All samples were measured in data independent mode (DIA). The analysis was performed with an Ultimate 3000 nano RSLC system coupled to an Orbitrap Fusion Lumos mass spectrometer (all Thermo Scientific). For each measurement, 1 μg of peptides from each sample was pre‐concentrated on a 100 μm × 2 cm C18 trapping column for 10 min using 0.1% TFA (v/v) at a flow rate of 20 μL/min, followed by separation on a 75 μm × 50 cm C18 main column (both Pepmap, Thermo Scientific) with a 120‐min LC gradient of 3%–35% B (84% ACN in 0.1% FA) at a flow rate of 250 nL/min. An appropriate amount of iRT standard peptides (Biognosys) was added to each sample before starting the measurement. MS survey scans were acquired from 300 to 1100 m/z at a resolution of 60,000 FWHM, followed by MS/MS using 24 DIA windows, each covering a range of 25 m/z (with 1 m/z overlap) at a resolution of 30,000. The polysiloxane ion at 445.12 m/z was used as a lock mass. The CID spectra were recorded with a normalized collision energy of 32% and an activation time of 10 ms.
Orbitrap fusion lumos mass
Manufactured by Thermo Fisher Scientific
The Orbitrap Fusion Lumos is a high-resolution mass spectrometer designed for advanced proteomics and metabolomics research. It utilizes an Orbitrap mass analyzer to provide accurate and sensitive mass measurements. The Orbitrap Fusion Lumos is capable of performing tandem mass spectrometry (MS/MS) experiments for in-depth structural analysis of complex samples.
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5 protocols using orbitrap fusion lumos mass
1
DIA-based Proteomic Analysis with Orbitrap
2
TMT-labeled Peptide Fractionation and LC-MS/MS
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The TMT-labelled peptides were fractionated by High performance liquid chromatography (HPLC). For LC-MS/MS analysis, peptides were separated using a 135-min gradient elution at a flow rate 0.3 μL/min with the Ultimate U3000 system, which was directly interfaced with the Thermo Orbitrap Fusion Lumos mass spectrometer. A detailed description of HPLC and LC-MS/MS experiments is given in Supplementary Materials and Methods.
3
Proteomic analysis of biological samples
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1 μg of the samples was analyzed using a self-made analytical column (75 μm × 150 mm, 1.9 μm) on an EASY-nLC1000 connected to an Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific). Peptides were eluted by using a binary solvent system with 99.9% H2O, 0.1% formic acid (phase A) and 80% ACN, 19.9% H2O, 0.1% formic acid (phase B). The following linear gradient was used: 2–5% B in 2 min, 5–10% B in 29 min, 10–20% B in 78 min, 20–28% B in 6 min, 28–95% B in 2 min, washed at 95% B for 3 min. The eluent was introduced directly to a mass spectrometer via EASY-Spray ion source. Source ionization parameters were as follows: spray voltage, 2.2 kV; capillary temperature, 320 °C; and decluttering potential, 100 V.
4
Targeted Proteomics of Post-DRE Urine
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SPE processed peptides were dried and suspended in 15 µL of 0.1% formic acid. Peptide concentrations were determined by NanoDrop and SIL peptides were spiked at detectable concentrations. For each injection, 2 µg of total peptide was loaded onto an in‐line EASY‐Spray 50 cm C18 column (Thermo Fisher ES803A) using an EASY‐nLC‐1200 UHPLC system. Data were acquired with both a full MS1 scan and an unscheduled PRM scan targeting both endogenous and SIL peptides of interest over a 140‐min gradient using a Thermo Fisher Orbitrap Fusion Lumos mass spectrometer. Targets were as follows: I179T variant, LQCVDLHVT
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Proteomic Analysis of MS Lesions
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All sections were dorsal–lateral frontal subcortical white matter. Subcortical white matter lesions and normal appearing white matter were dissected and β-mercaptoethanol-, sarkosyl-insoluble proteins were purified using the isolation procedure from [32 (link)], followed by sonication, tryptic enzymatic digestion, and nanoLC-ms/ms analysis of the peptides using an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific). Subcortical white matter lesions from five individuals with relapsing–remitting multiple sclerosis (RRMS) and normal-appearing subcortical white matter (NAWM) from non-neurologically impaired individuals were compared. RRMS and non-neurological controls (NNCs) were obtained from the Rocky Mountain MS Center Tissue Bank. Supplemental Table S1 lists the cases studied.
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