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

Proteome Science

Connie R Jiménez¹  Jaco C Knol¹  Inge de Reus¹  Meike de Wit¹  Marc O Warmoes¹  Sander R Piersma¹ 

[1] Department of Medical Oncology, OncoProteomics Laboratory, VU University Medical Center, Amsterdam, The Netherlands

关键词: Clinical proteomics;    GeLC-MS/MS;    In-gel digestion;   

Others  :  816986
DOI  :  10.1186/1477-5956-11-17

 received in 2012-11-16, accepted in 2013-04-11,  发布年份 2013

【 摘 要 】

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<20% on protein quantitation.

Conclusions

The whole gel procedure allows for reproducible large-scale differential GeLC-MS/MS experiments, without a prohibitive amount of manual processing and with similar performance as conventional in-gel digestion. This procedure will especially enable clinical proteomics for which GeLC-MS/MS is a popular workflow and sample numbers are relatively high.

【 授权许可】

   
2013 Piersma et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

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