Proteins are a class of molecule that are amenable to mass spectrometric fragmentation at the peptide backbone. These fragmentation pathways result in a series of ions differing in mass by one amino acid. The sequence of the peptide can then be determined by looking at the mass differences between fragment ions derived from the same fragmentation pathway.
Since most amino acids have different chemical structures and therefore different masses, the identity of the amino acid can be determined by the mass difference between associated fragment ions. The limiting factor in the effectiveness of this as an approach is the size of the peptides that can be readily fragmented. Energy considerations and the need to limit dissipation of energy through the ionized molecule to promote fragmentation mean that the maximum size for fragment ion generation is about 2-3kDa, which translates to a molecule of approximately 18-28 amino acids in length. Clearly, this is much smaller than most proteins of therapeutic value (e.g. IgG monoclonal antibodies, which are composed of two light chains of approximately 214 amino acids each and two heavy chains of approximately 450 amino acids each). Therefore, these proteins need to be reduced down to a mixture of constituent peptides to generate meaningful fragmentation data.
This peptide generation is achieved through the use of specific protease digests in conjunction with on-line liquid chromatographic (LC) separation of the digestion products and UV and mass spectrometric analysis of the separated components. The most useful form of ionization for mass spectrometric analysis of peptides is electrospray, which produces peptide ions with multiple degrees of charging that are amenable to fragmentation. The whole procedure is therefore described as on-line LC/ES-MS analysis.