Jan-Peter Hauschild

Mass Spectrometry-based Proteomics Using Q Exactive, a High-performance Benchtop Quadrupole Orbitrap Mass Spectrometer

Mass spectrometry-based proteomics has greatly benefitted from enormous advances in high resolution instrumentation in recent years. In particular, the combination of a linear ion trap with the Orbitrap analyzer has proven to be a popular instrument configuration. Complementing this hybrid trap-trap instrument, as well as the standalone Orbitrap analyzer termed Exactive, we here present coupling of a quadrupole mass filter to an Orbitrap analyzer. This “Q Exactive” instrument features high ion currents because of an S-lens, and fast high-energy collision-induced dissociation peptide fragmentation because of parallel filling and detection modes. The image current from the detector is processed by an “enhanced Fourier Transformation” algorithm, doubling mass spectrometric resolution. Together with almost instantaneous isolation and fragmentation, the instrument achieves overall cycle times of 1 s for a top10 higher energy collisional dissociation method. More than 2500 proteins can be identified in standard 90-min gradients of tryptic digests of mammalian cell lysate— a significant improvement over previous Orbitrap mass spectrometers. Furthermore, the quadrupole Orbitrap analyzer combination enables multiplexed operation at the MS and tandem MS levels. This is demonstrated in a multiplexed single ion monitoring mode, in which the quadrupole rapidly switches among different narrow mass ranges that are analyzed in a single composite MS spectrum. Similarly, the quadrupole allows fragmentation of different precursor masses in rapid succession, followed by joint analysis of the higher energy collisional dissociation fragment ions in the Orbitrap analyzer. High performance in a robust benchtop format together with the ability to perform complex multiplexed scan modes make the Q Exactive an exciting new instrument for the proteomics and general analytical communities.

The Q Exactive HF, a Benchtop Mass Spectrometer with a Pre-filter, High-performance Quadrupole and an Ultra-high-field Orbitrap Analyzer

The quadrupole Orbitrap mass spectrometer (Q Exactive) made a powerful proteomics instrument available in a benchtop format. It significantly boosted the number of proteins analyzable per hour and has now evolved into a proteomics analysis workhorse for many laboratories. Here we describe the Q Exactive Plus and Q Exactive HF mass spectrometers, which feature several innovations in comparison to the original Q Exactive instrument. A low-resolution pre-filter has been implemented within the injection flatapole, preventing unwanted ions from entering deep into the system, and thereby increasing its robustness. A new segmented quadrupole, with higher fidelity of isolation efficiency over a wide range of isolation windows, provides an almost 2-fold improvement of transmission at narrow isolation widths. Additionally, the Q Exactive HF has a compact Orbitrap analyzer, leading to higher field strength and almost doubling the resolution at the same transient times. With its very fast isolation and fragmentation capabilities, the instrument achieves overall cycle times of 1 s for a top 15 to 20 higher energy collisional dissociation method. We demonstrate the identification of 5000 proteins in standard 90-min gradients of tryptic digests of mammalian cell lysate, an increase of over 40% for detected peptides and over 20% for detected proteins. Additionally, we tested the instrument on peptide phosphorylation enriched samples, for which an improvement of up to 60% class I sites was observed.

Development of a GC/Quadrupole-Orbitrap Mass Spectrometer,Part I: Design and Characterization

Identification of unknown compounds is of critical importance in GC/MS applications (metabolomics, environmental toxin identification, sports doping, petroleomics, and biofuel analysis, among many others) and remains a technological challenge. Derivation of elemental composition is the first step to determining the identity of an unknown compound by MS, for which high accuracy mass and isotopomer distribution measurements are critical. Here, we report on the development of a dedicated, applications-grade GC/MS employing an Orbitrap mass analyzer, the GC/Quadrupole-Orbitrap. Built from the basis of the benchtop Orbitrap LC/MS, the GC/Quadrupole-Orbitrap maintains the performance characteristics of the Orbitrap, enables quadrupole-based isolation for sensitive analyte detection, and includes numerous analysis modalities to facilitate structural elucidation. We detail the design and construction of the instrument, discuss its key figures-of-merit, and demonstrate its performance for the characterization of unknown compounds and environmental toxins.