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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

Author:
Jin, Ya, Wen, Meiling, Yuan, Qi, Zhang, Jun, Tan, Wen
Source:
Journal of chromatography 2019 v.1110-1111 pp. 25-35
ISSN:
1570-0232
Subject:
cutters, dye binding, gels, heart, labor, liquid chromatography, molecular weight, peptides, polyacrylamide gel electrophoresis, proteins, proteomics, rats, slicing, staining, tandem mass spectrometry
Abstract:
Proteomic analysis by combining PAGE separation, gel slicing and slice-by-slice in-gel digestion and LC-MS/MS has been frequently reported in recent years. Since the MS analysis would provide identities and quantities of the proteins along the whole lane, gel visualization by dye staining could be skipped to save time and labor. In this work, we examined the effects of CBB R-250 staining on the performance of the method and the results showed actually better results were obtained with CBB staining than without, both in nondenaturing PAGE and SDS-PAGE. A primary examination was firstly performed with excised gel bands of purified proteins and the LC-MS/MS results showed that almost all the proteins were detected with higher sequence coverages and quantities from the stained bands than from the unstained. Then a proteomic sample of rat heart soluble proteins was examined for the complete workflow. The sample was separated by both nondenaturing PAGE and SDS-PAGE and the gels were divided to halves for CBB staining and fixation without staining, respectively. Multi-blade cutters were used to simultaneously cut lanes and then slice each lane into about forty squares of the same size. All the gel pieces were analyzed in standard procedures of in-gel digestion, peptide extraction and label-free quantitative LC-MS/MS. The results showed more proteins, about 40% in nondenaturing PAGE and 18% in SDS-PAGE, were detected from the CBB-stained lanes than from the unstained ones. Examination on the detected quantities and square numbers of individual proteins also confirmed about the better detection with CBB staining. As the data showed the detection of proteins with lower molecular masses (e.g. <30 kDa) were more benefited by the staining, we speculate the dye binding might help retaining of the proteins in the gel matrix.
Agid:
6331596