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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.
On this page
- Look inside the Orbitrap Eclipse MS
- Single-cell analysis with Orbitrap Eclipse Tribrid MS
- How Orbitrap Eclipse MS Real-Time Search works with TMT SPS MS3
- Native analysis of therapeutic proteins with Orbitrap Eclipse MS
- Characterization of native protein-ligand complexes with Orbitrap Eclipse Tribrid MS HMRn
- Reduce protein mass spectral complexity with PTCR
- Application-specific methods just one click away
- Optional features to maximize performance
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.
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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 |
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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. |
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.
Individual cells are isolated using FACS sorter, labeled, and analyzed using Orbitrap Eclipse MS
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Novel MS3 workflow with Real-Time Search detected more than 300 differentially expressed proteins in each individual cell
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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.
High-resolution classification of individual cells in an SPS MS3 experiment with Real-Time Search
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Confirmation of overexpression of known protein biomarkers in individual cells of each type underscores the sensitivity of the workflow
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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
Brochure: Go Beyond—Limited samples to single-cell proteomics
At Thermo Fisher Scientific, we’re proud to partner with life science innovators who are transforming the future of human health. Every single day.
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
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.
MS precursor spectrum
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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).
MS2
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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
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 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.
Simplify highly complex spectra with PTCR
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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.
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PTCR dispersed the selected ion population to a higher m/z range, revealing individual molecular ion species at lower charge states. The interpretation of this spectrum provided accurate information about the mixture of different proteoforms, which were undecipherable in the original spectrum.
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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)
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.
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Native spectrum acquired using the HMRn mode. The 27+ species showed baseline resolution of the major glycoforms.
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Representative MS2 spectra of the 27+ parent ion. The spectra were obtained by multiple fragmentation techniques and showed different fragment ion patterns, providing complementary information and increasing the primary amino acid sequence coverage for this protein.
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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.
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.
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Intact protein complexes are extracted from the membrane using detergent. Detergent molecules form a micelle layer around the protein complex that allows solubilization. The micelles are then ionized using nESI and introduced into the mass spectrometer.
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The intact membrane protein-ligand assembly is released from the detergent micelle using source-CID (pseudo MS2) and detected in the Orbitrap mass analyzer. The 17+ parent ion with associated ligands is isolated (gray bar) for the next MSn step.
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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).
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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
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.
PTCR enabled detection of more and larger proteoforms
PTCR resulted in a significant increase in the number of identified 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
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.
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.
Optional features to maximize performance
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.
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).
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 Thermo Scientific Orbitrap IQ-X Tribrid MS and/or Orbitrap Eclipse mass spectrometers with 1M.
What customers are saying:
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
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
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.
Recommended for the following workflows:
- Top-down protein MS/MS ID
- Elucidation of unknown PTMs
- Protein database curation
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)
Thermo Scientific Compound Discoverer software streamlines unknown identification, determination of real differences between samples, and elucidation of biological pathways
Recommended for the following workflows:
- Metabolomics
- Environmental Testing
- Food Safety
- Drug Development
- Forensic Toxicology
Thermo Scientific Mass Frontier Spectral Interpretation Software helps simplify management, evaluation, and interpretation of mass spectral data to provide greater insights.
Recommended for the following workflows:
- Untargeted Metabolomics
- Pharmaceutical Analysis
- Metabolite ID
- Impurity ID
Target applications for Obitrap Eclipse Tribrid mass spectrometer
Single-cell proteomics is now a reality
Single-cell proteomics is now a reality
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.
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