Orbitrap fusion lumos mass spectrometer

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany, Denmark

The Orbitrap Fusion Lumos mass spectrometer is a high-performance, hybrid mass spectrometer designed for advanced proteomics and metabolomics research. It combines the Orbitrap technology for high-resolution, accurate mass measurements with other mass analyzer technologies to provide comprehensive and sensitive analysis of complex samples.

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236 protocols using orbitrap fusion lumos mass spectrometer

Zebrafish Embryo Proteomics

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Total protein was extracted using cell lysis buffer (Sigma–Aldrich, C3228) from the head region of zebrafish embryos. After purification and trypsin (Promega, V5111) treatment, the peptides were desalted using Pierce C18 Spin Columns (Thermo Fisher, 89870). Proteomics was then performed on a Thermo Fisher Orbitrap Fusion Lumos Mass Spectrometer coupled with a Dionex UltiMate 3000 RSLCnano. Label-free relative quantification was processed with Progenesis QI software, and the abundance of various proteins was quantified based on three independent experiments and normalized according to the housekeeping protein glyceraldehyde 3-phosphate dehydrogenase (gapdh).

Chen X.K., Kwan J.S., Wong G.T., Yi Z.N., Ma A.C, & Chang R.C. (2022). Leukocyte invasion of the brain after peripheral trauma in zebrafish (Danio rerio). Experimental & Molecular Medicine, 54(7), 973-987.

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Proteomic and Behavioral Analysis of Zebrafish

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Total protein was extracted using cell lysis buffer (Sigma-Aldrich) from the head region of zebra sh embryos. After puri cation and trypsin (Promega) treatment, the peptides were desalted using Pierce C18 Spin Columns (Thermo sher). Proteomics was then performed on Thermo Fisher Orbitrap Fusion Lumos Mass Spectrometer coupled with Dionex UltiMate 3000 RSLCnano. Label-free relative quanti cation was processed with Progenesis QI software (http://www.nonlinear.com/progenesis/qi/) and the abundance of various proteins was quanti ed based on three independent experiments and normalized according to the housekeeping protein (gapdh).
Behavioral Assay of Locomotor Activity il1b Mut Zebra sh larvae at 7 dpf/4 dpa and their siblings with or without amputation were transferred into the 35 mm Petri dish containing 2 mL E3 medium. The protocol of locomotor activity was adapted from the Locomotion Assay with slight modi cation [34] . Zebra sh larvae from four groups was recorded for a 30-min period using a camera at the same time. A 10-min period after 10-min acclimation was analyzed using Fiji -Image J (https://imagej.net/Fiji) with tracking plugin and the total travel distance, active swim time and mean velocity were calculated.

Chen X., Kwan J., Wong G.T.C., H K.M.M., Yi Z., Chun‐Hang A., & Chang R.C. (2020). IL-1β-mediated macrophage infiltration into the brain after peripheral tissue injury.

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Quantitative Proteomics by LC-MS/MS

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Dried peptides were dissolved in 10 µl 0.1% formic acid (solvent A). Peptide separation was performed on a Dionex Ultimate nano HPLC system (Dionex GmbH, Idstein, Germany) coupled to an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). One µg peptides were injected onto a C18 analytical column (400 mm length, 100 µm inner diameter, ReproSil-Pur 120 C18-AQ, 3 µm).
The samples were analyzed by a standard data-dependent (DDA) method: Peptides were separated during a linear gradient from 5% to 35% solvent B (90% acetonitrile, 0.1% FA) at 300 nl/min within 120 min. Data-dependent acquisition was performed on ions between 330 and 1600 m/z scanned in the Orbitrap detector every 2.5 s (R=120,000, standard gain control and inject time settings). Polysiloxane (m/z 445.12002) was used for internal calibration. z>1 ions were subjected to higher-energy collision-induced dissociation (1.0 Da quadrupole isolation, threshold intensity 25,000, collision energy 28%) and fragments analyzed in the Orbitrap (R=15,000). Fragmented precursor ions were excluded from repeated analysis for 25 s.

