An instrument designed with your most difficult analytical challenges in mind, the Thermo Scientific Orbitrap Eclipse mass spectrometer incorporates the latest inventions in ion transmission and control, extended m/z range, and real-time decision making to expand the breadth of your work and push your science beyond today's discovery. This system is ideally suited for proteomics, structural biology, small-molecule, and biopharmaceutical characterization experiments.
Built on revolutionary Orbitrap Tribrid architecture, the Orbitrap Eclipse Tribrid mass spectrometer is a powerful, versatile mass spectrometry solution to help you accurately resolve subtle differences, to distinguish the right answer from many wrong ones, and to avoid costly dead ends.
High-Capacity Transfer Tube (HCTT)Increases ion flux into the mass spectrometer
Electrodynamic Ion FunnelFocuses ions after HCTT
EASY-IC/ETD/PTCR Ion SourceBased on Townsend discharge; reliable and easy to use
Advanced Active Ion Beam GuidePrevents contaminants from entering the mass resolving quadrupole
QR5 Segmented Quadrupole Mass Filter with Hyperbolic SurfacesImproves sensitivity with 0.4 m/z precursor isolation width
Ultra-High-Field Orbitrap Mass AnalyzerOffers resolution >500,000 FWHM (optionally > 1,000, 000 FWHM) and MSn acquisition rate up to 40 Hz; enables TurboTMT and HMRn
Ultra-High Vacuum (UHV) ManifoldReduces pressure in the UHV region; improves ion detection in the Orbitrap mass analyzer
Ion-Routing MultipoleEnable higher acquisition rates; performs HCD at any MSn stage; allows variable pressure (0.5–20 mTorr) for superior top-down performance
Real-Time SearchProvides on-the-fly peptide identification, increasing depth and accuracy of TMT quantitation
Modified Dual-Pressure Linear Ion TrapEnables MSn for ion detection in both ion trap and Orbitrap mass analyzers; sensitive mass analysis for multiple fragmentation modes: CID, HCD, ETD/EThcD/ETciD and UVPD; and precursor ion isolation for HMRn. The extended front section of the high-pressure cell improves control over ETD and PTCR reactions.
Comprehensive characterization of single-cell proteomes can provide a wealth of novel information about cellular development in the context of disease progression and in response to treatment as a function of cell type. Yet, proteome analysis with single-cell resolution remains an enormous challenge due to the analytical sensitivity this experiment demands. The Orbitrap Eclipse Tribrid mass spectrometer was developed with the capability to extract unrivaled quantitative data from ultra-low-level samples, including from individual cells.
The MS3-based Tandem Mass Tag (TMT) method, enhanced by Real-Time Search, provides the throughput and sensitivity to achieve the proteome coverage and the quantitative accuracy needed to differentiate cell types and to capture their heterogeneity. Further, using novel Thermo Scientific TMTpro 16plex Label Reagent Set, up to 16 single cells can be analyzed in one LC-MS run, providing quantitative comparison of thousands of proteins among individual cells.
TMT analysis of two cell types revealed more differentially expressed proteins when using SPS MS3 with Real-Time Search compared to the MS2-based experiment. The MS3 experiment allows for more accurate quantitation, enabling detection of subtle changes in more proteins for each individual cell.
PCA plot showing unsupervised classification of the three cell types using TMT data. Each point corresponds to the protein expression of a single cell. The plot shows clear classification of cell types and resolved heterogeneity within each type. This level of resolution is uniquely achievable using a combination of TMT-boost** and SPS MS3 with Real-Time Search data acquisition technologies.
* See below for details on TMT and Real-Time Search
** Budnik et al., Genome Biol. 2018,19(1); Zhu et al., 2018, Nature Comm., 2018, 9(882).
*** A total of 40 individual cells were analyzed in this study.
"A revolution in single-cell proteomics is just beginning. The combination of nanoPOTS with the Orbitrap Eclipse Tribrid mass spectrometer, TMT reagents, and SPS MS 3 with Real-Time Search provide the depth of coverage, quantitative accuracy, and throughput needed to propel this nascent field forward.”
Ryan Kelly, Professor, Brigham Young University, UT
At Thermo Fisher Scientific, we’re proud to partner with life science innovators who are transforming the future of human health. Every single day.
The standard for high-throughput quantitative comparisons of protein abundances is the TMT SPS MS3 workflow, unique to Orbitrap Tribrid mass spectrometers. A significant advancement of the Orbitrap Eclipse Tribrid mass spectrometer is Real-Time Search1, which can be used to identify peptide spectra on-the-fly to intelligently direct MS3 data acquisition, resulting in accurate quantitation to depths often exceeding 8,000 proteins in up to 16 samples per LC/MS analysis.
