Orbitrap Eclipse Tribrid Mass Spectrometer

Go beyond today's discovery

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.

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Explore the latest innovations to Thermo Scientific Orbitrap Tribrid technology that enable industry leading performance while delivering experimental versatility and usability for all user levels.


Look inside the Orbitrap Eclipse MS

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.

How the Orbitrap Eclipse Tribrid MS works
High-Capacity Transfer Tube (HCTT) Electrodynamic Ion Funnel EASY-IC/ETD/PTCR Ion Source Advanced Active Ion Beam Guide QR5 Segmented Quadrupole Mass Filter with Hyperbolic Surfaces

High-Capacity Transfer Tube (HCTT)

Increases ion flux into the mass spectrometer

Electrodynamic Ion Funnel

Focuses ions after HCTT

EASY-IC/ETD/PTCR Ion Source

Based on Townsend discharge; reliable and easy to use

Advanced Active Ion Beam Guide

Prevents contaminants from entering the mass resolving quadrupole

QR5 Segmented Quadrupole Mass Filter with Hyperbolic Surfaces

Improves sensitivity with 0.4 m/z precursor isolation width
Ultra-High-Field Orbitrap Mass Analyzer Ultra-High Vacuum (UHV) Manifold Ion-Routing Multipole Real-Time Search Modified Dual-Pressure Linear Ion Trap

Ultra-High-Field Orbitrap Mass Analyzer

Offers 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) Manifold

Reduces pressure in the UHV region; improves ion detection in the Orbitrap mass analyzer

Ion-Routing Multipole

Enable higher acquisition rates; performs HCD at any MSn stage; allows variable pressure (0.5–20 mTorr) for superior top-down performance

Real-Time Search

Provides on-the-fly peptide identification, increasing depth and accuracy of TMT quantitation

Modified Dual-Pressure Linear Ion Trap

Enables 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.

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Single-cell proteomics analysis

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

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How Real-Time Search works with TMT SPS MS3

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).

Sample preparation

Peptides from digested samples labeled with TMT pro and analyzed by LC/MS

LC/MS analysis

LC/MS analysis

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.

TMTpro reporter 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.

Quantified peptides in a 50-min LC run

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).

Quantified peptides per LC run

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)

Eclipse MS real time search increased accuracy

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

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Native analysis of therapeutic proteins

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 MS(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.

Simplify highly complex spectra with PTCR

Achieving high sequence coverage of CDR3 domain

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.

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Characterization of native protein-ligand complexes

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.

"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

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Reduce protein mass spectral complexity with PTCR

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).

PTCR elucidates novel protein charge state distributions

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.

Eclipse PTCR reduces spectra complexity

PTCR enabled detection of more and larger proteoforms

Eclipse PTCR parent ions detected

PTCR resulted in a significant increase in the number of identified proteoforms*

Eclipse PTCR unique proteoforms

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

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Application-specific methods just one click away

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.

Method Editor Orbitrap Eclipse Flow

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.

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Optional features to maximize performance

Thermo Scientific FAIMS Pro interface enhances precursor selectivity, improving qualitative and quantitative results for most peptide and protein applications while reducing time consuming sample preparation steps.

Identifying and characterizing proteins and post-translational modifications by bottom-up mass spectrometry relies on the acquisition of high-quality MS and MS/MS data. The FAIMS Pro interface increases analytical performance through gas phase fractionation and selective enhancement of peptidic compounds, reducing the complexity of the MS spectra, and improving analyte signal/noise ratio. The end result is greater proteome coverage.

Learn more

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Easy-IC Ion Source

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.

Thermo Scientific 1 Million (M) resolution

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).

Orbitrap Resolution

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.

What customers are saying:

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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.

Testimonial

Testimonial

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Monitor your mass spectrometry analyses anywhere, anytime.

Monitor your mass spectrometry analyses anywhere, anytime.

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.

Key Almanac Tools

  • Instrument Connect Platform: manage your systems and data in a single location
  • Instrument Status: see what is happening in real time
  • Instrument Logbook and Events: easily see what has happened and log important information
  • Instrument Utilization Reports: evaluate system usage over time
  • Instrument Scheduler: manage system access and utilization
  • Push to Service: minimize time taken to diagnose issues
Almanac Workflow

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.

Free download

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:

  • Top-down proteomics
  • TMT SPS MS3
  • Label-free quantitation
  • Cross-linking
  • Glycopeptide identification and quantification
  • Phosphopeptide ID and quantification
  • Iterative search strategies
  • Multi-search engine comparison

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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:

  • Intact analysis
  • Top-down and middle-down analysis
  • Peptide mapping
  • Multi-attribute method (MAM)

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Target applications for Obitrap Eclipse Tribrid mass spectrometer

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