Use of Ultraviolet Photodissociation Coupled with Ion Mobility Mass Spectrometry To Determine Structure and Sequence from Drift Time Selected Peptides and Proteins

Abstract

We demonstrate the capabilities of a laser-coupled ion mobility mass spectrometer for analysis of peptide sequence and structure showing ultraviolet photodissociation (UVPD) spectra of mass and mobility selected ions. A Synapt G2-S mass spectrometer has been modified to allow photointeraction of ions post the mobility cell. For this work, we have employed a single wavelength laser, which irradiates at 266 nm. We present the unique capabilities of this instrument and demonstrate several key features. Irradiation of luteinizing hormone releasing hormone (LHRH), growth hormone releasing hexapeptide (GHRP-6), and TrpCage (sequence NLYIQWLKDGGPSSGRPPPS) yields extensive b- and y-type fragmentation as well as a- and c-type ions. In addition, we observe side chain losses, including the indole group from tryptophan, and immonium ions. For negatively charged ions, we show the advantage of using collision-induced dissociation (CID) post-UVPD: radical ions are produced following irradiation, and these fragment with higher efficiency. Further, we have incorporated ion mobility and subsequent drift time gating into the UVPD method allowing the separate analysis of m/z-coincident species, both conformers and multimers. To demonstrate, we selectively dissociate the singly charged dimer or doubly charged monomer of the peptide gramicidin A and conformers of the [M + 5H] form of the peptide melittin. Each mobility selected form has a different "fingerprint" dissociation spectrum, both predominantly containing b and y fragments. Differences in the intensities of various loss channels between the two species were revealed. The smaller conformer of melittin has fewer cleavage sites along the peptide backbone than the larger conformer suggesting considerable structural differences. For gramicidin, a single laser shot UVPD discriminates between primary photodissociation and subsequent fragmentation of fragments. We also show how this modified instrument facilitates activated electron photodissociation. UVPD-IM-MS analysis serves both as a method for peptide sequencing for peptides of similar (or identical) m/z and a method for optical analysis of mobility separated species.

Initial Protein Unfolding Events in Ubiquitin, Cytochrome c and Myoglobin Are Revealed with the Use of 213 nm UVPD Coupled to IM-MS

“…Ly and Julian utilised 266 nm UVPD to cleave a carbon-iodide bond incorporated into Tyr59 of ubiquitin and subsequent radical-directed dissociation cleavage sites provided distance constraints for molecular modelling conformational searches, yielding structures that were distinctly different to the crystal structure and in agreement with data from ECD [ 25 , 61 ]. We have also previously observed differences in the fragmentation pattern of two different, distinct conformational families of 5+ melittin as probed by 266 nm UVPD [ 62 ]. Overall, there is increasing evidence that ultraviolet photodissociation, especially in combination with IM-MS, is a promising avenue for protein-structural characterisation.…”

Collision-induced dissociation of doubly-charged barium-cationized lipids generated from liquid samples by atmospheric pressure matrix-assisted laser desorption/ionization provides structurally diagnostic product ions

“…UVPD has recently been implemented on commercial instrumentation, initially requiring significant non-trivial instrument modifications [34,35]. It differs from collision-or electron-mediated dissociation methods in that the fragmentation energy is delivered by photon absorption.…”

Coupling 193 nm Ultraviolet Photodissociation and Ion Mobility for Sequence Characterization of Conformationally-Selected Peptides

“…UVPD of drift time-selected ions has been accomplished previously with 266 nm 30 and 213 nm 24 wavelengths. Here, we present the modification of a commercial instrument platform to incorporate 193 nm UVPD following IM separation and characterize its performance for a series of model peptides.…”