This new high-throughput workflow offers increased proteome coverage with improved accuracy and precision, boosting the number of quantifiable low-level peptides.
1. Erickson et al. J Proteome Res. 2019, 18(3).
Base peak extracted ion chromatogram of the multiplexed TMT sample. The shaded region in the LC-MS analysis image below highlights a selected MS spectrum.
When analyzing complex peptide mixtures, coisolated ion interferences can occur despite using narrow precursor isolation. Co-fragmentation of precursors of interest and interfering ions negatively impacts the quantitative accuracy of the TMT-based experiment.2
2. Ting et al. Nat. Methods, 2011, 8(11).
Every MS2 spectrum is interrogated against a database of choice using Real-Time Search in parallel with the acquisition of the next MS2 scan. If the search results in a peptide match, the instrument is directed to perform an SPS MS3 scan using only the matched fragment ions that carry the TMT tags, while avoiding any fragments that may have originated from the interfering ions. For YLYEIAR, the TMT tag was found only on the N-terminus, so the SPS MS3 is performed only on five b-ions.
With Real-Time Search data acquisition, MS3 scans are only triggered if a peptide-spectrum match (PSM) is identified from the preceding MS2. This reduces the number of MS3 events, increases the precursor sampling rate and results in a greater number of quantified peptides; improving experimental speed.
With Real-Time Search, 38% more peptides and 53% more proteins were quantified versus a classic SPS MS3 experiment, approaching the results of the classic MS2 experiment, but with much higher precision and accuracy. Shown here are data for a TKO yeast standard (Thermo Fisher Scientific).
Real-Time Search directs the mass spectrometer to perform MS3 only on identified precursors. As a result, it doubles the throughput of SPS MS3 experiments. Shown here are data for an HHM sample: three human cell lines labeled as biological replicates in 10plex (3-3-4). (Data courtesy Devin Schweppe and Qing Yu, Harvard Medical School)
For each TMT channel, 4–100 pmol of a six-protein digest was added to 40 µg of HeLa digest, and then labeled, resulting in a spiked-in standard mixed in ratios of 2–24 (2, 4, 8, 12, 16 and 24). Only combined averages for the expected ratios of 2 and 24 are shown here. Higher accuracy afforded by Real-Time Search is indispensable for teasing out subtle differences in protein abundances, including when sub-classifying individual cells.
"The Orbitrap Eclipse Tribrid mass spectrometer provides several exciting advances which allow us to perform our analyses 100% faster with significantly improved quantification accuracy. Instead of 36 hours to perform a typical proteome-wide analysis, we can accomplish it now in 18 hours to reach between 8,000 and 10,000 quantified proteins in as many as 16 samples. This new mass spectrometer acts as if it were two instruments, collecting more accurate data in half the time, but at the same or even better depth.”
Steven P. Gygi, Professor, Harvard University, MA
Therapeutic proteins are successfully used to treat various cancers and a wide range of autoimmune diseases. However, their structural characterization presents a significant challenge because, unlike small molecule-based drugs, they exist as a heterogeneous mixture containing numerous modified proteoforms. Their highly complex ESI spectra can be simplified and made interpretable by increasing analyte m/z. This can be achieved by performing the ESI LC-MS experiment under native conditions where unfolded proteins accept fewer charges, or by performing precursor ion charge reduction in the mass spectrometer, or by both. For complete structural characterization, the mass spectrometer is required to not only detect ions within a higher m/z range, but to also fragment the selected precursor efficiently. The Orbitrap Eclipse Tribrid mass spectrometer is equipped with High Mass Range MSn (HMRn), Proton Transfer Charge Reduction (PTCR) and a multitude of dissociation techniques, including CID, HCD, ETD, EThcD and UVPD, making it the most powerful system available for comprehensive characterization of therapeutic proteins.
Native MS spectrum of intact desalinated Cytokine Fc-fusion protein after sialidase treatment containing six N-linked glycosylation sites. The raw spectrum was highly complex due to the presence of multiple overlapping glycosylated proteoforms, making it challenging to interpret. The highlighted 80 m/z window represents the precursor ions that were isolated for subsequent PTCR.
After deconvolution of the PTCR spectrum shown (ReSpect™ algorithm), several glycoforms of Cytokine Fc-fusion protein were identified. PTCR analysis of the entire native charge state envelope (m/z 4,000–7,000) resulted in the identification of >30 distinct glycoforms of this protein (data not shown)
Multiple dissociation techniques available on the Orbitrap Eclipse MS can provide complementary information about the primary sequence, resulting in 100% coverage across the CDR3 motifs, increasing the confidence in antibody characterization and providing insight into its reactivity with different antigens at a proteoform level.
