Shotgun proteomics

The most common method of shotgun proteomics starts with the proteins in the mixture being digested and the resulting peptides are separated by liquid chromatography.

In 1975, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) was described by O’Farrell and Klose with the ability to resolve complex protein mixtures.

[9] It also avoids the modest separation efficiency and poor mass spectral sensitivity associated with intact protein analysis.

[1] The dynamic exclusion filtering that is often used in shotgun proteomics maximizes the number of identified proteins at the expense of random sampling.

The "fingerprint" of each peptide's fragmentation mass spectrum is used to identify the protein from which they derive by searching against a sequence database with commercially available software (e.g. Sequest or Mascot).

Additionally, some proteome samples of vertebrates have a large number of paralogs, and alternative splicing in higher eukaryotes can result in many identical protein subsequences.

[14][15][16][17] With the human genome sequenced, the next step is the verification and functional annotation of all predicted genes and their protein products.

[2] Vaisar et al. uses shotgun proteomics to implicate protease inhibition and complement activation in the antiinflammatory properties of high-density lipoprotein.