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.

Figure 10.25.1

Bottom-up proteomics workflow. Proteins are enzymatically or chemically cleaved into peptides which are then analyzed in the mass spectrometer. The proteins and peptides for analysis are often derived from separation/purification methods such as gel electrophoresis (UNIT 10.2A), liquid chromatography (UNITS 10.9–10.11A), and affinity chromatography (UNIT 10.11B).

Figure 10.25.2

Strategies for the enrichment of phosphopeptides prior to MS analysis. It is possible to perform either IMAC enrichment or TiO₂ enrichment alone, or in combination (SIMAC strategy). Following digestion of proteins, enrichment of phosphopeptides using the SIMAC strategy is achieved first by IMAC enrichment. Some phosphorylated peptides may not be captured by IMAC (IMAC unbound) and should therefore be enriched further by TiO₂. This sample can be analyzed directly by MS/MS. Singly phosphorylated (singly PO₄) peptides are eluted from IMAC (IMAC elution 1), then further enriched by TiO₂ and analyzed by MS/MS. Finally, multiply phosphorylated peptides are eluted from IMAC and analyzed directly by MS/MS.

Figure 10.25.3

200 μg of whole-tissue extract from mouse myocardium run on pH 4 to 7 two-dimensional gel and visualized with an MS-compatible silver stain. Protein spots that will likely provide reliable protein identification from a single gel (circled), as well as protein spots that will likely need to be pooled from multiple gels (squares), are indicated. Also shown are protein spots (triangles) that may or may not contain sufficient protein for identification, depending upon the MS instrumentation used.