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Photoinitiated radical chemistry has proven to be useful for breaking covalent bonds within many biomolecules in the gas phase. Herein, we demonstrate that radical chemistry is useful for bond synthesis in the gas phase. Single peptides containing two cysteine residues capped with propylmercaptan (PM) often form disulfide bonds following ultraviolet excitation at 266 nm and loss of both PM groups. Similarly, noncovalently bound peptide pairs where each peptide contains a single cysteine residue can be induced to form disulfide bonds. Comparison with disulfide bound species sampled directly from solution yields identical collisional activation spectra, suggesting that native disulfide bonds have been recapitulated in the gas phase syntheses. Another approach utilizing radical chemistry for covalent bond synthesis involves creation of a reactive diradical that can first abstract hydrogen from a target peptide, creating a new radical site, and then recombine the second radical with the new radical to form a covalent bond. This chemistry is illustrated with 2-(hydroxymethyl-3,5-diiodobenzoate)-18-crown-6 ether, which attaches noncovalently to protonated primary amines in peptides and proteins. Following photoactivation and crosslinking, the site of noncovalent adduct attachment can frequently be determined. The ramifications of these observations on peptide structure and noncovalent attachment of 18-crown-6-based molecules is discussed.

Ultraviolet photodissociation or UVPD is an increasingly popular option for tandem-mass spectrometry experiments. UVPD can be carried out at many wavelengths, and it is important to understand how the results will be impacted by this choice. Here, we explore the utility of 213nm photons for initiating bond-selective fragmentation. It is found that bonds previously determined to be labile at 266nm, including carbon-iodine and sulfur-sulfur bonds, can also be cleaved with high selectivity at 213nm. In addition, many carbon-sulfur bonds that are not subject to direct dissociation at 266nm can be selectively fragmented at 213nm. This capability can be used to site-specifically create alaninyl radicals that direct backbone dissociation at the radical site, creating diagnostic d-ions. Furthermore, the additional carbon-sulfur bond fragmentation capability leads to signature triplets for fragmentation of disulfide bonds. Absorption of amide bonds can enhance dissociation of nearby labile carbon-sulfur bonds and can be used for stochastic backbone fragmentation typical of UVPD experiments at shorter wavelengths. Several potential applications of the bond-selective fragmentation chemistry observed at 213nm are discussed. Graphical Abstract ᅟ.