Orbitrap eclipse mass spectrometer

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Orbitrap Eclipse mass spectrometer is a high-resolution, high-mass-accuracy mass analyzer designed for advanced proteomics, metabolomics, and other analytical applications. It utilizes Orbitrap technology to provide precise mass measurements and high-resolution separation of complex samples.

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23 protocols using orbitrap eclipse mass spectrometer

Comparative Mass Spectrometry of Osteoblast Differentiation

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Analysis used liver or stromal stem cells differentiated to form osteoblasts, from wild type, ClCN3, and ClCN5 knockout animals by mass spectrometry (Puig et al., 2023 (link)). Briefly, tandem mass tag labeled peptides on a PepMap RSLC C18 column were eluted by gradients. Sample eluate was electrosprayed into an Orbitrap Eclipse Mass Spectrometer (ThermoFisher Scientific) for analysis. Spectrometry files were processed in Proteome Discoverer v. 2.5 (ThermoFisher Scientific). MS spectra were searched against the Mus musculus SwissProt database (http://www.ebi.ac.uk/swissprot). Proteins of high confidence were retained for analyses and protein values were log2 transformed prior to analysis to detect differentially expressed (DE) proteins in the knockout and wild type.

Tourkova I.L., Larrouture Q.C., Liu S., Luo J., Shipman K.E., Onwuka K.M., Weisz O.A., Riazanski V., Nelson D.J., MacDonald M.L., Schlesinger P.H, & Blair H.C. (2024). Chloride/proton antiporters ClC3 and ClC5 support bone formation in mice. Bone Reports, 21, 101763.

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Serum Glycoproteome Profiling by Mass Spectrometry

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Serum samples from volunteer donors were first enriched for 14 abundant serum proteins and digested with trypsin. Glycopeptides were enriched from the peptide mixture using either size exclusion chromatography or mixed-mode anion exchange cartridge (MAX), and analyzed by mass spectrometry (MS) in data dependent acquisition mode an Orbitrap Eclipse mass spectrometer (Thermo Fisher Scientific) [11 (link), 16 (link), 17 (link)]. Data was searched in pGlyco3 [18 (link)]. Commercial bovine (Thermo Scientific) and rabbit (Sigma) serum albumin were digested followed by glycopeptide enrichment using MAX. Details of sample preparation and MS analysis are provided in Additional file 1: Supplemental Methods.

Garapati K., Jain A., Madden B.J., Mun D.G., Sharma J., Budhraja R, & Pandey A. (2024). Defining albumin as a glycoprotein with multiple N-linked glycosylation sites. Journal of Translational Medicine, 22, 454.

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Peptide Analysis by Stepped HCD-MS

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Peptides were dried then resuspended in 0.1% formic acid and analyzed by nanoLC-ESI MS with an Ultimate 3000 HPLC (Thermo Fisher Scientific) system coupled to an Orbitrap Eclipse mass spectrometer (Thermo Fisher Scientific) using stepped higher energy collision-induced dissociation (HCD) fragmentation. Peptides were separated using an EasySpray PepMap RSLC C18 column (75μm × 75cm). A trapping column (PepMap 100 C18 3μM 75μM x 2cm) was used in line with the LC prior to separation with the analytical column. LC conditions were as follows: 280 minute linear gradient consisting of 4–32% ACN in 0.1% formic acid over 260 minutes, followed by 20 minutes of alternating 76% ACN in 0.1% formic acid and 4% ACN in 0.1% formic acid to ensure all the sample elutes from the column. The flow rate was set to 300nL/min. The spray voltage was set to 2.7 kV and the temperature of the heated capillary was set to 40°C. The ion transfer tube temperature was set to 275°C. The scan range was 375−1500 m/z. Stepped HCD collision energy was set to 15%, 25% and 45% and the MS2 for each energy was combined. Precursor and fragment detection were performed with an Orbitrap at a resolution MS1 = 120,000, MS2 = 30,000. The AGC target for MS1 was set to standard and injection time set to auto which involves the system setting the two parameters to maximize sensitivity while maintaining cycle time.

