Dataset Information

BAC-DROP: Rapid digestion of proteome fractionated via dissolvable polyacrylamide gel electrophoresis and its application to bottom-up proteomics workflow

ABSTRACT: The GeLC-MS workflow, which combines low-cost, easy-to-use SDS-polyacrylamide gel electrophoresis (SDS-PAGE) with liquid chromatography-mass spectrometry (LC-MS), is very popular in current bottom-up proteomics. However, GeLC-MS requires that PAGE-separated proteins undergo overnight enzymatic digestion in a gel, resulting in more than 20 hours of sample preparation for LC-MS. In this study, we overcame the limitations of GeLC-MS by developing a rapid digestion workflow for PAGE separation proteins using N,N'-bis(acryloyl)cystamine (BAC) cross-linked gels that can be solubilized by reductive treatment.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Nobuaki Takemori

PROVIDER: PXD021523 | JPOST Repository | Fri Sep 17 00:00:00 BST 2021

REPOSITORIES: jPOST

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	Cjo119v2_001_A1_2uL_T80_3SGrp.mgf	Mgf
	Cjo119v2_001_A1_2uL_T80_3SGrp.msf	Msf
	Cjo119v2_001_A1_2uL_T80_3SGrp.raw	Raw
	Cjo119v2_002_A2_2uL_T80_3SGrp.mgf	Mgf
	Cjo119v2_002_A2_2uL_T80_3SGrp.msf	Msf

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Publications

BAC-DROP: Rapid Digestion of Proteome Fractionated via Dissolvable Polyacrylamide Gel Electrophoresis and Its Application to Bottom-Up Proteomics Workflow.

Takemori Ayako A Ishizaki Jun J Nakashima Kenji K Shibata Takeshi T Kato Hidemasa H Kodera Yoshio Y Suzuki Tetsuro T Hasegawa Hitoshi H Takemori Nobuaki N

Journal of proteome research 20201224 3

The GeLC-MS workflow, which combines low-cost, easy-to-use sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE) with liquid chromatography-mass spectrometry (LC-MS), is very popular in current bottom-up proteomics. However, GeLC-MS requires that PAGE-separated proteins undergo overnight enzymatic digestion in a gel, resulting in more than 20 h of sample preparation for LC-MS. In this study, we overcame the limitations of GeLC-MS by developing a rapid digestion workflow for ...[more]

PMID: 33356312

Similar Datasets

Project description:Proteomic analysis by combining PAGE separation, gel slicing and slice-by-slice 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, visualization by dye staining could be skipped to save time and labor and also in some reports assumed to improve MS identification and sensitivity. In this work, we examined the effect 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. A primary examination was firstly performed with gel bands of purified proteins, in which eight protein bands were excised, each from both the CBB-stained and unstained gel parts, and then analyzed by in-gel digestion and quantitative LC-MS/MS. Almost all the proteins were detected with higher sequence coverages and quantities from the stained gel bands than from the unstained. Then a proteomic sample of rat heart soluble proteins was examined for the complete workflow. The sample was firstly separated by nondenaturing PAGE and the gel was divided to two halves, with one CBB-stained and the other unstained. Laboratory-made tools were used to simultaneously cut duplicate lanes from each gel half and then slice each lane into about 39 pieces of the same size (1.1 mm 椋?1.1 mm 椋?1 mm thick). All the gel square pieces were analyzed in standardized procedures of in-gel digestion, peptide extraction and label-free quantitative LC-MS/MS. The results showed an average of 1434 proteins were detected in CBB-stained lanes, 40% higher than the 1013 in unstained lanes. When proteins detected in both conditions were compared, most of them were detected in higher quantities and in more gel squares with CBB staining. The comparison was also performed for SDS-PAGE and similarly advantageous results were obtained from the CBB-stained lanes, in both the detected numbers of proteins and peptides and the detected quantities and square numbers of individual proteins. The data also showed the proteins with lower molecular masses (e.g. < 30 kDa) were more benefited by the staining, probably because the dye binding helped to retain the proteins in gel matrix. In short, though dye staining is no longer a requisite when PAGE separation is followed by whole-gel LC-MS/MS analysis, CBB staining is still recommended for the better detection in proteomic analysis. All the raw and search files of the LC-MS/MS analysis, for (8*4=) 32 gel squares of HMW and LMW markers and (39*4+41*4=) 320 squares of rat heart soluble proteins (totally 704 files), as well as the gel patterns (2 files) and the summaries of the protein-level search results (3 files), are deposited in this project.