Schneider S., Kovacevic A., Mayer M., Dicke A.K., Arévalo L., Koser S.A., Hansen J.N., Young S., Brenker C., Kliesch S., Wachten D., Kirfel G., Strünker T., Tüttelmann F, & Schorle H. (2023). Cylicins are a structural component of the sperm calyx being indispensable for male fertility in mice and human. eLife, 12, RP86100.

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Proteomic and Phosphoproteomic Analysis

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The pooled proteomics fractions were
separated using a Waters nano-Aquity UPLC system equipped with a homemade
75 μm I.D. × 50 cm length C18 PicoFrit (New Objective)
column packed with ReproSil-Pur 120 Å, C18-AQ, 1.9 μm.
A 110 min gradient of 100% mobile phase A [0.1% (v/v) formic acid
in water] to 60% (v/v) mobile phase B [0.1% (v/v) FA in acetonitrile]
was applied to each fraction. The column was equipped with a 20 cm
Nanospray column heater (Phoenix S & T). The separation was coupled
to an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher) for
MS/MS analysis. MS spectra were collected from 350 to 1800 m/z at an MS1 resolution setting of 60 000,
with a maximum injection time of 50 ms and the Orbitrap AGC set to
400 000. The top 20 most intense ions were selected with an
isolation width of 0.7 m/z for higher
energy collision dissociation (HCD); +1 charged species were excluded,
and the dynamic exclusion window was set at 45 s. MS2 spectra were
acquired at a mass resolution of 50 000, with a maximum injection
time of 105 ms and the Orbitrap AGC set to 100 000. Phosphoproteomics
fractions were separated as described above, with the LC gradient
length extended to 200 min for each fraction. The separation was coupled
to the same Lumos mass spectrometer with the same acquisition method.

Sanford J.A., Wang Y., Hansen J.R., Gritsenko M.A., Weitz K.K., Sagendorf T.J., Tognon C.E., Petyuk V.A., Qian W.J., Liu T., Druker B.J., Rodland K.D, & Piehowski P.D. (2021). Evaluation of Differential Peptide Loading on Tandem Mass Tag-Based Proteomic and Phosphoproteomic Data Quality. Journal of the American Society for Mass Spectrometry, 33(1), 17-30.

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Shotgun Proteomics Analysis Pipeline

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Shotgun proteomics analyses were performed using an EASY-nLC^TM 1200 UHPLC system (Thermo Fisher Scientific) coupled to an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific) operating in the data-dependent acquisition (DDA) mode. A sample volume corresponding to 2 μg of total peptides reconstituted in 0.1% FA was injected onto an Acclaim PepMap100 C18 Nano-Trap column (2 cm × 100 μm, 5 μm; Dionex, Sunnyvale, USA). Peptides were separated on a Reprosil-Pur 120 C18-AQ analytical column (15 cm × 150 μm, 1.9 μm; Dr. Maisch HPLC GmbH, Ammerbuch-Entringen, Germany) using a 75 min linear gradient from 5 to 100% eluent B (0.1% FA in 80% acetonitrile) in eluent A (0.1% FA in H₂O) at a flow rate of 600 nl/min. For DDA, the Orbitrap Fusion Lumos mass spectrometer was operated in positive polarity mode with spray voltage of 2.3 kV and capillary temperature of 320 °C. Full mass spectrometry (MS) scans from 300 to 1500 m/z were acquired at a resolution of 60,000 resolving power (at 200 m/z) with an AGC target value of 4 × 105 and a maximum ion injection time of 50 ms. The MS2 scans were acquired at a resolution of 15,000 resolving power (at 200 m/z) with an automatic gain control (AGC) target value of 5 × 104, a maximum ion injection time of 35 ms, and a normalized collision energy of 36%.

