Dried fractions were resuspended in 0.1% FA and separated on a 500 mm, 0.075 mm Acclaim PepMap 100 C18 HPLC column (ThermoFisher, #164942) and analyzed on a Orbitrap Lumos Tribrid mass spectrometer (ThermoFisher) equipped with a FAIMS device (ThermoFisher) that was operated in two compensation voltages, −50 V and −70 V. Synchronous precursor selection based MS3 was used for TMT reporter ion signal measurements. Peptides were separated by EASY-nLC1200 using a 90 min linear gradient from 6% to 31% buffer; buffer A was 0.1% FA, buffer B was 0.1% FA, 80% ACN. The analytical column was operated at 50 °C. Raw files corresponding to the mouse livers were split on the basis of the FAIMS compensation voltage using FreeStyle (ThermoFisher). Fly fat body proteomics data were analyzed using Proteome Discoverer (version 2.4.1.15); mouse liver proteomics data were analyzed using MaxQuant (version 1.6.17.0). Isotope purity correction factors, provided by the manufacturer, were included in the analysis.
Orbitrap lumos tribrid mass spectrometer
Manufactured by Thermo Fisher Scientific
The Orbitrap Lumos Tribrid mass spectrometer is a high-resolution, high-mass-accuracy mass analyzer designed for advanced proteomics and metabolomics applications. It combines an Orbitrap mass analyzer with a linear ion trap and an optional quadrupole mass filter, providing a versatile platform for a wide range of analytical workflows.
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Lab products found in correlation
Faims device, by Thermo Fisher Scientific (3 mentions)
Ultimate 3000, by Thermo Fisher Scientific (3 mentions)
Acclaim pepmap 100 c18 hplc column, by Thermo Fisher Scientific (2 mentions)
Reprosil c18 resin, by Dr. Maisch (2 mentions)
Reprosil pur c18, by Dr. Maisch (1 mentions)
Easy nanolc 1200, by Thermo Fisher Scientific (1 mentions)
Xcalibur software, by Thermo Fisher Scientific (1 mentions)
Anti flag m2 affinity gel, by Merck Group (1 mentions)
8 protocols using orbitrap lumos tribrid mass spectrometer
1
FAIMS-Enabled Quantitative Proteomics
2
Orbitrap Lumos LC-MS/MS Proteomics Protocol
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An Easy nanoLC 1200 liquid chromatography system connected to an Orbitrap Lumos Tribrid mass spectrometer (Thermo Fisher Scientific) was used for proteomics analyses. Peptides were first introduced onto a trap column (PepMap 100 C18, 5 μm, 0.1 × 20 mm) and then separated on an in-house packed analytical column (ReproSil-Pur C18, 3 μm, 0.075 × 330 mm, Dr Maisch) using gradient (0.2% FA in water as phase A; 0.2% FA in acetonitrile as phase B) running from 6% to 35% B in 167 min and from 35% to 100% B in 3 min, at a flow rate of 300 nl/min. Positive ion MS scans were acquired at a resolution of 120,000 within m/z range of 400–1,600 using AGC target 5 × 105 and maximum injection time 50 ms. MS/MS analysis was performed in data-dependent mode with a top speed cycle of 1 s for the most intense multiply charged precursor ions. MS precursors above 50,000 threshold were isolated by quadrupole using 0.7 m/z isolation window and then fragmented in ion trap by collision-induced dissociation at a collision energy of 35%. Dynamic exclusion was set to 60 s with 10 ppm tolerance. MS/MS spectra were acquired by ion trap using AGC target 5 × 105 and maximum injection time 35 ms.
3
Orbitrap Lumos FAIMS Mass Spectrometry
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Dried fractions were resuspended in 0.1% formic acid and separated on a 50 cm, 75 μm Acclaim PepMap column (164942; Thermo Fisher Scientific) and analyzed on an Orbitrap Lumos Tribrid mass spectrometer (Thermo Fisher Scientific) equipped with a FAIMS device (Thermo Fisher Scientific). The FAIMS device was operated in two compensation voltages, −50 and −70 V. Synchronous precursor selection based on MS3 was used for the acquisition of the TMT reporter ion signals. Peptide separation was performed on an EASY-nLC1200 using a 90 min linear gradient from 6–31% buffer; buffer A was 0.1% formic acid, and buffer B was 0.1% formic acid with 80% acetonitrile. The analytical column was operated at 50°C. Raw files were split based on the FAIMS compensation voltage using FreeStyle (Thermo Fisher Scientific).
