Preparation of Proteins and Peptides for Mass Spectrometry Analysis in a Bottom‐Up Proteomics Workflow

Abstract

This unit outlines the steps required to prepare a sample for MS analysis following protein separation or enrichment by gel electrophoresis, liquid chromatography, and affinity capture within the context of a bottom-up proteomics workflow in which the protein is first broken up into peptides, either by chemical or enzymatic digestion, prior to MS analysis. Also included are protocols for enrichment at the peptide level, including phosphopeptide enrichment and reversed-phase chromatography for sample purification immediately prior to MS analysis. Finally, there is a discussion regarding the types of MS technologies commonly used to analyze proteomics samples, as well as important parameters that should be considered when analyzing the MS data to ensure stringent and robust protein identifications and characterization. Keywordsin-solution digestion; in-gel digestion; peptide desalting proteomics; mass spectrometry Thingholm et al., 2008. See above. This is a landmark paper in which both IMAC and TiO 2 are combined in a sequential manner to allow expanded phosphopeptide enrichment. Shevchenko et al., 1996. See above. This is the most commonly used silver staining and peptide extraction method for MS compatible samples. Key References Internet Resources

Identification and Selected Reaction Monitoring (SRM) Quantification of Endocytosis Factors Associated with Numb

“…The gel was fixed in 50% methanol/7% acetic acid and then stained with GelCode Blue (Thermo Scientific). Gel bands were excised and digested and extracted as described (36).…”

Anti-influenza Activity of a Bacillus subtilis Probiotic Strain

“…In-gel enzymatic digestion was performed according to published protocols (38,39). Briefly, protein bands were excised from the gel, stained with Coomassie blue, and cut into cubes (1 mm by 1 mm).…”

Top-down proteomic analysis of protein pharmaceuticals, mixtures of protein complexes, and ribosomal protein extracts by capillary zone electrophoresis-mass spectrometry

“…Native polyacrylamide gel electrophoresis (Native PAGE) separates proteins according to electrophoretic mobility under near-physiological conditions [165]. However, typically for SDS-PAGE, 2D SDS-PAGE, and Native PAGE, following separation, individual bands of interest are manually excised from the gel, and the gel polymer is removed prior to analysis by ESI-MS [166]. Conversely, gel elution liquid fraction entrapment electrophoresis (GELFrEE) enables separation of intact proteins in a gel-based format but allows for liquid phase recovery of separated species, thereby bypassing the requirement for further sample pretreatment prior to MS analysis [167].…”