Dataset Information

Hit-Gel: Streamlining in-gel protein digestion for high-throughput proteomics experiments.

ABSTRACT: Traditional in gel-digestion procedures require extensive sample handling, are prone to contamination and not compatible with high-throughput sample preparation. To address these shortcomings, we have modified the conventional in-gel digestion procedure for high-throughput proteomics studies. The modified method, termed “High Throughput in Gel digestion” (HiT-Gel), is based on a 96-well plate formatwhich results in a drastic reduction in labour intensity and sample handling. Direct comparison revealed that HiT-Gel reduces technical variation and significantly decreases sample contamination over the conventional in-gel digestion method. HiT-Gel also produced superior results when a single protein band was excised from a gel and processed by in-gel digestion.Moreover, we applied Hit-Gel for a mass spectrometry analysis of Arabidopsis thalianaprotein complexesseparated by native PAGE in 24 fractions and four biological replicates. We show that the high throughput capacity ofHiT-Gel facilitates large scale studies with high sample replication or detailed fractionation. Our method can easily be implemented as it does not require specialised laboratory equipment.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Rosette

SUBMITTER: Michal Gorka

LAB HEAD: Dr. Alexander Graf

PROVIDER: PXD009485 | Pride | 2018-10-16

REPOSITORIES: Pride

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Hit-Gel: Streamlining in-gel protein digestion for high-throughput proteomics experiments.

Swart Corné C Martínez-Jaime Silvia S Gorka Michal M Zander Kerstin K Graf Alexander A

Scientific reports 20180605 1

In-gel digestion has been used as a standard method for the preparation of protein samples for mass spectrometry analysis for over 25 years. Traditional in gel-digestion procedures require extensive sample handling, are prone to contamination and not compatible with high-throughput sample preparation. To address these shortcomings, we have modified the conventional in-gel digestion procedure for high-throughput proteomics studies. The modified method, termed "High Throughput in Gel digestion" (H ...[more]

PMID: 29872109

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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.

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	Fr11_Rep1.raw	Raw

Dataset Information

Hit-Gel: Streamlining in-gel protein digestion for high-throughput proteomics experiments.

Dataset's files

Publications

Hit-Gel: Streamlining in-gel protein digestion for high-throughput proteomics experiments.

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