De novo sequencing of peptides using selective 351 nm ultraviolet photodissociation mass spectrometry

Although in silico database search methods remain more popular for shotgun proteomics methods, de novo sequencing offers the ability to identify peptides derived from proteins lacking sequenced genomes and ones with subtle splice variants or truncations. Ultraviolet photodissociation (UVPD) of peptides derivatized by selective attachment of a chromophore at the N-terminus generates a characteristic series of y ions. The UVPD spectra of the chromophore-labeled peptides are simplified and thus amenable to de novo sequencing. This method resulted in an observed sequence coverage of 79% for cytochrome C (eight peptides), 47% for β-lactoglobulin (five peptides), 25% for carbonic anhydrase (six peptides), and 51% for bovine serum albumin (33 peptides). This strategy also allowed differentiation of proteins with high sequence homology as evidenced by de novo sequencing of two variants of green fluorescent protein.