Cascade dissociations of peptide cation-radicals. Part 2. Infrared multiphoton dissociation and mechanistic studies of z-ions from pentapeptides.

International audienceDissociations of z(4) ions from pentapeptides AAXAR where X=H, Y, F, W, and V produce dominant z(2) ions that account for >50 % of the fragment ion intensity. The dissociation has been studied in detail by experiment and theory and found to involve several isomerization and bond-breaking steps. Isomerizations in z(4) ions proceed by amide trans→cis rotations followed by radical-induced transfer of a β-hydrogen atom from the side chain, forming stable C(β) radical intermediates. These undergo rate-determining cleavage of the C(α)-CO bond at the X residue followed by loss of the neutral AX fragment, forming x(2) intermediates. The latter were detected by energy-resolved resonant excitation collision-induced dissociation (CID) and infrared multiphoton dissociation (IRMPD) experiments. The x(2) intermediates undergo facile loss of HNCO to form z(2) fragment ions, as also confirmed by energy-resolved CID and IRMPD MS(4) experiments. The loss of HNCO from the x(2) ion from AAHWR is kinetically hampered by the Trp residue that traps the OCNH radical group in a cyclic intermediate

Tyrosine Deprotonation Yields Abundant and Selective Backbone Cleavage in Peptide Anions upon Negative Electron Transfer Dissociation and Ultraviolet Photodissociation

Tyrosine deprotonation in peptides yields preferential electron detachment upon NETD or UVPD, resulting in prominent N–Cα bond cleavage N-terminal to the tyrosine residue. UVPD of iodo-tyrosine-modified peptides was used to generate localized radicals on neutral tyrosine side chains by homolytic cleavage of the C–I bond. Subsequent collisional activation of the radical species yielded the same preferential cleavage of the adjacent N-terminal N–Cα bond. LC-MS/MS analysis of a tryptic digest of BSA demonstrated that these cleavages are regularly observed for peptides when using high-pH mobile phases

Infrared Multiphoton Dissociation for Quantitative Shotgun Proteomics

We modified a dual-cell linear ion trap mass spectrometer to perform infrared multiphoton dissociation (IRMPD) in the low-pressure trap of a dual-cell quadrupole linear ion trap (dual-cell QLT) and perform large-scale IRMPD analyses of complex peptide mixtures. Upon optimization of activation parameters (precursor <i>q</i>-value, irradiation time, and photon flux), IRMPD subtly, but significantly, outperforms resonant-excitation collisional-activated dissociation (CAD) for peptides identified at a 1% false-discovery rate (FDR) from a yeast tryptic digest (95% confidence, <i>p</i> = 0.019). We further demonstrate that IRMPD is compatible with the analysis of isobaric-tagged peptides. Using fixed QLT rf amplitude allows for the consistent retention of reporter ions, but necessitates the use of variable IRMPD irradiation times, dependent upon precursor mass to charge (<i>m</i>/<i>z</i>). We show that IRMPD activation parameters can be tuned to allow for effective peptide identification and quantitation simultaneously. We thus conclude that IRMPD performed in a dual-cell ion trap is an effective option for the large-scale analysis of both unmodified and isobaric-tagged peptides