Whole gel processing procedure for GeLC-MS/MS based proteomics

Autor: Meike de Wit, Connie R. Jimenez, Sander R. Piersma, Jaco C. Knol, Marc O. Warmoes, Inge de Reus
Přispěvatelé: Medical oncology laboratory, CCA - Disease profiling
Rok vydání: 2013
Předmět:
Zdroj: Piersma, S R, Warmoes, M O, de Wit, M, de Reus, I, Knol, J C & Jimenez, C R 2013, ' Whole gel processing procedure for GeLC-MS/MS based proteomics ', Proteome Science, vol. 11, 17 . https://doi.org/10.1186/1477-5956-11-17
Proteome Science, 11:17. BioMed Central
Proteome Science
ISSN: 1477-5956
DOI: 10.1186/1477-5956-11-17
Popis: Background SDS-PAGE followed by in-gel digestion (IGD) is a popular workflow in mass spectrometry-based proteomics. In GeLC-MS/MS, a protein lysate of a biological sample is separated by SDS-PAGE and each gel lane is sliced in 5–20 slices which, after IGD, are analyzed by LC-MS/MS. The database search results for all slices of a biological sample are combined yielding global protein identification and quantification for each sample. In large scale GeLC-MS/MS experiments the manual processing steps including washing, reduction and alkylation become a bottleneck. Here we introduce the whole gel (WG) procedure where, prior to gel slice cutting, the processing steps are carried out on the whole gel. Results In two independent experiments human HCT116 cell lysate and mouse tumor tissue lysate were separated by 1D SDS PAGE. In a back to back comparison of the IGD procedure and the WG procedure, both protein identification (>80% overlap) and label-free protein quantitation (R2=0.94) are highly similar between procedures. Triplicate analysis of the WG procedure of both HCT116 cell lysate and formalin-fixed paraffin embedded (FFPE) tumor tissue showed identification reproducibility of >88% with a CV
Databáze: OpenAIRE
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