Combined fragmentation maps of intact trastuzumab from the native HCD, ETD, EThcD and UVPD MS2 data. Sequence coverage for light and heavy chains was 58% and 36% respectively, with combined coverage of 43% (fragment ion RMS error <3.7 ppm). Complementarity Determining Regions (CDR) 3 of the light and heavy chains are highlighted in red and blue respectively in the maps, showing 100% sequence coverage. Validation of CDR sequences is essential for studying binding affinity and efficacy of the antibody.
Ligands are inextricably linked to the regulation and function of the proteins to which they are bound. However, determining the identity of small molecule ligands associated with membrane proteins is a significant challenge, underscored by the high prevalence of electron microscopy data with unassigned or poorly resolved ligand density. Direct MS identification of the ligands without losing ligand-protein complex associations is difficult because it requires a very wide mass range while retaining optimal ion transmission of intact protein assemblies of (hundreds of thousands of Daltons) and accurate detection of small-molecule ligands and their fragments (often smaller than 100 Daltons).
The High Mass Range MSn capability of the Orbitrap Eclipse Tribrid mass spectrometer facilitates identification of ligands bound to membrane proteins, allowing ellucidation of specific ligands interacting with specific proteins. This approach helps to elucidate a ligand’s influence on the cascade of protein interactions that underlie cellular mechanisms, including mechanisms of cancer, diabetes, and Alzheimer’s.
MS3-based isolation and activation of the intact membrane protein-ligand assembly promote the release of the ligand (m/z 350.0). This singly-charged ion is isolated (gray bar) for MS4 step to allow full structural characterization. (Note: in this experiment the ligand at m/z 350.0 was detected in the ion trap. Both the Orbitrap and the linear ion trap mass analyzers can be used to detect ions across the entire m/z range).
The MS4 spectrum contains ligand fragments detected in the ion trap. Unambiguous identification of the endogenous ligand bound to the protein is performed using established small-molecule characterization techniques. Here, ampicillin was identified bound to E. coli Outer Membrane Porin F (OmpF), forming a 111 kDa protein-ligand complex that mediates its intake. Further characterization of the protein’s primary structure is also possible using this novel top-down MSn approach.
"The Orbitrap Eclipse Tribrid mass spectrometer brings a new dimension to native MS, enabling us to discover and chemically define the ligands, lipids, and drugs that regulate the function of membrane protein assemblies within one single experiment.”
Dr. Joseph Gault, University of Oxford, the UK
PTCR formed lower charge state distributions of the parent ions that overlapped less and were easier to interpret. This increased the number of identifiable precursor ions from two species in the original full MS spectrum to 32 species identified after PTCR of a 3 m/z range. The newly identified masses were distributed over 10–70 kDa range (see left histogram below).
Top-down mass spectrometry is used to directly characterize intact proteoforms. The ESI spectral complexity of proteoform mixtures, even after LC separation, is often very high due to the multitude of modified forms that overlap in the m/z domain. Proton Transfer Charge Reduction (PTCR) technology, unique to the Orbitrap Eclipse Tribrid mass spectrometer, reduces the average charge of the parent-ion distributions, shifting them to higher m/z. For overlapping indistinguishable proteoforms this reduces the signal overlap, and, as a result, easier-to-interpret spectra are obtained, enabling the proteoform mass calculation. For automated top-down experiments, this approach increases the number of distinguishable precursors available for data-dependent MSn, ultimately increasing the number of characterized proteoforms.
The co-elution of multiple proteoforms increases spectral complexity, particularly when analyzing proteins >25 kDa. These spectra are often impossible to interpret fully as few charge state distributions are distinguishable by deconvolution software (e.g., only two distinct masses of 16 and 25 kDa were identified in this MS spectrum at 25 min). The 800–900 m/z region shows the underlying spectral complexity. PTCR was performed on the precursors in two 1.5 m/z windows, highlighted in gray and teal.
Huguet, R. et al. Anal. Chem.2019, 91(24).
"PTCR is required to study large proteoforms that previously would remain uncharacterized in LC-MS experiments. Unveiling the true complexity of the intact proteome beyond the 30 kDa barrier represents a substantial step forward for the proteomics community.”
Luca Fornelli, Professor, University of Oklahoma, OK
The intuitive method editor features a drag-and-drop user-friendly interface with predefined, optimized method templates for a wide range of applications. This allows scientists to generate high-quality data easily for the most popular experiments, such as Glycosylation, Metabolites, Peptide ID, Peptide Quantitation, PTM, Advanced TMT, SureQuant and more.
The common method editor combines both instrument control and data acquisition parameters, through the use of extensive templates for the most common experiments to allow you to acquire high-quality data from the start, or use drag-and-drop to rapidly create and visualize your own experiments.
