To mark Orbitrap’s tenth anniversary, Thermo Scientific’s Shannon Eliuk and Alexander Makarov recently reviewed the evolution of this prominent technology as well as its potential trajectory.1
Although the first Orbitrap-based mass spectrometer didn’t enter the market until 2005, the technological principles that underlay its conception date back to 1923, when Kingdon first described orbital trapping using an enclosed metal can and charged wire. The mass analyzers that eventually emerged all carried significant performance issues: the Fourier transform ion cyclotron resonance (FT-ICR) machines were large and complex; the orthogonal time-of-flight (TOF) instruments bore problems of sensitivity, dynamic range and resolution; and ion trap platforms offered limited mass accuracy.
This analytical reality brought the first incarnation of the Orbitrap mass analyzer to birth. Makarov’s goal was three-fold: enhance the trapping field, harness an external ion source for ion injection, and create a suitable detection scheme. To do this, he incorporated a spindle-shaped central electrode and two symmetrical outer electrodes. Then, he employed a compensation electrode (deflector) to minimize ion loss as a pulsed laser injected ions via an opening to the orbital trap. Once captured, these ions oscillated around the central electrode, and receiver plates detected the image current, allowing for the generation of a mass spectrum via Fourier transform and simple two-point calibration. This innovative device eventually received the moniker Orbitrap.
In its infancy, Orbitrap mass spectrometer technology had challenges to overcome before it could emerge as a true option for mass analysis—namely, poor control over the quantity of ions entering the trap, a propensity toward metastable decay of peptide ions, and inadequate ion transmission and mass range. The development of the C-trap external storage device, which allowed ions sourced from electrosprays to accumulate before injection into the Orbitrap mass analyzer, was another challenging piece.
Three families: LTQ Orbitrap, Q Exactive, and Orbitrap Fusion mass spectrometers
In 2005 Thermo Electron introduced the LTQ Orbitrap mass spectrometer. It had become clear that the research community would enthusiastically receive an instrument with tandem mass spectrometry (MS/MS) capability that met the then-current standard for resolution, mass accuracy and speed while eliminating the issues that came with FT-ICR machines (i.e., maintenance and space issues). This first commercial incarnation of Orbitrap technology combined high-resolution, accurate mass (HRAM) detection with the benefits of a linear ion trap: sensitive ion detection, precursor isolation and fragmentation capabilities. HRAM capability allowed researchers to detect multiply charged species in complex mixtures and improved database searching with accurate precursor mass detection. For discovery, scientists could harness this instrument’s full scan mode to compile a list of precursors suitable for MS/MS by collision-induced dissociation (CID) within a single run.
The LTQ Orbitrap mass spectrometer offered decent scan rates (4–5 Hz MS/MS with nominal mass detection and 3 Hz with accurate mass detection), narrow extraction windows, and high-resolution detection capable of resolving interference peaks for accurate quantitation, rendering it suitable for identifying even low-level analytes. Using automatic gain control (AGC), the instrument prescanned the linear trap to determine the appropriate number of ions to store to ensure high sensitivity without space-charging issues. The instrument’s limitations included only one fragmentation option (CID) that was not well-suited for modified peptides and a relatively slow accurate mass MS/MS scan rate.
The next step in Orbitrap mass spectrometer evolution, the LTQ Orbitrap XL mass spectrometer, reflected this focus on expanding fragmentation options as well as C-trap improvement for higher transmission. This involved the addition of a gas-filled quadrupole after the C-trap for higher energy collision-induced dissociation (HCD) and a reagent ion source behind the HCD cell for electron transfer dissociation (ETD). The manufacturer also developed an optimized data-dependent decision tree protocol to direct specific ions to CID or ETD on the basis of charge and m/z. The expansion of the Orbitrap mass spectrometer to include ETD was vital to the positioning of MS as the go-to technique for proteomics.
