Combining UV photodissociation with electron transfer for peptide structure analysis Christopher J. Shaffer, a Ales Marek, b Robert Pepin, a Kristina Slovakova c and Frantisek Turecek a * The combination of near-UV photodissociation with electron transfer and collisional activation provides a new tool for structure investigation of isolated peptide ions and reactive intermediates. Two new types of pulse experiments are reported. In the first one called UV/Vis photodissociation–electron transfer dissociation (UVPD-ETD), diazirine-labeled peptide ions are shown to un- dergo photodissociation in the gas phase to form new covalent bonds, guided by the ion conformation, and the products are an- alyzed by electron transfer dissociation. In the second experiment, called ETD-UVPD wherein synthetic labels are not necessary, electron transfer forms new cation–peptide radical chromophores that absorb at 355 nm and undergo specific backbone photo- dissociation reactions. The new method is applied to distinguish isomeric ions produced by ETD of arginine containing peptides. Copyright © 2015 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: electron transfer dissociation; laser photodissociation; peptide ions; cation radical; chromophores; isomer distinction Protein analysis by mass spectrometry relies on the determination of amino acid sequence through backbone dissociations of gas- phase ions. Traditional methods of peptide ion structure analysis utilize collision-induced dissociation (CID) or infrared multiphoton dissociation (IRMPD). More recently, electron transfer dissociation (ETD) [1] and UV/Vis photodissociation (UVPD) [2] have been explored for peptide and protein analysis. Principally, ETD proceeds by radical-driven backbone dissociations producing closed-shell N- terminal cations and C-terminal cation radicals. UVPD of peptide ions requires short wavelengths (<240 nm) [3] to excite the natural chromophores or the presence of auxiliary chromophores that are introduced into the peptide by chemical modifications prior to mass spectrometric analysis. [2,4] Whereas CID and IRMPD are mostly insensitive to the peptide ion 3D structure, backbone cleavages by ETD have been shown to de- pend on the peptide ion conformation, as reviewed. [5] This warrants development of new methods for determining peptide ion 3D structure, as well as the electronic structure of ions produced by ETD. We now report a method that combines the features of UVPD and ETD into two complementary experiments aimed at elucidat- ing peptide ion structures. The first experiment, called UVPD-ETD, (Fig. 1, top panel) probes the 3D structure by photolysis of a chromophore-tagged peptide ion, first by mass selection, storage in a linear ion trap and irradiation by a train of 3–6-ns laser pulses, followed by ETD analysis of selected photodissociation products. The chromophore that we use is a diazirine ring that is built into the peptide as an L-2-amino-4,4-azi-pentanoic acid (photoleucine, L*) residue absorbing at 340–360 nm. [6] Photoleucine is inserted in the peptide sequence by standard solid-phase synthesis or by in vivo protein expression to replace the natural Leu residues. [7] Nat- ural amino acid residues do not absorb light in this wavelength region, and thus, the initial electronic excitation is localized in the diazirine ring. Photolysis of the diazirine ring at 355 nm (3.49-eV photon energy) results in nitrogen expulsion, forming a highly reac- tive carbene intermediate. This can either undergo insertion into a sterically proximate X–H bond forming a new covalent C–X bond [8,9] or undergo competitive isomerization to a non-reactive olefin. [10–12] Insertion forms a new ring structure in the peptide that can be probed by ETD (Scheme 1). The second experiment, which we refer to as ETD-UVPD, does not require an auxiliary chromophore in the peptide ion to be ana- lyzed. This claim is based on our discovery that peptide–cation rad- icals produced by electron transfer become absorbed at 355 nm and can be probed by UVPD. In this pulse sequence, the precursor peptide dication is first selected by mass, stored in the ion trap and allowed to react with an electron donor such as fluoranthene anion radical. The ETD product of interest is then selected by mass and ir- radiated by a train of laser pulses to achieve photodissociation (Fig. 1, bottom panel). Selected ETD-UVPD fragment ions can be iso- lated again and further analyzed by CID or ETD. Experiments of the first type, UVPD-ETD, can be carried out with several peptide ions of different chain length, folding pattern and amino acid composition. Here, it is illustrated with a dication generated by electrospray ionization of diazirine-labeled peptide GL*GGK (m/z 222, M 2+ , Fig. 2, top). Irradiation with a single laser pulse of 16-mJ energy achieved ca 10% conversion by loss of N 2 (m/z 208) and backbone dissociation forming the y 3 fragment ion. The conversion is consistent with the relatively low molar ab- sorptivity of the diazirine chromophore (ε max = 50–100). [13] The * Correspondence to: Frantisek Turecek, Department of Chemistry, Bagley Hall, Box 351700, University of Washington, Seattle, WA, 98195–1700, USA. E-mail: turecek@chem.washington.edu a Department of Chemistry, University of Washington, Bagley Hall, Box 351700, Seattle, WA, 98195-1700, USA b Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague, Czech Republic c Department of Analytical Chemistry, Palacky University, Olomouc, Czech Republic J. Mass Spectrom. 2015, 50, 470–475 Copyright © 2015 John Wiley & Sons, Ltd. Accelerated communication Received: 31 October 2014 Revised: 18 November 2014 Accepted: 18 November 2014 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/jms.3551 470