Facile identification of photocleavable reactive metabolites and oxidative stress biomarkers in proteins via mass spectrometry

Abstract

Described herein is a method which combines bond selective fragmentation by photodissociation with online liquid chromatographic separation and mass spectrometric analysis. Photoexcitation of proteins or peptides with 266-nm light does not normally yield abundant fragmentation; however, incorporation of a suitable carbon-sulfur or carbon-halogen bond that is proximal to a chromophore allows access to direct dissociation pathways, resulting in homolytic cleavage of these bonds. Radicals generated through this process can cause further dissociation of the peptide backbone, which is useful for site specifically identifying the point of modification. Two specific applications of this technique for peptide analysis in model systems are presented: (1) identification of reactive metabolites which covalently modify cysteine residues, and (2) characterization of halogenated tyrosine residues which are biomarkers related to oxidative stress. In both cases, these naturally occurring post translational modifications create photocleavable bonds which can be fragmented by 266-nm light. The selectivity offered by photodissociation allows facile identification of the peptides of interest even in complex mixtures, and subsequent selective radical directed backbone fragmentation pinpoints the site of modification. This combination greatly simplifies data analysis and provides more confident assignments.

The Mechanism Behind Top-Down UVPD Experiments: Making Sense of Apparent Contradictions

“…Well-documented examples of this type of chemistry include fragmentation of carbon-iodine, carbon-sulfur, and sulfur-sulfur bonds. 19–21 Indeed, direct dissociation is arguably a feature unique to UVPD, though proponents of the ‘nonergodic’ mechanism 22 for ECD/ETD might argue otherwise.…”

Identification of Amino Acid Epimerization and Isomerization in Crystallin Proteins by Tandem LC-MS

“…In a RDD LC-MS run, the laser pulses were triggered during the MS 2 (scan event 3) and the CID was performed as a pseudo-MS 3 step (scan event 4). 36 Since the photodissociation of 4-iodo-benzoic labeled peptide will always produce the radical peptide as the major product, the precursor ion of CID in the MS 3 step is the radical species rather than the original peptide. The exclusion time was 60 s for the identification of peptides and 16 s for the isomer discrimination.…”

Tautomerization and Dissociation of Molecular Peptide Radical Cations

“…As mentioned, E(C/T)D techniques yield hydrogen‐rich radical cations of peptides/proteins in multiply protonated forms by adding a low‐energy electron. Hydrogen‐deficient peptide radical cations can also be produced; for example, through photo‐dissociation of protonated peptides derivatized with photo‐labile covalent bonds, under ultraviolet (UV) or vacuum ultraviolet (VUV) irradiation . Peptide dissociation behavior in tert ‐butyl peroxycarbamate–, Vazo 68–, and TEMPO– (2,2,6,6‐tetramethylpiperidine‐1‐oxyl) based FRIPS (free radical–initiated peptide sequencing) MS has been analyzed in both positive‐ and negative‐ion modes for a number of peptides.…”