Indeed, the evolving needs of proteomics as a discipline led to the next goal of enabling direct analysis of complex samples without intensive pre-fractionation while also improving sensitivity and scan speeds. The 2009 release of the LTQ Orbitrap Velos hybrid mass spectrometer platform answered these challenges with enhanced sensitivity (3- to 5-fold in full scan and up to 10-fold in MS/MS) via the S-lens ion guide. The instrument also used a novel dual-pressure ion trap mass analyzer with a high-pressure cell for ion isolation and fragmentation and a low-pressure cell for accelerated ion analysis for faster acquisition without compromising spectral quality. These advances, plus predictive AGC, produced enhanced scan rates (up to 10 Hz MS/MS). In addition, as the trend in proteomics turned toward quantitative analysis to monitor subtle changes in protein abundance, the LTQ Orbitrap Velos mass spectrometer also received a relocated HCD cell to simplify the interface with the C-trap and a drag field in the HCD cell to aid in the ejection of ions from the cell.
True accessibility to Orbitrap mass analyzer technology came in the form of the Exactive mass spectrometer. The elimination of the ion trap mass analyzer reduced cost and complexity, producing a bench-top instrument that could perform full-scan detection and HCD without precursor selection. This led to the development of protocols for Orbitrap-only detection, and enabled HRAM screening of known and unknown analytes with very high selectivity (<5 ppm). It also facilitated retrospective analysis of full scan data. The Q Exactive hybrid quadrupole-Orbitrap mass spectrometer allowed precursor ion isolation via a mass filtering quadrupole and faster acquisition rates as a result of the S-lens, efficient quadrupole isolation and Orbitrap mass analyzer detection with enhanced Fourier transform (eFT; up to 12 Hz accurate mass MS/MS).
In 2011, the Orbitrap Elite hybrid ion trap-Orbitrap mass spectrometer introduced a compact, high-field Orbitrap mass analyzer later found on the Q Exactive HF and Orbitrap Fusion mass spectrometer platforms as well. This analyzer plus eFT effectively quadruples the resolving power when compared to the first commercially available machines. Additionally, a preview mode that generates precursors for partially parallelized data-dependent analysis maintains fast scan rates, providing deeper penetration, more complete characterizations and more overall identifications. The Fusion instrument combines ion trap Orbitrap and quadrupole Orbitrap hybrid technologies to enhance overall performance, including full parallelization and efficient, flexible implementation of complex modes to answer difficult structural questions. This machine also offers a top speed mode, which allows users to specify the cycle time when choosing precursors for data-dependent analysis.
The Orbitrap Fusion mass spectrometer employs HCD in either the ion trap or Orbitrap mass analyzers at any level of MSn. This plus a novel ETD reagent ion source render the instrument ideal for characterizing and profiling small molecules for –omics studies (glycomics, metabolomics, lipidomics, etc) and also makes it easier to maintain and tune. Other advances include the option to preferentially select ions with greater susceptibility to ETD to increase acquisition rates and to use synchronous precursor selection (SPS) to enhance sensitivity when using TMT MS3. Data-independent acquisition modes for the identification and quantification of peptides in complex mixtures continues to be developed on the Orbitrap Fusion mass spectrometer.
Looking toward the future, Eliuk and Makarov highlight a few potential areas of advancement that are, like the historical changes, drawn from research feedback:
Continued upgrading to attain previously out of reach resolving powers sufficient for demanding tasks like element counting, NeuCode quantitation, and petroleomics.
Further advances in high m/z mass selection and innovative top-down techniques for native protein complexes.
Potential for signal processing methods to move resolving power and quantitation capacity beyond the limit of Fourier transformation technology (although possibly with increased signal-to-noise ratios).
Broader menu options for ion sources (e.g., electron impact, inductively coupled plasma, glow discharge) for new applications.
Altogether, the rich history and development trajectory of this flexible technology position it as a key partner for the scientific community.
1 Eliuk, S. and Makarov, A. (2015) “Evolution of Orbitrap mass spectrometry instrumentation,” The Annual Review of Analytical Chemistry, 8 (pp. 61–80), doi: 10.1146/annurev-anchem-071114-040325