4
High-throughput Fly Brain Proteomics
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Dried fractions were re-suspended in 0.1% FA and separated on a 50 cm, 0.075 mm Acclaim PepMap 100 C18 HPLC column (ThermoFisher, #164942) and analysed on a Orbitrap Lumos Tribrid mass spectrometer (ThermoFisher) equipped with a FAIMS device (ThermoFisher) that was operated in two compensation voltages, − 50 V and − 70 V. Synchronous precursor selection based MS3 was used for TMT reporter ion signal measurements. Peptides were separated by EASYnLC1200 using a 90 min linear gradient from 6 to 31% buffer; buffer A was 0.1%
FA, buffer B was 0.1% FA, 80% ACN. The analytical column was operated at 50 °C. Fly brain proteomics data were analysed using Proteome Discoverer (version 2.4.1.15). Isotope purity correction factors, provided by the manufacturer, were included in the analysis.
FA, buffer B was 0.1% FA, 80% ACN. The analytical column was operated at 50 °C. Fly brain proteomics data were analysed using Proteome Discoverer (version 2.4.1.15). Isotope purity correction factors, provided by the manufacturer, were included in the analysis.
5
Orbitrap Lumos Tribrid LC-MS/MS
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1μg of each sample was loaded on a PepMap RSLC C18 easy-spray column (3um, 100A, 75um*15 cm) using Easy-nLC 1200 coupled to an Orbitrap Lumos Tribrid Mass Spectrometer (Thermo Scientific). The peptides were separated using a 3-hour gradient of Mobile phase A (0.1% Formic acid in 95:5 Water: Acetonitrile) and Mobile Phase B (0.1% Formic acid in 80:20 Acetonitrile: Water). The following gradient program was used for peptide elution: 2 to 30% B in 140 minutes, 30 to 95% B in 30 minutes and 95–100%B in 10 minutes. The mass spectrometer data was acquired in MS1 scan mode (m/z = 375–1800) at a resolution of 120,000 with Automatic Gain Control (AGC) of 4.0 × 105 and Maximum injection time of 50 ms. MS/MS data acquisition was done using HCD mode at a resolution of 30,000 with an AGC target of 5.0 × 104 and maximum injection time of 54 ms. All the data acquisition was done using Thermo Scientific Xcalibur software.
6
Identification of SLC27A5-Interacting Proteins
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PLC/PRF/5 cells, which were pre‐transfected with SLC27A5‐TO4‐3Flag or TO4‐3Flag plasmid, were incubated overnight at 4°C with ANTI‐FLAG M2 affinity gel (A2220, Sigma). Coomassie blue was used to stain the protein complexes. The excised protein gel bands were submitted to Shanghai Applied Protein Technology Co., Ltd. for identification of proteins that bind to SLC27A5. After undergoing reduction reaction and alkylation treatment, the samples were incubated with trypsin (mass ratio, 1:50). The digested samples were then dried and analyzed by mass spectrometry after injecting 10 or 20 µL of each sample. Mass spectrometry (MS) analysis was performed on a Lumos Tribrid Orbitrap Mass Spectrometer (Thermo Scientific) equipped with Ultimate 3000 (Thermo Scientific) nano‐high‐performance liquid chromatograph. The raw MS data were analyzed using Mascot 2.2 software to identify the immunoprecipitating proteins. Further, Gene Ontology enrichment analysis was performed using clusterProfiler package in R, with Fisher's Exact Test, and the significant p value cutoff was set at 0.05.
7
Proteomic Analysis by LC-MS/MS on Orbitrap
8
LC-MS/MS Analysis of Peptides
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The LC-MS/MS experiment was performed on a Lumos Tribrid Orbitrap Mass Spectrometer (Thermo Fischer) equipped with Ultimate 3000 (Thermo Fisher) nano-HPLC. Peptides were separated onto a 150μm inner diameter microcapillary trapping column packed first with approximately 2cm of C18 Reprosil resin (5μm, 100 Å, Dr. Maisch GmbH, Germany) followed by PharmaFluidics (Gent, Belgium) 50cm analytical column. Separation was achieved by applying a gradient from 5–27% acetonitrile in 0.1% formic acid over 90 min at 200 nl/min. Electrospray ionization was enabled by applying a voltage of 2 kV using a homemade electrode junction at the end of the microcapillary column and sprayed from metal tips (PepSep, Denmark). The mass spectrometry survey scan was performed in the Orbitrap in the range of 400–1,800 m/z at a resolution of 6×104, followed by the selection of the twenty most intense ions (TOP20) for CID-MS2 fragmentation in the Ion trap using a precursor isolation width window of 2 m/z, AGC setting of 10,000, and a maximum ion accumulation of 100 ms. Singly charged ion species were not subjected to CID fragmentation. The normalized collision energy was set to 35 V and an activation time of 10 ms. Ions in a 10 ppm m/z window around ions selected for MS2 were excluded from further selection for fragmentation for 60s.
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