Streamlining Bottom-Up Protein Identification Based on Selective Ultraviolet Photodissociation (UVPD) of Chromophore-Tagged Histidine- and Tyrosine-Containing Peptides

We report a fast and highly efficient diazonium reaction that couples a nitroazobenzene chromophore to tyrosine and histidine residues, thus endowing peptides with high photoabsorption cross sections at 351 nm in the gas phase. Only the tagged peptides undergo ultraviolet photo-dissociation (UVPD) at 351 nm, as demonstrated for several Tyr- and His-containing peptides from protein digests. Additional selectivity is achieved by the integration of the LTVPD-MS method with an in silico database search restricted to Tyr- and His-containing peptides. A modified Mass Matrix algorithm condenses analysis by filtering the input database file to include Tyr/His-containing peptides only, thus reducing the search space and increasing confidence. In summary, derivatization of specific amino acid residues in conjunction with selective activation of the derivatized peptides provides a streamlined approach to shotgun proteomics.