The Impact II,a Very High-Resolution Quadrupole Time-of-Flight Instrument (QTOF) for Deep Shotgun Proteomics

Hybrid quadrupole time-of-flight (QTOF) mass spectrometry is one of the two major principles used in proteomics. Although based on simple fundamentals, it has over the last decades greatly evolved in terms of achievable resolution, mass accuracy, and dynamic range. The Bruker impact platform of QTOF instruments takes advantage of these developments and here we develop and evaluate the impact II for shotgun proteomics applications. Adaption of our heated liquid chromatography system achieved very narrow peptide elution peaks. The impact II is equipped with a new collision cell with both axial and radial ion ejection, more than doubling ion extraction at high tandem MS frequencies. The new reflectron and detector improve resolving power compared with the previous model up to 80%, i.e. to 40,000 at m/z 1222. We analyzed the ion current from the inlet capillary and found very high transmission (>80%) up to the collision cell. Simulation and measurement indicated 60% transfer into the flight tube. We adapted MaxQuant for QTOF data, improving absolute average mass deviations to better than 1.45 ppm. More than 4800 proteins can be identified in a single run of HeLa digest in a 90 min gradient. The workflow achieved high technical reproducibility (R2 > 0.99) and accurate fold change determination in spike-in experiments in complex mixtures. Using label-free quantification we rapidly quantified haploid against diploid yeast and characterized overall proteome differences in mouse cell lines originating from different tissues. Finally, after high pH reversed-phase fractionation we identified 9515 proteins in a triplicate measurement of HeLa peptide mixture and 11,257 proteins in single measurements of cerebellum—the highest proteome coverage reported with a QTOF instrument so far.Building on the fundamental advance of the soft ionization techniques electrospray ionization and matrix-assisted laser desorption/ionization (1, 2), MS-based proteomics has advanced tremendously over the last two decades (3–6). Bottom-up, shotgun proteomics is usually performed in a liquid chromatography-tandem MS (LC-MS/MS)¹ format, where nanoscale liquid chromatography is coupled through electrospray ionization to an instrument capable of measuring a mass spectrum and fragmenting the recognized precursor peaks on the chromatographic time scale. Fundamental challenges of shotgun proteomics include the very large numbers of peptides that elute over relatively short periods and peptide abundances that vary by many orders of magnitude. Developments in mass spectrometers toward higher sensitivity, sequencing speed, and resolution were needed and helped to address these critical challenges (7, 8). Especially the introduction of the Orbitrap mass analyzers has advanced the state of the art of the field because of their very high resolution and mass accuracy (9, 10). A popular configuration couples a quadrupole mass filter for precursor selection to the Orbitrap analyzer in a compact benchtop format (11–13).In addition to the improvements in MS instrumentation, there have been key advances in the entire proteomics workflow, from sample preparation through improved LC systems and in computational proteomics (14–16). Together, such advances are making shotgun proteomics increasingly comprehensive and deep analyses can now be performed in a reasonable time (13, 17–19). Nevertheless, complete analysis of all expressed proteins in a complex system remains extremely challenging and complete measurement of all the peptides produced in shotgun proteomics may not even be possible in principle (20, 21). Therefore, an urgent need for continued improvements in proteomics technology remains.Besides the Orbitrap analyzer and other ion trap technologies, the main alternative MS technology is time-of-flight, a technology that has been used for many decades in diverse fields. The configuration employed in proteomics laboratories combines a quadrupole mass filter via a collision cell and orthogonal acceleration unit to a reflectron and a multichannel plate (MCP) detector (22). TOF scans are generated in much less than a millisecond (ms), and a number of these “pulses” are added to obtain an MS or MS/MS spectrum with the desired signal to noise ratio. Our own laboratory has used such a quadrupole time-of-flight (QTOF) instrument as the main workhorse in proteomics for many years, but then switched to high-resolution trapping instruments because of their superior resolution and mass accuracy. However, TOF technology has fundamental attractions, such as the extremely high scan speed and the absence of space charge, which limits the number of usable ions in all trapping instruments. In principle, the high spectra rate makes TOF instruments capable of making use of the majority of ions, thus promising optimal sensitivity, dynamic range and hence quantification. It also means that TOF can naturally be interfaced with ion mobility devices, which typically separate ions on the ms time scale. Data independent analysis strategies such as MS^E, in which all precursors are fragmented simultaneously (23, 24) or SWATH, in which the precursor ion window is rapidly cycled through the entire mass range (25), also make use of the high scanning speed offered by QTOF instruments. It appears that QTOFs are set to make a comeback in proteomics with recent examples showing impressive depth of coverage of complex proteomes. For instance, using a variant of the MS^E method, identification of 5468 proteins was reported in HeLa cells in single shots and small sample amounts (26). In another report, employing ion mobility for better transmission of fragment ions to the detector led to the identification of up to 7548 proteins in human ovary tissue (27).In this paper, we describe the impact II™, a benchtop QTOF instrument from Bruker Daltonics, and its use in shotgun proteomics. This QTOF instrument is a member of an instrument family first introduced in 2008, which consists of the compact, the impact, and the maXis. The original impact was introduced in 2011 and was followed by the impact HD, which was equipped with a better digitizer, expanding the dynamic range of the detector. With the impact II, which became commercially available in 2014, we aimed to achieve a resolution and sequencing speed adequate for demanding shotgun proteomics experiments. To achieve this we developed an improved collision cell, orthogonal accelerator scheme, reflectron, and detector. Here we measure ion transmission characteristics of this instrument and the actually realized resolution and mass accuracy in typical proteomics experiments. Furthermore, we investigated the attainable proteome coverage in single shot analysis and we ask if QTOF performance is now sufficient for very deep characterization of complex cell line and tissue proteomes.