy is accompanied by reactive myofibrosis, fat substitution, and chronic inflammation. Fibrotic changes and reduced tissue elasticity correlate with the loss in motor function in this X-chromosomal disorder. Thus, although dystrophinopathies are due to primary abnormalities in the DMD gene causing the almost-complete absence of the cytoskeletal Dp427-M isoform of dystrophin in voluntary muscles, the excessive accumulation of extracellular matrix proteins presents a key histopathological hallmark of muscular dystrophy. Animal model research has been instrumental in the characterization of dystrophic muscles and has contributed to a better understanding of the complex pathogenesis of dystrophinopathies, the discovery of new disease biomarkers, and the testing of novel therapeutic strategies. In this article, we review how mass-spectrometry-based proteomics can be used to study changes in key components of the endomysium, perimysium, and epimysium, such as collagens, proteoglycans, matricellular proteins, and adhesion receptors. The mdx-4cv mouse diaphragm displays severe myofibrosis, making it an ideal model system for large-scale surveys of systematic alterations in the matrisome of dystrophic fibers. Novel biomarkers of myofibrosis can now be tested for their appropriateness in the preclinical and clinical setting as diagnostic, pharmacodynamic, prognostic, and/or therapeutic monitoring indicators.

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“…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.…”

Section: Introductionmentioning

confidence: 99%

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

2022

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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.

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Beneficial effects of Coomassie staining on proteomic analysis employing PAGE separation followed with whole-gel slicing, in-gel digestion and quantitative LC-MS/MS

Cited by 8 publications

References 33 publications

Complementarity of Different SDS‐PAGE Gel Staining Methods for the Identification of Short Open Reading Frame‐Encoded Peptides

Complementarity of Different SDS‐PAGE Gel Staining Methods for the Identification of Short Open Reading Frame‐Encoded Peptides

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

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

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