Alexander Makarov

Parts per Million Mass Accuracy on an Orbitrap Mass Spectrometer via Lock Mass Injection into a C-trap

Mass accuracy is a key parameter of mass spectrometric performance. TOF instruments can reach low parts per million, and FT-ICR instruments are capable of even greater accuracy provided ion numbers are well controlled. Here we demonstrate sub-ppm mass accuracy on a linear ion trap coupled via a radio frequency-only storage trap (C-trap) to the orbitrap mass spectrometer (LTQ Orbitrap). Prior to acquisition of a spectrum, a background ion originating from ambient air is first transferred to the C-trap. Ions forming the MS or MSn spectrum are then added to this species, and all ions are injected into the orbitrap for analysis. Real time recalibration on the “lock mass” by corrections of mass shift removes mass error associated with calibration of the mass scale. The remaining mass error is mainly due to imperfect peaks caused by weak signals and is addressed by averaging the mass measurement over the LC peak, weighted by signal intensity. For peptide database searches in proteomics, we introduce a variable mass tolerance and achieve average absolute mass deviations of 0.48 ppm (standard deviation 0.38 ppm) and maximal deviations of less than 2 ppm. For tandem mass spectra we demonstrate similarly high mass accuracy and discuss its impact on database searching. High and routine mass accuracy in a compact instrument will dramatically improve certainty of peptide and small molecule identification.

Higher-energy C-trap dissociation for peptide modification analysis

Peptide sequencing is the basis of mass spectrometry–driven proteomics. Here we show that in the linear ion trap–orbitrap mass spectrometer (LTQ Orbitrap) peptide ions can be efficiently fragmented by high-accuracy and full-mass-range tandem mass spectrometry (MS/MS) via higher-energy C-trap dissociation (HCD). Immonium ions generated via HCD pinpoint modifications such as phosphotyrosine with very high confidence. Additionally we show that an added octopole collision cell facilitates de novo sequencing.

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.

Dynamic range of mass accuracy in LTQ orbitrap hybrid mass spectrometer

Using a novel orbitrap mass spectrometer, the authors investigate the dynamic range over which accurate masses can be determined (extent of mass accuracy) for short duration experiments typical for LC/MS. A linear ion trap is used to selectively fill an intermediate ion storage device (C-trap) with ions of interest, following which the ensemble of ions is injected into an orbitrap mass analyzer and analyzed using image current detection and fast Fourier transformation. Using this technique, it is possible to generate ion populations with intraspectrum intensity ranges up to 104. All measurements (including ion accumulation and image current detection) were performed in less than 1 s at a resolving power of 30,000. It was shown that 5-ppm mass accuracy of the orbitrap mass analyzer is reached with >95% probability at a dynamic range of more than 5000, which is at least an order of magnitude higher than typical values for time-of-flight instruments. Due to the high resolving power of the orbitrap, accurate mass of an ion could be determined when the signal was reliably distinguished from noise (S/Np-p>2…3).

Ultra high resolution linear ion trap Orbitrap mass spectrometer (Orbitrap Elite) facilitates top down LC MS/MS and versatile peptide fragmentation modes

Although only a few years old, the combination of a linear ion trap with an Orbitrap analyzer has become one of the standard mass spectrometers to characterize proteins and proteomes. Here we describe a novel version of this instrument family, the Orbitrap Elite, which is improved in three main areas. The ion transfer optics has an ion path that blocks the line of sight to achieve more robust operation. The tandem MS acquisition speed of the dual cell linear ion trap now exceeds 12 Hz. Most importantly, the resolving power of the Orbitrap analyzer has been increased twofold for the same transient length by employing a compact, high-field Orbitrap analyzer that almost doubles the observed frequencies. An enhanced Fourier Transform algorithm—incorporating phase information—further doubles the resolving power to 240,000 at m/z 400 for a 768 ms transient. For top-down experiments, we combine a survey scan with a selected ion monitoring scan of the charge state of the protein to be fragmented and with several HCD microscans. Despite the 120,000 resolving power for SIM and HCD scans, the total cycle time is within several seconds and therefore suitable for liquid chromatography tandem MS. For bottom-up proteomics, we combined survey scans at 240,000 resolving power with data-dependent collision-induced dissociation of the 20 most abundant precursors in a total cycle time of 2.5 s—increasing protein identifications in complex mixtures by about 30%. The speed of the Orbitrap Elite furthermore allows scan modes in which complementary dissociation mechanisms are routinely obtained of all fragmented peptides.

High-sensitivity Orbitrap mass analysis of intact macromolecular assemblies

The analysis of intact protein assemblies in native-like states by mass spectrometry offers a wealth of information on their biochemical and biophysical properties. Here we show that the Orbitrap mass analyzer can be used to measure protein assemblies of molecular weights approaching one megadalton with sensitivity down to the detection of single ions. Minor instrumental modifications enabled the measurement of various protein assemblies with outstanding mass-spectral resolution.

Orbitrap mass spectrometry.

Orbitrap is the newest addition to the family of high-resolution mass spectrometry analyzers. With its revolutionarily new, miniature design, Orbitrap combines high speed with excellent quantification properties, ranking favorably in many analytical applications.

Coupling liquid chromatography to Orbitrap mass spectrometry.

The Orbitrap mass analyzer has become a mainstream mass spectrometry technique. In addition to providing a brief introduction to the Orbitrap technology and its continuing development, this article reviews the most recent publications quoting the use of the Orbitrap detection for a variety of chromatographic separation techniques. Its coupling to reversed-phase liquid chromatography (LC) represents undoubtedly the most ubiquitous approach to both small molecule and proteomic analyses. Multi-dimensional LC separations have an important role to play in the proteomics applications while an ultra-high-pressure LC is more frequently encountered in the area of metabolomics and metabolite analysis. Recently, special chromatographic techniques such as hydrophilic interaction chromatography and its variations have also been also cited with the Orbitrap detection.

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.

Performance Evaluation of a High-field Orbitrap Mass Analyzer

A new design of the Orbitrap™ mass analyzer is presented. Higher frequencies of ion oscillations and hence higher resolving power over fixed acquisition time are achieved by decreasing the gap between the inner and outer Orbitrap electrodes, thus providing higher field strength for a given voltage. Experimental results confirm maximum FWHM resolving power in excess of 350,000 at m/z 524 and 600,000 at m/z 195, isotopic resolution of proteins above 40 kDa, and a single-shot dynamic range of 25,000. It was also found that mass shifts in the new design depend very little on space charge inside the analyzer. This performance was achieved using higher voltages and by careful balancing of construction tolerances and operation parameters, which appeared to vary in narrower ranges of tuning than for a standard Orbitrap analyzer.