Shotgun proteomics: biochemistry and sample preparation

     Shotgun proteomics is a discovery-based platform, which has evolved to yield comprehensive proteomes. Commencing with sample preparation, proteins are isolated from a variety of biological materials, including cells, tissues, or body fluids, using customizable extraction buffers. Extraction protocols may require extensive tissue disruption or the simple solubilization and denaturation of proteins by using a chaotropic agent (e.g. urea) or by boiling in a detergent (e.g. sodium dodecyl sulfate, SDS). The extracted proteins are then subjected to digestion by sequence-specific proteases, such as trypsin, for the generation of peptides, followed by purification.

     Shotgun proteomics workflows include the investigation of protein-protein interactions (PPIs), enrichment of post-translational modifications (PTMs), and fractionation using chromatography-based separation techniques. For PPIs, ‘bait’ proteins are attached to a matrix and incubated with a sample containing ‘prey’ protein(s) to capture interacting partners. Considerations pertaining to detergent selection may influence the retainment of strong versus weak interactions and the utilization of chemical cross-linkers may permit capturing of transient interactions. Conversely, the investigation of PTMs captures peptides on an antibody-based matrix, matrices with affinities to distinct peptide properties such as titanium dioxide (TiO2) beads, or an immobilized metal affinity chromatography (IMAC) for the biochemical enrichment of the modified peptides. Lastly, peptides can be fractionated based on their biophysical and chemical properties by methods including cation exchange (SCX), size-exclusion (SEC), and high pH chromatography. The application of fractionation techniques often leads to increased proteomic depth at the expense of increased measurement time.

     For the absolute and relative quantification of proteins or peptides, different strategies include metabolic and chemical labeling, as well as label free methods (discussed in the next post). For example, metabolic labeling, including stable isotope labeling of amino acids in cell culture (SILAC) and 15N involves the incorporation of isotopically stable and non-radioactive forms of amino acids into proteins at the cellular or organizational levels. Alternatively, chemical labeling such as tandem mass tags (TMT), isobaric tags for relative and absolute quantification (iTRAQ), and dimethyl are based on the chemical derivatization of peptides.

Suggested reading:

Mann, M., Kulak, N.A., Nagaraj, N. & Cox, J. The coming age of complete, accurate, and ubiquitous proteomes. Mol Cell 49, 583-90 (2013).

Liu, F. & Heck, A.J. Interrogating the architecture of protein assemblies and protein interaction networks by cross-linking mass spectrometry. Curr Opin Struct Biol 35, 100-8 (2015).

Thingholm, T.E. & Jensen, O.N. Enrichment and characterization of phosphopeptides by immobilized metal affinity chromatography (IMAC) and mass spectrometry. Methods Mol Biol 527, 47-56, xi (2009).

Doll, S. & Burlingame, A.L. Mass spectrometry-based detection and assignment of protein posttranslational modifications. ACS Chem Biol 10, 63-71 (2015).

Humphrey, S.J., Azimifar, S.B. & Mann, M. High-throughput phosphoproteomics reveals in vivo insulin signaling dynamics. Nat Biotechnol 33, 990-5 (2015).

Batth, T.S., Francavilla, C. & Olsen, J.V. Off-line high-pH reversed-phase fractionation for in-depth phosphoproteomics. J Proteome Res 13, 6176-86 (2014).

Mohammed, S. & Heck, A., Jr. Strong cation exchange (SCX) based analytical methods for the targeted analysis of protein post-translational modifications. Curr Opin Biotechnol 22, 9-16 (2011).

Ong, S.E., Foster, L.J. & Mann, M. Mass spectrometric-based approaches in quantitative proteomics. Methods 29, 124-30 (2003).

Gouw, J.W., Krijgsveld, J. & Heck, A.J. Quantitative proteomics by metabolic labeling of model organisms. Mol Cell Proteomics 9, 11-24 (2010).

Li, Z. et al. Systematic comparison of label-free, metabolic labeling, and isobaric chemical labeling for quantitative proteomics on LTQ Orbitrap Velos. J Proteome Res 11, 1582-90 (2012).

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