Zhang F.K., Hu R.S., Elsheikha H.M., Sheng Z.A., Zhang W.Y., Zheng W.B., Zhu X.Q, & He J.J. (2019). Global serum proteomic changes in water buffaloes infected with Fasciola gigantica. Parasites & Vectors, 12, 281.

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TMT-based Quantitative Proteomics of XAB2 Knockdown in HeLa Cells

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HeLa cells were plated in six-well plate and transfected with control or XAB2 siRNA-1 for 48 h. Then cells were harvested and subjected to TMT-based quantitative proteome analysis by Jingjie PTM Biolabs (China). In brief, cell pellets were sonicated in lysis buffer (8 M urea, 1% protease inhibitor cocktail), and the extracted protein was quantified with BCA kit. Then the protein solution was digested with trypsin for two times and labeled with TMT kit (Thermo) according to the manufacturer's instructions. The tryptic peptides were fractionated into 60 fractions by high pH reverse-phase HPLC, combined into 9 fractions, and then analyzed by EASY-nLC 1000 UPLC system and Orbitrap Fusion Lumos mass spectrometer (Thermo). Raw MS files were analyzed using MaxQuant software (version 1.5.2.8). Tandem mass spectra were searched against SwissProt Human database (20,130 protein entries). TMT-6plex was specified as fixed modifications, and the false discovery rate was set at 0.01 for both protein and peptide identification. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (46 (link)) partner repository with the dataset identifier PXD012552.

Hou S., Qu D., Li Y., Zhu B., Liang D., Wei X., Tang W., Zhang Q., Hao J., Guo W., Wang W., Zhao S., Wang Q., Azam S., Khan M., Zhao H., Zhang L, & Lei H. (2019). XAB2 depletion induces intron retention in POLR2A to impair global transcription and promote cellular senescence. Nucleic Acids Research, 47(15), 8239-8254.

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LC-MS Analysis of ShHTL7 Enzymatic Reactions

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For LC-MS analysis: a 200 μL reaction solution (100 mM HEPES, 150 mM NaCl, pH 7.0) containing 10, or 400 μM of tested samples 4a, (R)- and (S)-4a, and 4g-i, and 100 μg of recombinant ShHTL7 or its mutants was incubated at 25 °C for 30 min. Subsequently, the solution was filtered and analyzed by a nanoACQUITY UPLC system, which was directly interfaced with an SYNAPT-G2-Si mass spectrometer produced by Waters Company. For LC-MS/MS analysis, the above chemicals treated ShHTL7 reaction solution was subjected to the SDS-PAGE analysis. The corresponding protein band around 30 KDa was excised, respectively, then digested with trypsin (Promega) in NH₄HCO₃ solution (50 mM) at 37 °C for 24 h^{23 (link),26 (link)–28 (link)}. The covalent modification of C₅H₅O₂/C₆H₆O on the peptide was analyzed by a Thermo-Dionex Ultimate 3000 HPLC system, which was directly interfaced with a Thermo Orbitrap Fusion Lumos mass spectrometer. MS-MS spectra are generated by pLabel software^{53 (link),54 (link)}.

Wang D., Pang Z., Yu H., Thiombiano B., Walmsley A., Yu S., Zhang Y., Wei T., Liang L., Wang J., Wen X., Bouwmeester H.J., Yao R, & Xi Z. (2022). Probing strigolactone perception mechanisms with rationally designed small-molecule agonists stimulating germination of root parasitic weeds. Nature Communications, 13, 3987.

