ABSTRACT
Vibrational ion spectroscopy techniques coupled with mass spectrometry are applied to standard metabolites as a proof-of-principle demonstration for the structural identification of unknown metabolites. The traditional room temperature infrared multiple photon dissociation (IRMPD) spectroscopy technique is shown to differentiate chemical moieties in isobaric and isomeric variants. These results are compared to infrared spectra of cryogenically cooled analyte ions, showing enhanced spectral resolution, and thus also improved differentiation between closely related molecules, such as isomers. The cryogenic spectroscopy is effected in a recently developed mass-selective cryogenic linear ion trap, which is capable of high sensitivity and the ability to measure the IR spectra of multiple analytes simultaneously.
ABSTRACT
We report on the rearrangement chemistry of model phosphorylated peptides during collision-induced dissociation (CID), where intramolecular phosphate group transfers are observed from donor to acceptor residues. Such "scrambling" could result in inaccurate modification localization, potentially leading to misidentifications. Systematic studies presented herein provide mechanistic insights for the unusually high phosphate group rearrangements presented some time ago by Reid and coworkers (Proteomics 2013, 13 [6], 964-973). It is postulated here that a basic residue like histidine can play a key role in mediating the phosphate group transfer by deprotonating the serine acceptor site. The proposed mechanism is consistent with the observation that fast collisional activation by collision-cell CID and higher-energy collisional dissociation (HCD) can shut down rearrangement chemistry. Additionally, the rearrangement chemistry is highly dependent on the charge state of the peptide, mirroring previous studies that less rearrangement is observed under mobile proton conditions.