Protein Identification by Mass Spectrometry: Proteomics

Abstract

Identification, characterization, and quantitation of changes of proteins induced by various types of external and/or internal stimuli are the major objectives of proteomics. Because proteins consist of 20 amino acids, their sequencing is more complicated than nucleic acids, which are a combination of four nucleotides. Proteins and peptides cannot be amplified and/or hybridized in vitro as nucleic acids. Moreover, proteins may have multiple posttranslationally modified sites and multiple types of modifications, such as phosphorylation, nitration, acetylation, and methylation, just to mention a few. These facts have a decisive impact on analytical methods used for protein sequencing and characterization. Before the development of soft ionization (the formation of ions without breaking any chemical bonds) techniques, proteins and peptides were sequenced using the Edman degradation method, based on the sequential removal of N-terminal amino acids (Edman 1970). This method, although still used (Mari et al. 2010), has many limitations such as an inability to sequence peptides that have the N-terminus blocked (Chen et al. 2008). Fragmentation of relatively short peptides (usually 6–30 amino acids in length) in gas phase in mass spectrometers exponentially increased the confidence with which we are able to determine peptide sequence and reveal other structural features, including posttranslational modifications (Biemann 1988; Strachunskii et al. 1992; Appella et al. 2000; Meng et al. 2005; Paizs and Suhai 2005). A number of specific proteolytic enzymes are available to fragment larger proteins into shorter peptides more suitable for high-quality analysis in tandem mass spectrometry (MS/MS) (see Fig. 28.3). The most commonly used enzyme, trypsin, specifically cleaves the peptide bond C-terminal to arginine and lysine residues with any amino acid excluding proline (Thiede et al. 2005). The Arg/Lys–Pro peptide bond is cleaved at a very low rate; however, if such a peptide is well ionized, it still can be detected and sequenced by mass spectrometry (Pottiez et al. 2010)