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Proteomic Analysis by Orbitrap MS/MS

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The MS/MS spectra was acquired in a positive ion mode in a data-dependent manner on an Orbitrap Fusion Lumos Mass Spectrometer (Thermofisher Scientific, USA) coupled to a liquid chromatography instrument UltiMate 3000 RSLCnano (Thermofisher Scientific, USA). The liquid chromatography instrument was equipped with a reverse phase C18 column for fractionation of the peptides before being analyzed in the mass spectrometry instrument. In the mass spectrometry instrument, the MS/MS analysis was conducted through a survey MS1 scan in the mass range 400 to 1,400 m/z, followed by the selection of the 10 most abundant ions for MS/MS fragmentation analysis. The fragmentation of precursor ions was achieved through collision-induced dissociation (CID). For each parent ion, the maximum signal accumulation time was 100 ms before being dynamically excluded for 12 secs and reconsidered.

Rajwani R., Galata C., Lee A.W., So P.K., Leung K.S., Tam K.K., Shehzad S., Ng T.T., Zhu L., Lao H.Y., Chan C.T., Leung J.S., Lee L.K., Wong K.C., Yam W.C, & Siu G.K. (2022). A multi-omics investigation into the mechanisms of hyper-virulence in Mycobacterium tuberculosis. Virulence, 13(1), 1088-1100.

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Label-free Quantification of Exosomal Proteins

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Three biological exosome samples (20 μg/sample) were separated on 4–15% mini-protein TGX precast protein gels (Bio-Rad) and subsequently stained with Coomassie Blue, then each sample lane was excised and digested with trypsin and spiked with 0.2 pmol of ADH peptides (YEAST Alcohol dehydrogenase 1) at the Mass Spectrometry Facility at the University of Massachusetts Medical School. The samples were then injected into Orbitrap Fusion Lumos Mass Spectrometer (Thermo Fisher Scientific) in technical triplicates for label-free quantitation (LFQ) analysis. The data were searched against Swiss-Prot Mouse protein database using Mascot search engine through Proteome Discoverer software. The data was exported and normalized as intensity-based absolute quantification (iBAQ) quantitative values in Scaffold (version Scaffold_4.10, Proteome software). The selected parameters for protein identification were the following: Protein Threshold >95%; minimum 3 peptides per candidate protein; Peptide Threshold >90%; >1 × 10⁵ iBAQ value in at least one of samples. The iBAQ value of the housekeeping protein ADH was used for normalization of biological replicates.

Jin S., Chen X., Tian Y., Jarvis R., Promes V, & Yang Y. (2023). Astroglial exosome HepaCAM signaling and ApoE antagonization coordinates early postnatal cortical pyramidal neuronal axon growth and dendritic spine formation. Nature Communications, 14, 5150.

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Comprehensive LC-MS/MS Proteomics Analysis

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For LC-MS/MS analysis, digested CSF and saliva samples were resuspended in a mobile phase A solution (2% ACN/water, 0.1% formic acid) and then separated by reversed-phase chromatography using a Dionex Ultimate 3000 RSLC nano UPLC system on a homemade 75μm ID×50 cm C18 capillary column (Reprosil-Pur AQ 120 Å, 1.9μm) in line connected with an Orbitrap Fusion Lumos Mass-Spectrometer (Thermo Fisher Scientific, Waltman, MA, USA). Human CSF and saliva peptides were separated by applying a nonlinear 150 min gradient ranging from 99% solvent A (2% ACN and 0.1% FA) to 90% solvent (90% ACN and 0.1% FA) at a flow rate of 250 nl/min.
MS acquisition was performed using a data-dependent acquisition. First MS scans were acquired with a resolution of 120,000 (at 200 m/z) and the 10 most intense parent ions were selected and fragmented by High energy Collision Dissociation (HCD) with a Normalized Collision Energy (NCE) of 32% using an isolation window of 2.0 m/z. Fragmented ions were then excluded for the following 45 s.

Eldem E., Barve A., Sallin O., Foucras S., Annoni J.M., Schmid A.W, & Alberi Auber L. (2022). Salivary Proteomics Identifies Transthyretin as a Biomarker of Early Dementia Conversion. Journal of Alzheimer's Disease Reports, 6(1), 31-41.

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