TMT-labeled peptides were reconstituted in 0.1% formic acid and delivered to an Acclaim PepMap 75 μm × 2 cm NanoViper C18, 3 μm column (Thermo Scientific) with mobile phase containing 0.1% formic acid at 8 μl/min flow rate by EASY-nLC 1200 system. Then, the trap column was switched to a PepMap RSLC C18, 2 μm, 75 μm × 25 cm column (Thermo Scientific). Peptides were separated in the second dimension using a 194 min acetonitrile gradient 6 to 90% buffer (0.1% formic acid in 80% acetonitrile) at 300 nl/min flow rate. Tandem MS data were collected using an Orbitrap Fusion Lumos Tribrid instrument (Thermo Scientific). The general mass spectrometric settings included MS1 orbitrap resolution = 120,000; MS2 orbitrap resolution = 50,000; high-energy collision dissociation fragmentation (for MS/MS), and 37% collision energy.
Orbitrap fusion lumos tribrid instrument
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Orbitrap Fusion Lumos Tribrid instrument is a high-performance mass spectrometer designed for advanced analytical applications. It combines multiple mass analyzer technologies, including an Orbitrap mass analyzer, to provide high-resolution, accurate mass measurements. The core function of the instrument is to enable the sensitive and selective detection and identification of a wide range of analytes in complex samples.
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3 protocols using orbitrap fusion lumos tribrid instrument
1
Targeted Peptide Separation and Analysis
2
Orbitrap Fusion Lumos Tribrid Peptide Analysis
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TMT-labeled peptides were analyzed on an Orbitrap Fusion Lumos Tribrid instrument (Thermo Scientific). Peptides were dissolved in 0.2% formic acid, detached in mobile phase containing 0.1% formic acid and eluted using a nonlinear 194 min acetonitrile gradient of 6%-90% buffer (0.1% formic acid with 80% acetonitrile) at 300 nL/min. The settings of MS parameters were as follows: MS1 resolution at 120,000, mass scan of 300-1500 m/z, automatic gain control (AGC) target at 1 × 10 6 , maximum injection time at 100 ms, and 30% of radio frequency, MS2 mass resolution at 50,000, high-energy collision dissociation for MS/MS, 37% of collision energy, normalized AGC target at 1 × 10 5 , 1.2 m/z isolation width, 30 s dynamic exclusion [17] .
3
Orbitrap Fusion Lumos Tribrid Mass Spectrometry Protocol
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All fractions were analyzed on an Ultimate 3000-nLC coupled to an Orbitrap Fusion Lumos Tribrid instrument (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a nanoelectrospray source. Peptides were separated on an EASY-Spray C18 column (75 μm x 50 cm inner diameter, 2 μm particle size and 100 Å pore size, Thermo Fisher Scientific, Waltham, MA, USA). Peptide fractions were placed in an autosampler and separation was achieved by 125 min gradient from 3-24% buffer B (100% ACN and 0.1% formic acid) at a flow rate of 300 nL/min. An electrospray voltage of 1.9 kV was applied to the eluent via the EASY-Spray column electrode. The Lumos was operated in positive ion data-dependent mode, using Synchronous Precursor Selection (SPS-MS3) (56) . Full scan MS1 was performed in the Orbitrap with a precursor selection range of 380-1,500 m/z at nominal resolution of 1. performed by the quadrupole with 0.7 m/z transmission window, followed by CID fragmentation in the linear ion trap with 35% normalized collision energy in rapid scan mode and parallelizable time option was selected. SPS was applied to co-select 10 fragment ions for HCD-MS 3 analysis.
SPS ions were all selected within the 400-1,200 m/z range and were set to preclude selection of the precursor ion and TMTC ion series (57) . The AGC target and maximum accumulation time were set to 1 x 10
SPS ions were all selected within the 400-1,200 m/z range and were set to preclude selection of the precursor ion and TMTC ion series (57) . The AGC target and maximum accumulation time were set to 1 x 10
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