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

Mass spectrometry-based proteomics often involves the analysis of complex mixtures of proteins derived from cell or tissue lysates or from body fluids, posing tremendous analytical challenges (1–3). After proteolytic digestion, the resulting peptide mixtures are separated by liquid chromatography and online electrosprayed for mass spectrometric (MS) and tandem mass spectrometric (MS/MS) analysis. Because tens of thousands of peptides elute over a relatively short time and with ion signals different by many orders of magnitude (4, 5), mass spectrometers have been pushed to even higher sensitivity, sequencing speed, and resolution (6, 7). In current shotgun proteomics there are mainly four mass spectrometric separation principles: quadrupole mass filters, time of flight (TOF)1 mass analyzers, linear ion traps, and Orbitrap™ analyzers. These are typically combined in hybrid configurations. Quadrupole TOF instruments use a quadrupole mass filter to either transmit the entire mass range produced by the ion source (for analysis of all ions in MS mode) or to transmit only a defined mass window around a precursor ion of choice (MS/MS mode). In the latter case ions are activated in a collision cell and resulting fragments are analyzed in the TOF part of the instrument with very high repetition rate. This TOF part of quadrupole TOF instruments replaces the final quadrupole section of triple quadrupole instruments, which are today mainly used for targeted proteomics (8–10). The quadrupole TOF instruments achieve peptide separation “in space”, meaning the ions are separated nearly instantaneously by passing through either the quadrupole section, in which only a chosen small mass range has stable trajectories, or by traversing the TOF section. In contrast, trapping instruments such as linear ion traps separate ions “in time” by applying external RF-DC fields to a stationary ion population that allow only a certain ion population to stably remain in the trap (see ref (11)) for the concept of separation and fragmentation in time versus in space). The Orbitrap mass analyzer was developed about ten years ago by Makarov. It consists of a small electrostatic device into which ion packets are injected at high energies to orbit around a central, spindle-shaped electrode (12–14). The image current of the axial motion of the ions is picked up by the detector and this signal is Fourier transformed (FT) to yield high resolution mass spectra. Commercially, the Orbitrap analyzer was first introduced in 2005 in a hybrid instrument (15). In proteomics and related fields, this combination of a low resolution linear ion trap with the high resolution Orbitrap analyzer—termed “LTQ Orbitrap”—has now become widespread (16, 17). The LTQ Orbitrap instruments represent a multistage trap combination (Fig. 1). In MS mode the linear trap performs the function of collecting the ion population, passing them on to an intermediate C-trap for injection and analysis in the Orbitrap analyzer at high resolution. In MS/MS mode the linear ion trap only retains a chosen mass window, which is activated by a supplemental RF field leading to fragmentation of the trapped precursor ions, and records the signal of a mass dependent scan at low resolution. Note that the high resolution MS scan can be performed at the same time as the low resolution MS/MS scans in the linear ion trap. Recently, an improved linear ion trap Orbitrap analyzer combination termed “LTQ Orbitrap Velos” has been introduced (18). It features an S-lens with up to 10-fold improved ion transmission from the atmosphere, a dual linear ion trap, and a more efficient Higher energy Collisional Dissociation (HCD) cell interfaced directly to the C-trap (18). HCD fragmentation is similar to the fragmentation in triple quadrupole or quadrupole TOF instruments and its products are analyzed with high mass accuracy in the Orbitrap analyzer (19). Thus, the LTQ Orbitrap or LTQ Orbitrap Velos instruments offer versatile fragmentation modes depending on the analytical problem (20–22). Fig. 1. Mass spectrometers incorporating an Orbitrap analyzer. The Exactive is a standalone instrument without mass selection. The total ion population is collected in the C-trap and injected into the Orbitrap analyzer (see text and Fig. 2 for details on detector ... Taking advantage of the small size of the Orbitrap analyzer a standalone benchtop instrument termed “Exactive” has been introduced mainly for small molecule applications. However, because of the absence of mass selection, its use in proteomics is limited to non-mass selective fragmentation of the entire mass range (called “All Ion Fragmentation” (AIF) on this instrument (23)). The combination of a quadrupole mass filter with an Orbitrap analyzer has not yet been reported. We reasoned that such a quadrupole trap combination might offer unique and complementary advantages to the hybrid mass spectrometers described above. In particular, a quadrupole Exactive instrument or “Q Exactive” would be able to select ions virtually instantaneously because of the fast switching times of quadrupoles and it would be able to fragment peptides in HCD mode on a similarly fast time scale. Furthermore, because of the small size and mature technology used in current quadrupole mass filters, this analyzer combination should have a small footprint and be particularly robust. Finally, the ability to separate “in space” and analyze MS and MS/MS ranges at high resolution in the Orbitrap analyzer offers the promise of enabling efficient multiplexed scan modes not currently applied in proteomics research using trapping instruments.