Tong T., D’Addabbo A., Xu J., Chawla H., Nguyen A., Ochoa P., Crispin M, & Binley J.M. (2023). Impact of stabilizing mutations on the antigenic profile and glycosylation of membrane-expressed HIV-1 envelope glycoprotein. PLOS Pathogens, 19(8), e1011452.

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LC-MS/MS Analysis of HeLa Digest

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Final LC–MS/MS
analysis for method evaluation was performed using an Orbitrap Eclipse
mass spectrometer (Thermo Scientific, San Jose, CA, USA) coupled to
an UltiMate 3000 LC system (Thermo Fisher Scientific, Germering, Germany).
For these short gradients (5, 10, and 15 min), a self-packed trap
column (XBridge Premier Peptide BEH C18, 2.5 μm, 150 μm
i.d., 3 cm length, 130 Å; Waters, Milford, MA, USA) and a self-packed
analytical column (XBridge Premier Peptide BEH C18, 2.5 μm,
75 μm i.d., 5 cm length, 130 Å; Waters, Milford, MA, USA)
were used. The mobile phases were as follows: for the (i) positive
ion mode, (A) 0.1% FA in H₂O and (B) 0.1% FA and 95% ACN
in water and for the (ii) negative ion mode, (A) 2.5 mM imidazole
and 3% IPA in water and (B) 2.5 mM imidazole, 3% IPA, and 95% ACN
in water. The loading solvent was in both cases mobile phase A. The
gradient was from 2 to 40% B in the corresponding minutes of the gradient
(5, 10, or 15 min) at a flow rate of 1.5 μL/min. Full MS scans
were acquired with 240,000 resolution. For the Thermo Scientific Pierce
HeLa protein digest standard, 500 ng of the sample was injected. In
the case of the digestion with the different enzymes, 1 μg of
HeLa digest was loaded on the column. For each condition (positive
and negative), each chromatogram time (5, 10, and 15 min), and each
enzyme (trypsin, GluC, LysC, and AspN), the sample was injected in
quadruplicate.

Penanes P.A., Gorshkov V., Ivanov M.V., Gorshkov M.V, & Kjeldsen F. (2023). Potential of Negative-Ion-Mode Proteomics: An MS1-Only Approach. Journal of Proteome Research, 22(8), 2734-2742.

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Lipidomic analysis by LC-MS/MS

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The extracted lipids were dissolved in 20 μL sample buffer (50 % acetonitrile, 30 % H2O and 20 % isopropanol) and sonicated for 10 min. For LC-MS/MS analysis, lipids were loaded on a C18 column (Acclaim PepMap 100, C18, 75 μm × 150 mm, 3 μm, Thermo Scientific) by a Dionex UltiMate 3000 RSLC nano System connected to an Orbitrap Eclipse mass spectrometer (Thermo Scientific). A binary buffer system was used with buffer A of acetonitrile:H2O (60:40), 10 mM ammonium formate, 0.1 % formic acid and buffer B of isopropanol:acetonitrile (90:10), 10 mM ammonium formate, 0.1 % formic acid. Lipids were separated at 40 °C with a gradient of 32 % to 99 % buffer B at a flow rate of 300 nL/min over 33 min. Spray voltage was set to 2.2 kV and heated capillary temperature was at 320 °C. For data-dependent acquisition, full MS scan mass range was set to 300 to 2000 with a resolution of 120000. MS/MS spectra were acquired using higher-energy collision-induced dissociation (HCD) fragmentation with a stepped collision energy with 25 and 30. Phospholipid identification was performed using LipiDex (version 1.1) ⁷¹. MS and MS/MS search tolerances were set to 0.01 Th with LipiDex_HCD_Formic and LipiDex_Splash_ISTD_Formic libraries. Phospholipid quantification was performed using MZmine (version 2.53) ⁷². The peak detection and integration for PE and PG lipids were manually checked in MZmine.

