Peptides shotgun proteomics

Cargile, B.J., Talley, D.L., Stephenson, J.L., Jr. (2004b). Immobilized pH gradients as a first dimension in shotgun proteomics and analysis of the accuracy of pi predictability of peptides. Electrophoresis 25, 936-945. 

We therefore sought to evaluate reproducibility of shotgun proteomics in studies of archival FFPE tissue. Because FFPE samples are more complex than non-cross-linked samples, we evaluated FFPE human liver for analytical reproducibility and confidence in protein assignments.20 This complexity strengthens the argument for using high-resolution separations to maximize analyte concentration and minimize matrix effects. In this case, we used transient capillary isotachophoresis/capillary zone electrophoresis (cITP/cZE) in place of IEF to help address this effect. cITP/cZE has a resolution superior even to cIEF (90% of identified peptides in 1 fraction, 95% in 2 fractions or less for cITP/cZE, vs. 75% and 80%, respectively, for cIEF). 

Figure 20.11 Coverage of protein ErbB2 by shotgun proteomic discovery of sample fixed for various times, including fresh. The color gradient represents the increasing abundance of the peptides. All were identified at an FDR <1%. Reproduced with permission from Reference 20.

In the gel-free approaches, also referred as shotgun proteomics, proteins in a mixture are digested and the peptides released separated by reverse-phase HPLC, either alone or combined with strong cation exchange chromatography... 

Co-immunoprecipitation is a common technique to enrich protein interaction complexes. This method uses an antibody to bind a known protein, which in turn has known or unknown binding partners. Shotgun proteomics is then performed to determine the enriched proteins. Quantitative MS is used to validate bound proteins and measure binding stoichiometry for proteins in the complexes prepared via IP (127,128). Quantitation approaches used include SILAM (129), label-free (130), and AQUA peptides with a normalization step (131). 

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