Philip M. Remes

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. Further, these improvements to the acquisition rate continue to enhance proteome coverage and general experimental throughput.

Evaluation of Front-End Higher Energy Collision-Induced Dissociation on a Benchtop Dual-Pressure Linear Ion Trap Mass Spectrometer for Shotgun Proteomics

We report the implementation of front-end higher energy collision-induced dissociation (fHCD) on a benchtop dual-pressure linear ion trap. Software and hardware modifications were employed, described in detail vide-infra, to allow isolated ions to undergo collisions with ambient gas molecules in an intermediate multipole (q00) of the instrument. Results comparing the performance of fHCD and resonance excitation collision-induced dissociation (RE-CID) in terms of injection time, total number of scans, efficiency, mass measurement accuracy (MMA), unique peptide identifications, and spectral quality of labile modified peptides are presented. fHCD is approximately 23% as efficient as RE-CID, and depending on the search algorithm, it identifies 6.6% more or 15% less peptides (q < 0.01) from a soluble whole-cell lysate ( Caenorhabditis elegans ) than RE-CID using Mascot or Sequest search algorithms, respectively. fHCD offers a clear advantage for the analysis of phosphorylated and glycosylated (O-GlcNAc) peptides as the average cross-correlation score (XCorr) for spectra using fHCD was statistically greater (p < 0.05) than for spectra collected using RE-CID.

On The Time Scale of Internal Energy Relaxation of AP-MALDI and nano-ESI Ions in a Quadrupole Ion Trap

Recently reported results (Konn et al. [14]) on the collisional cooling of atmospheric pressure matrix assisted laser desorption ionization (AP-MALDI) and nano-electrospray ionization (nano-ESI) generated ions in a quadrupole ion trap mass spectrometer (QITMS) are inconsistent with measured collisional cooling rates. The work reported here presents a re-examination of those previous results. Collision induced dissociation (CID) has been used to probe various properties of ions contained in a QITMS. It is shown experimentally that when trapping large numbers of ions, an effective dc trapping voltage is induced that varies with changes in the size of the ion cloud. A decrease in the resonant frequency for maximum CID efficiency is observed as the cool time between parent ion isolation and CID is increased. Ion trajectories in a QITMS are simulated to demonstrate how ion density changes over the course of parent ion isolation. The effect of space charge on ion motion is simulated, and Fourier transformations of ion axial motion plus simple calculations corroborate the experimentally observed transient frequency shifts. The relative stability of ions formed by AP-MALDI and nano-ESI is compared under low charge density conditions. These data show that the ions have reached equilibrium internal energy and, thus, that differences in dissociation onsets and “50% fragmentation efficiency points” between the ionization mechanisms are due to the formation of distinct ion conformations as previously shown in reference [28]. The conclusions of Konn et al. [14] are based on invalid experimental procedures as well as inappropriate comparisons of QITMS data to low-pressure FT-ICR data.