Shotgun Proteomics

Abstract Investigating how the protein machinery in cells functions is fundamental to biological enquiry. Technology that can identify and quantitate a multitude of unknown proteins can facilitate such investigations. Although techniques such as Western blotting are able to provide limited but useful information about a known protein if a suitable antibody is available, mass spectrometry provides a platform for the large‐scale identification and quantitation of the many unknown proteins in a typical sample. In particular, shotgun proteomics techniques have been used for studying global changes in whole proteomes, probing the subunit composition of protein complexes, and for mapping post‐translational modifications. The advent of whole genome sequencing has enabled recent advances in shotgun proteomics technology. Multidimensional protein identification technology (MudPIT), which couples mass spectrometry and multidimensional chromatography, achieves exquisitely sensitive protein detection. There are a variety of methods used to quantify the identified proteins, from isotopic labelling to ‘label free’ spectral counting. Key Concepts: ‘Shotgun proteomics’ aims to identify all proteins present in a sample using mass spectrometry to make measurements that depend on a protein's mass. Before mass spectrometry, proteins in a sample are digested with a protease to create a complex peptide mixture for analysis. Peptides between approximately 10 and 30 amino acids long can generate characteristic fragmentation patterns that can be used to identify their sequences. Chromatography is used to enrich peptides of the same kind; if enough of these enter the mass spectrometer concurrently, they can be characterised. These homogeneous populations of peptides are isolated and fragmented inside the mass spectrometer; as these fragments are detected, MS/MS spectra are generated. The patterns described by these MS/MS spectra are peptide ‘fingerprints’ which can be matched to predicted spectral patterns; genomic nucleic acid sequences are used to predict these spectral patterns. Protein quantitation is possible; strategies used include counting the number of MS/MS spectra per protein obtained during the analysis (label free), or labelling proteins/peptides from two samples with different isotopes.