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

Area of Science:

• Proteomics
• Biochemistry
• Analytical Chemistry

Background:

• Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by in-gel digestion (IGD) is a common workflow in mass spectrometry-based proteomics.
• The conventional GeLC-MS/MS workflow involves slicing gels into multiple pieces for IGD, which presents a manual processing bottleneck in large-scale experiments.

Purpose of the Study:

• To introduce and evaluate a novel whole gel (WG) procedure for streamlining GeLC-MS/MS workflows.
• To assess the performance of the WG procedure in terms of protein identification and quantification compared to the traditional IGD method.

Main Methods:

• The WG procedure involves performing sample processing steps (washing, reduction, alkylation) on the entire gel prior to slicing.
• Comparative analysis was conducted using 1D SDS-PAGE separation of human HCT116 cell lysate and mouse tumor tissue lysate.
• Protein identification and label-free quantification were performed using LC-MS/MS analysis of samples processed by both WG and IGD methods.

Main Results:

• The WG procedure demonstrated high similarity in protein identification (>80% overlap) and label-free protein quantification (R²=0.94) when compared to the conventional IGD procedure.
• Reproducibility analysis of the WG procedure on HCT116 cell lysate and formalin-fixed paraffin-embedded (FFPE) tumor tissue showed identification reproducibility of >88% with a coefficient of variation <20% for protein quantification.

Conclusions:

• The WG procedure offers a reproducible and efficient alternative for large-scale differential GeLC-MS/MS experiments, significantly reducing manual processing.
• This method maintains comparable performance to conventional IGD, making it particularly suitable for clinical proteomics applications requiring high sample throughput.