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

Abstract

BackgroundSDS-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.ResultsIn 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.ConclusionsThe 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.

Chitosan enhances rice seedling growth via gene expression network between nucleus and chloroplast

“…In order to overcome these limitations and reduce the complexity of proteins, the use of gel-based LC-MS/MS (GeLC-MS/MS) has been developed. Total proteins are first separated by SDS-PAGE then the entire gel is sliced into pieces according to protein marker sizes before digestion and analysis by LC-MS/MS followed by mapping of the peptide sequences onto the corresponding proteins using computer algorithms (Piersma et al 2013). Shotgun proteomics has proven to be more reproducible in terms of peptide identification and protein quantitation than other techniques (Delmotte et al 2009;Piersma et al 2010;Tabb et al 2010).…”

The beauty of being (label)-free: sample preparation methods for SWATH-MS and next-generation targeted proteomics

“…These approaches are compatible with multiple sample extraction buffers, can easily be combined with gel staining that does not interfere with protein digestion 34– 36 , and thus provide a visual quality control over the protein sample. However, casting and running the gels render these protocols time consuming; hence the protocols are of relatively low throughput.…”

Serum Carbonic Anhydrase 1 is a Biomarker for Diagnosis of Human Schistosoma mansoni Infection

“…19 Each lane was sliced into 20 horizontal bands that were processed for proteomic analysis as previously described. 20 Briefly, bands were placed in 1.5-mL tubes and exposed to 40 μL of 100 mM NH 4 HCO 3 for 5 minutes followed by centrifugation at 14,000 × g for 10 minutes. After aspiration of the liquid, the gel pieces were vortexed with 40 μL of 100 mM NH 4 HCO 3 /50% ACN (Sigma-Aldrich, St. Louis, MO) for 10 minutes to shrink the gel.…”