Paul P., Rouas-Freiss N., Khalil-Daher I., Moreau P., Riteau B., Le Gal F.A., Avril M.F., Dausset J., Guillet J.G, & Carosella E.D. (1998). HLA-G expression in melanoma: A way for tumor cells to escape from immunosurveillance. Proceedings of the National Academy of Sciences of the United States of America, 95(8), 4510-4515.

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Protein Identification via Mass Spectrometry

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Affinity-purified pLVX samples were separated on SDS/PAGE gels, and pcDNA3 samples were prepped using S-Trap microcolumns (Protifi) according to the manufacturer’s instructions. The samples were reduced, alkylated, digested with trypsin, and desalted as previously described (26 (link), 27 (link)). An aliquot of each sample was loaded onto an Acclaim PepMap trap column (75 μm in diameter [ID] × 2 cm, 3-μm bead size, 100-Å pore size) in line with an EASY-Spray PepMap analytical column (75 μm ID × 50 cm C18, 2-μm bead size, 100-Å pore size) using the autosampler of an EASY-nLC 1000 high-performance liquid chromatography (HPLC) (Thermo Fisher) and solvent A (2% acetonitrile, 0.5% acetic acid). The peptides were eluted into the Orbitrap QExactive (Thermo Fisher Scientific) or the Orbitrap Eclipse Mass Spectrometer (Thermo Fisher Scientific) using the following gradient: 5 to 35% solvent B (80% acetonitrile, 0.5% acetic acid) over 60 min, followed by an increase from 35 to 45% solvent B over 10 min, followed by an increase of 45 to 100% solvent B in 10 min.

Mena E.L., Donahue C.J., Vaites L.P., Li J., Rona G., O’Leary C., Lignitto L., Miwatani-Minter B., Paulo J.A., Dhabaria A., Ueberheide B., Gygi S.P., Pagano M., Harper J.W., Davey R.A, & Elledge S.J. (2021). ORF10–Cullin-2–ZYG11B complex is not required for SARS-CoV-2 infection. Proceedings of the National Academy of Sciences of the United States of America, 118(17), e2023157118.

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Tandem mass spectrometry pipeline

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TMT labeled peptides (~1 µg) were loaded onto a heated PepMap RSLC C18 column (2 µm, 100 Å, 75 µm × 50 cm; ThermoScientific), then eluted by gradients optimized for each high pH reverse-phase fraction [27 (link)]. Sample eluate was electrosprayed (2000 V) into an Orbitrap Eclipse Mass Spectrometer (MS; ThermoFisher Scientific) for analysis. MS1 spectra were acquired at a resolving power of 120,000. MS2 spectra were acquired in the Ion Trap with CID (35%) in centroid mode. Real-time search (RTS) (max search time = 34 s; max missed cleavages = 1; Xcorr = 1; dCn = 0.1; ppm = 5) was used to select ions for SPS for MS3. MS3 spectra were acquired in the Orbitrap with HCD (60%) with an isolation window = 0.7 m/z and a resolving power of 60,000, and a max injection time of 400 ms.

Puig S., Xue X., Salisbury R., Shelton M.A., Kim S.M., Hildebrand M.A., Glausier J.R., Freyberg Z., Tseng G.C., Yocum A.K., Lewis D.A., Seney M.L., MacDonald M.L, & Logan R.W. (2023). Circadian rhythm disruptions associated with opioid use disorder in synaptic proteomes of human dorsolateral prefrontal cortex and nucleus accumbens. Molecular Psychiatry, 28(11), 4777-4792.

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Tryptic Peptide Separation and Mass Spectrometry

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The tryptic peptides were dissolved
in 10 μL of 4% acetonitrile, 0.1% formic acid, and 1.5 μL
was loaded and separated on an Acclaim PepMap RSLC column (75 μm
× 25 cm, C18, 2 μm, 100 Å, Thermo) with a 50 min 5–35%
linear gradient of mobile phase B (80% aqueous acetonitrile containing
0.1% formic acid) in mobile phase A (0.1% formic acid in water), followed
by a 10 min isocratic 95% B. The gradient was delivered by an Easy-nLC
1200 system (Thermo) at 300 nL/min. An Orbitrap Eclipse mass spectrometer
(Thermo Scientific) data acquisition method was based on the “Single
Cell LFQ” template with the following modifications: cycle
time 2 s, maximum injection time for MS2 250 ms, charge states 2–6.

