Structure verification and purity: Chemically synthesized molecules, such as peptides and oligonucleotides are often characterized by liquid chromatography and mass spectrometry. Suppose a chemically synthesized peptide, DAKLRYFNQP gives a MALDI mass spectrum as shown in Figure 13.3; the monoisotopic mass of the peptide is 1250.63 Da. The peak at 1180.59 is due to deletion of alanine.

Figure 13.3 A hypothesized MALDI mass spectrum of the synthesized peptide, DAKLRYFNQP (theoretically calculated mass: 1250.63 Da)

Identification of chemical modifications: Biomolecules, especially proteins, can undergo a variety of chemical modifications such as phosphorylation, acetylation, methylation, fatty acylation, glycosylation, etc. These modifications are involved in biological processes like regulation of enzyme activity, signal transduction, gene expression, etc. It is therefore important to identify these species for understanding their function. Owing to its sensitivity and resolution, mass spectrometry has emerged as the method of choice for identification of small molecule modifications in biomolecules.

Protein sequencing:

Proteins are usually ionized using soft ionization techniques, MALDI and ESI. These methods yield quasimolecular ions that allow identification of proteins in complex mixtures. For determining their sequences, however, proteins need to be fragmented. The idea behind protein sequencing using MS is very straight forward and is summarized in Figure 13.4. Briefly, a protein quasimolecular ion is selected using a mass analyzer. The selected ion is then fragmented, typically in a collision cell (collision induced dissociation). Collision induced dissociation results in a large number of fragments that also have overlapping amino acid sequences. These daughter ions are then detected by a second mass analyzer. Mass of a fragment comprises the information for its amino acid composition. Masses of the overlapping fragments allow sequencing of the complete molecule.