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.

Keywords: shotgun proteomics; peptide; mass spectrometry; multidimensional protein identification technology; microcapillary chromatography; spectral counting

Figure 1.

MudPIT workflow. (a) Proteins extracted from cells are digested into peptides, which are then fractionated using a combination of ion exchange and reverse phase chromatography. The peptides are sprayed directly into the mass spectrometer. After the MudPIT ‘run,’ experimentally generated MS/MS scans are analysed computationally and peptides/proteins identified. (b) Shotgun proteomics methods experimentally characterise short peptides rather than intact proteins. Such peptides dissociate inside the mass analyser in a predictable way to generate N‐terminal (y) or C‐terminal (b) ions. The mass to charge ratio of these ions results in a pattern in the MS/MS spectra that depends on the peptide sequence.

Figure 2.

Microcapillary chromatography. (a) Peptides are separated chromatographically using approximately 15 cm long silica microcapillary columns. The tip of the column is pulled to approximately 5 μm and columns packed with approximately 5 μm diameter chromatography resin. (b) Columns are packed with three regions of reverse phase (RP) or strong cation exchange (SCX) resin. The column is connected to an HPLC pump and the peptides are eluted across a approximately 5 mm gap into the mass spectrometer. A potential difference of approximately 2 kV between the buffer in the column and the mass spectrometer results in solvent free peptide ions (electrospray ionisation (ESI)).

Figure 3.

Resolving peptides with multidimensional chromatography. (a) During the initial 120 min chromatography step, a gradient of the organic solvent acetonitrile is used to disrupt hydrophobic interactions between peptides and the first region of reverse phase (RP) resin. Peptides can then bind to the strong cation exchange (SCX) resin. (b) In subsequent 120 min steps a salt ‘bump’ is used to transfer some peptides to the second region of reverse phase resin. These peptides are then gradually eluted into the mass spectrometer with an acetonitrile gradient. The salt concentration is increased in each successive salt bump.

Figure 4.

Generating MS/MS spectra. (a) An initial ‘MS’ scan is used to survey the different species of peptides entering the mass spectrometer. A sample of peptides is trapped and then peptides are gradually ejected through slits on two of the poles of the mass analyser. Peptides with lower m/q (mass/charge) values are ejected first. Data from this MS scan is used to select a particular m/q value for the subsequent ‘MS/MS’ analysis. (b) A fraction of a second after the MS scan, more peptides are trapped. Peptides with a chosen value of m/q (the red peptides) are then isolated. These (red) peptides collide with helium atoms in the mass analyser, absorb energy and dissociate. The fragments with different m/q are gradually ejected for detection as before. The resulting MS/MS spectrum can be used to identify the peptide. The data from each MS scan can be used to collect several MS/MS spectra.

Figure 5.

Labelling samples for quantitation. Proteins can be labelled with either heavy or light amino acids in the growth medium during culture (SILAC). After lysis, proteins are mixed prior to analysis. Peaks from peptides with either heavy or light amino acids are slightly offset in the MS spectrum; the relative intensity of the MS peaks can be used for quantitating the relative abundances of the parent proteins in the two samples. Alternative techniques include labelling proteins (ICAT) or peptides from digested proteins (iTRAQ).

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Further Reading

Banks CAS, Kong SE and Washburn MP (2012) Affinity purification of protein complexes for analysis by multidimensional protein identification technology. Protein Expression and Purification 86(2): 105–119.

Cravatt BF, Simon GM and YatesIII JR (2007) The biological impact of mass‐spectrometry‐based proteomics. Nature 450: 991–1000.

Fournier ML, Gilmore JM, Martin‐Brown SA and Washburn MP (2007) Multidimensional separations‐based shotgun proteomics. Chemical Reviews 107: 3654–3686.

Gingras A‐C, Gstaiger M, Raught B and Aebersold R (2007) Analysis of protein complexes using mass spectrometry. Nature Reviews Molecular Cell Biology 8: 645–654.

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Banks, Charles AS, Lakshminarasimhan, Mahadevan, and Washburn, Michael P(Oct 2014) Shotgun Proteomics. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0006197.pub2]