Sample preparation for proteomic analysis using a GeLC-MS/MS strategy
Year: 2016
Type: article
Abstract: In-gel digestion coupled with mass spectrometric analysis (GeLC-MS/MS) is a cornerstone for protein identification and characterization. Here I review this versatile approach which combines classical and modern biochemistry strategies and allows for targeted and proteome-wide analyses. Starting with any protein sample, reduced and alkylated proteins are precipitated prior to fractionation by SDS-PAGE. Proteins are in-gel digested and the resulting peptides are extracted and desalted for downstream LC-MS/MS analysis. GeLC-MS/MS leverages the advantages of both traditional SDS-PAGE visualization and protein fractionation with the robust protein and post-translational modification identification and quantitation capabilities of state-of-the-art mass spectrometry-based technology. As such, this strategy allows for the visible assessment of protein amount and quality, prior to analysis via virtually any mass spectrometry platform. Moreover, gel extracted peptides may be derived from any sample type—e.g., from cell culture, tissue, body fluid, or recombinantly-expressed protein—and are fully compatible with isobaric tagging. GeLC-MS/MS is an invaluable technique for proteomic analyses.
Source: Journal of Biological Methods
Author: Joao A. Paulo
Institution: Harvard University
Language: English
Subfield: Spectroscopy
Field: Chemistry
Domain: Physical Sciences
Open Access status: diamond
Award: G2559312920
OpenAlex ID: W2464017205