Ultraviolet photodissociation mass spectrometry of bis-aryl hydrazone conjugated peptides.

Ultraviolet photodissociation (UVPD) at 355 nm was used to rapidly identify peptides which had been chemically conjugated through bis-aryl hydrazone (BAH) moieties. The two biomolecules of interest were separately tagged to introduce either an aldehyde or a hydrazine and then conjugated together through these functional groups to from the UV-chromogenic BAH-group. In a mock mixture of peptides, UVPD was used to screen for the BAH-conjugated peptides in direct infusion ESI-UVPD-MS and online LC-UVPD-MS methods by comparing the abundances of the ions with the laser off and with the laser on. Only the BAH-conjugated peptides were observed to photodissociate upon exposure to UV irradiation, thus affording excellent selectivity for the pinpointing the relevant conjugated peptides in a complex mixture of nonconjugated peptides. UVPD analysis of conjugated model peptides indicated that the UVPD efficiencies of these species were charge state dependent. BAH-conjugated peptides that had a mobile proton which could protonate the basic BAH-moiety underwent more efficient photodissociation than the peptide ions with sequestered protons. Ultraviolet photodissociation of BAH-cross-linked peptides also yielded more diagnostic sequence ions than CID to unambiguously locate the site of conjugation.