Chauhan S.S., Marotta N.J., Karls A.C, & Weinert E.E. (2022). Binding of 2′,3′-Cyclic Nucleotide Monophosphates to Bacterial Ribosomes Inhibits Translation. ACS Central Science, 8(11), 1518-1526.

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Orbitrap Eclipse Mass Spectrometry Protocol

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Mass spectra were collected on an Orbitrap Eclipse mass spectrometer (Thermo Fisher Scientific) coupled to a Proxeon EASY-nLC 1200 LC pump (Thermo Fisher Scientific). Peptides were separated on a 35-cm in-house packed column (inner diameter 100 μm, Accucore, 2.6 μm, 150 Å) using a 150-minute gradient (from 5%–30% acetonitrile with 0.1% formic acid) at 500 nl/minute. MS1 data were collected using the Orbitrap (120,000 resolution; maximum injection time 50 ms; AGC 4× 10⁵, 400–1,400 m/z). Determined charge states between 2 and 5 were required for sequencing, and a 90-second dynamic exclusion window was used. MS2 scans consisted of collision-induced dissociation (CID), multi-stage activation CID at 97.9763 Da, or higher-energy-collision-induced dissociation (HCD) of precursors using a Top10 method. High-resolution MS2 scans were collected in the Orbitrap with an isolation window of 0.7 Da, q value of 0.25, AGC target of 50,000, resolution of 50,000, and maximum injection time of 86 ms. Low-resolution MS2 scans were collected in the ion trap with an isolation window of 1.3 Da, q value of 0.25, AGC target of 7,500, and maximum ion injection time of 60 ms. Supplementary Table 3 contains the metadata on all included raw files (including labeling state, collision mode, resolution, FAIMS CVs, and so on).

Gassaway B.M., Li J., Rad R., Mintseris J., Mohler K., Levy T., Aguiar M., Beausoleil S.A., Paulo J.A., Rinehart J., Huttlin E.L, & Gygi S.P. (2022). A multi-purpose, regenerable, proteome-scale, human phosphoserine resource for phosphoproteomics. Nature methods, 19(11), 1371-1375.

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High-Resolution Proteome Profiling using FAIMS

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Data were collected on an Orbitrap Eclipse mass spectrometer (ThermoFisher Scientific) coupled to a Proxeon EASY-nLC 1200 LC pump (ThermoFisher Scientific). Peptides were separated on a 35 cm column (i.d. 100 μm, Accucore, 2.6 μm, 150 Å) packed in-house using a 90 min gradient at 550 nl/min. High-field asymmetric-waveform ion mobility spectrometry (FAIMS) was enabled during data acquisition with compensation voltages (CVs) set as −40V, −60V, and −80V (Schweppe et al., 2019 (link)). MS1 data were collected using the Orbitrap (120,000 resolution; maximum injection time 50 ms; AGC 4×10⁵). Determined charge states between 2 and 5 were required for sequencing, and a 120 s dynamic exclusion window was used. MS2 scans were performed in the ion trap with CID fragmentation (isolation window 0.5 Da; Turbo; NCE 35%; maximum injection time 35 ms; AGC 1×10⁴). An on-line real-time search algorithm (Orbiter) was used to trigger MS3 scans for quantification (Schweppe et al., 2020 (link)). MS3 scans were collected in the Orbitrap using a resolution of 50,000, NCE of 45%, maximum injection time of 150 ms and AGC of 1.5×10⁵. The close out was set at two peptides per protein per fraction (Schweppe et al., 2020 (link)).

Li J., Cai Z., Vaites L.P., Shen N., Mitchell D.C., Huttlin E.L., Paulo J.A., Harry B.L, & Gygi S.P. (2021). Proteome-wide mapping of short-lived proteins in human cells. Molecular cell, 81(22), 4722-4735.e5.

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