A crown ether based, photolabile radical precursor which forms noncovalent complexes with peptides has been prepared. The peptide/precursor complexes can be electrosprayed, isolated in an ion trap, and then subjected to laser photolysis and collision induced dissociation to generate hydrogen deficient peptide radicals. It is demonstrated that these peptide radicals behave very differently from the hydrogen rich peptide radicals generated by electron capture methods. In fact, it is shown that side chain chemistry dictates both the occurrence and relative abundance of backbone fragments that are observed. Fragmentation at aromatic residues occurs preferentially over most other amino acids. The origin of this selectivity relates to the mechanism by which backbone dissociation is initiated. The first step is abstraction of a β-hydrogen from the side chain, followed by beta-elimination to yield primarily a-type fragment ions. Calculations reveal that those side chains which can easily lose a β-hydrogen correlate well with experimentally favored sites for backbone fragmentation. In addition, radical mediated side chain losses from the parent peptide are frequently observed. Eleven amino acids exhibit unique mass losses from side chains which positively identify that particular amino acid as part of the parent peptide. Therefore, side chain losses allow one to unambiguously narrow the possible sequences for a parent peptide, which when combined with predictable backbone fragmentation should lead to greatly increased confidence in peptide identification.

Novel p-iodobenzoate-based labelling reagents are shown to be effective photocaged precursors for synthesizing biomolecular radicals site-selectively in the gaseous and condensed phases. In vacuo, a single pulse of UV photons (266 nm) is sufficient to quantitatively photolyse the C-I bond. In aqueous solutions, the photolysis half-life is estimated to be 2.5 minutes when irradiating with a 15 W compact fluorescent lamp (254 nm).

Iodination of tyrosine residues in proteins has many uses in chemistry, biology, and medicine. Site specific identification of the sites of iodination is important for many of these uses. Reported herein is a facile method employing photodissociation and mass spectrometry to localize sites of iodination in whole proteins. Absorption of ultraviolet photons by iodotyrosine results in loss of iodine via homolytic bond dissociation. The resulting protein radical fragments in the vicinity of the iodotyrosine upon collisional activation. Analysis of the fragments within the vicinity of each tyrosine residue in the protein enables quantitative evaluation of the likelihood for iodination at each site. The results are compared with both traditional bottom up and top down mass spectrometric methods. Radical directed dissociation yields results in agreement with traditional approaches but requires significantly less effort and is inherently more sensitive. One limitation occurs when multiple tyrosine residues are in close proximity, in which case the extent of iodination at each residue may be difficult to determine. This limitation is frequently problematic for traditional approaches as well.