The Thermo Scientific FAIMS Pro Duo interface extends orthogonal selectivity to a broad range of applications, increasing experimental signal-to-noise ratios, expanding sample coverage, and maximizing throughput while reducing time-consuming sample preparation steps.
Identifying, characterizing, and quantifying compounds of interest by mass spectrometry relies on the acquisition of high-quality MS and MSn data in increasingly complex samples. The FAIMS Pro Duo interface improves the analytical performance of next-generation mass spectrometers through gas-phase fractionation resulting in selectivity enhancement of target compound classes. The reduction in spectral complexity and increased signal-to-noise ratios for target analytes results in greater sample coverage and higher data confidence, regardless of the chromatographic flow rates or loading amounts.
Improve mass-to-charge (m/z) ratio assignment accuracy to less than 1 ppm with the Thermo Scientific Easy-IC ion source, which incorporates a secondary ion source that delivers a regulated number of calibrant ions into the analyte ions. This feature enables real-time fine adjustment of the instrument's m/z calibration, correcting otherwise uncompensated errors due to temperature fluctuations and scan-to-scan variations.
Resolve isobaric components, obtain fine isotope information, and achieve confident assignment of the elemental composition of small molecule analytes with the ultra-high 1 Million (1M) resolution setting of the Orbitrap Eclipse Tribrid Mass Spectrometer. The 1M resolution option aids the characterization and quantitation of structurally diverse targets by obtaining mass measurements at ultra-high resolution (1,000,000 FWHM at m/z 200).
A mass spectrum of Irganox 1035 acquired at the 1,000,000 (1M) resolution setting (m/z 200) with a 2 s transient. The right panel shows the fine structure for the A+2 isotope acquired at the 500,000 versus 1,000,000 resolution setting. The 18O and 2 13C isotopes can be resolved using the Orbitrap Fusion Lumos and/or Orbitrap Eclipse mass spectrometers with 1M.
Achieve unambiguous characterization of lipids, modified peptides and intact proteins, even in complex mixtures, and obtain unique structurally diagnostic information (e.g., double bonds) that cannot be obtained with CID, HCD, ETD or EThcD. UVPD enhances unique sequence coverage over 20%, generating MSn fragments in the linear ion trap that can be detected by either the ion trap or Orbitrap mass analyzer.
Using the Thermo Scientific Almanac web- and smartphone-based application gives you the ability to remotely access and manage your LC-MS systems, freeing you to focus on other important tasks during your day.
Check real-time system status and acquisitions, set up automated e-mails to notify you of a completed acquisition or error, schedule instrument access, monitor utilization, or send service files to aid in system diagnostics and maximize up-time.
Connect your laboratory: Always know what’s going on within your laboratory with any cloud-connected device. Maximize system utilization with long-term trending of system usage and sample throughput, through to checking data acquisition and multiple other productivity tools.
Proteomics research requires more than just simple identification. Thermo Scientific Proteome Discoverer software offers a full suite of analysis tools with the flexibility to address multiple research workflows, and an easy-to-use, wizard-driven interface. Confidently interpret your data with sophisticated statistical and visualization tools unique to Proteome Discoverer software, and utilize third-party algorithms to take your analysis to the next level.
Recommended for the following workflows:
Thermo Scientific ProSightPD software adds support for middle-down and bottom-up experiments, making it an all-around good tool for identification and characterization of both intact proteins and peptides.
Complete protein characterization can be challenging, whether you are performing intact protein mass analysis, top- and middle-down analysis, peptide mapping or multi-attribute method (MAM) workflows. Access workflows to facilitate comprehensive interpretation and data visualization, confidently characterizing your biologics with speed and ease. Thermo Scientific BioPharma Finder software helps you choose the right path for confident protein characterization.
Recommended for the following workflows:
Thermo Scientific Compound Discoverer software streamlines unknown identification, determination of real differences between samples, and elucidation of biological pathways
Thermo Scientific Mass Frontier Spectral Interpretation Software helps simplify management, evaluation, and interpretation of mass spectral data to provide greater insights.
By collaborating with the scientific community, Thermo Fisher Scientific has developed innovations that allow you to perform single-cell proteomics analysis. Access our collection of materials dedicated to single-cell proteomics, including white papers, webinars, poster notes, and more.
A well-thought-out and detailed grant proposal is essential. Our technical experts are with you every step of the way in the grant-writing process to help you reach new heights.
The Thermo Scientific AccelerOme automated sample preparation platform, with factory-supplied reagents and kits, intuitive software and a unique experimental design experience transforms proteomics sample preparation.
Our LC-MS on-demand webinar series expands your application knowledge for pharma, biopharma, environmental, omics, food, forensics and clinical research and more. Learn how the latest high-performance mass spectrometry solutions can enable your laboratory to solve your most pressing analytical challenges, regardless of sample type.