Novel parallelized quadrupole/linear ion trap/Orbitrap tribrid mass spectrometer improving proteome coverage and peptide identification rates.

Proteome coverage and peptide identification rates have historically advanced in line with improvements to the detection limits and acquisition rate of the mass spectrometer. For a linear ion trap/Orbitrap hybrid, the acquisition rate has been limited primarily by the duration of the ion accumulation and analysis steps. It is shown here that the spectral acquisition rate can be significantly improved through extensive parallelization of the acquisition process using a novel mass spectrometer incorporating quadrupole, Orbitrap, and linear trap analyzers.

High resolution electron transfer dissociation studies of unfractionated intact histones from murine embryonic stem cells using on-line capillary LC separation: determination of abundant histone isoforms and post-translational modifications.

Epigenetic regulation of chromatin is dependent on both the histone protein isoforms and state of their post-translational modifications. The assignment of all post-translational modification sites for each individual intact protein isoform remains an experimental challenge. We present an on-line reversed phase LC tandem mass spectrometry approach for the separation of intact, unfractionated histones and a high resolution mass analyzer, the Orbitrap, with electron transfer dissociation capabilities to detect and record accurate mass values for the molecular and fragment ions observed.

High-resolution mass spectrometry analysis of protein oxidations and resultant loss of function.

MS, with or without pre-analysis peptide fractionation, can be used to decipher the residues on proteins where oxidative modifications caused by peroxynitrite, singlet oxygen or electrophilic lipids have occurred. Peroxynitrite nitrates tyrosine and tryptophan residues on the surface of actin. Singlet oxygen, formed by the interaction of UVA light with tryptophan, can oxidize neighbouring cysteine, histidine, methionine, tyrosine and tryptophan residues.

active site modifications of the brain isoform of creatine kinase by 4-hydroxy-2-nonenal correlate with reduced enzyme activity: mapping of modified sites by Fourier transform-ion cyclotron resonance mass spectrometry.

Creatine kinase reversibly catalyzes the transfer of the high-energy phosphoryl group from phosphocreatine to MgADP for rapid regeneration of ATP. It is hypothesized that factors which perturb creatine kinase activity, such as reactive oxygen species resulting from oxidative stress, could have a major role in the pathogenesis of diseases, particularly in the brain, where the level of ATP utilization is high. The reactive aldehyde 4-hydroxy-2-nonenal is a major secondary product of lipid peroxidation caused by oxidative stress; the levels of both free and protein-bound 4-hydroxy-2-nonenal are increased in Alzheimer's disease brain.