Proteome Analysis Using Gel‐LC‐MS/MS

This article describes processing of protein samples using 1D SDS gels prior to protease digestion for proteomics workflows that subsequently utilize reversed‐phase nanocapillary ultra‐high‐pressure liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS). The resulting LC‐MS/MS data are used to identify peptides and thereby infer proteins present in samples ranging from simple mixtures to very complex proteomes. Bottom‐up proteome studies usually involve quantitative comparisons across several or many samples. For either situation, 1D SDS gels represent a simple, widely available technique that can be used to either fractionate complex proteomes or rapidly clean up low microgram samples with minimal losses. After gel separation and staining/destaining, appropriate gel slices are excised, and in‐gel reduction, alkylation, and protease digestion are performed. Digests are then processed for LC‐MS/MS analysis. Protocols are described for either sample fractionation with high‐throughput processing of many samples or simple cleanup without fractionation. An optional strategy is to conduct in‐solution reduction and alkylation prior to running gels, which is advantageous when a large number of samples will be separated into large numbers of fractions. Optimization of trypsin digestion parameters and comparison to in‐solution protease digestion are also described. © 2019 by John Wiley & Sons, Inc.