An Orbitrap Fusion (Thermo Fisher Scientific) and RSLC Ultimate 3000 nano-UPLC (Dionex) were used to acquire the data. For superior quantitation accuracy, we used multinotch-MS3 59 (link). Two microliters of each fraction was resolved on a nanocapillary reverse-phase column (75 μm internal diameter by 50 cm; PepMap RSLC C18 column, Thermo Fisher Scientific) at a flowrate of 300 nL/min using 0.1% formic acid/acetonitrile gradient system (2 to 22% acetonitrile in 110 min; 22 to 40% acetonitrile in 25 min; 6-min wash at 90% acetonitrile; 25 min re-equilibration) and directly sprayed onto the Orbitrap Fusion using EasySpray source (Thermo Fisher Scientific). The mass spectrometer was set to collect one MS1 scan [Orbitrap; 120,000 resolution; AGC target 2 × 105; max IT (maximum ionization time) 50 ms] and data-dependent, “Top Speed” (3 s) MS2 scans [collision-induced dissociation; ion trap; NCE (normalized collision energy) 35; AGC (automatic gain control) 5 × 103; max IT 100 ms]. For multinotch-MS3, the top 10 precursors from each MS2 were fragmented by high energy collisional dissociation (HCD), followed by Orbitrap analysis (NCE 55; 60,000 resolution; AGC 5 × 104; max IT 120 ms, 100 to 500 mass/charge ratio scan range).
Protocol detail
Multinotch-MS3 Quantification Pipeline
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Corresponding Organization : University of Michigan–Ann Arbor
Other organizations : The University of Texas Health Science Center at Houston, City of Hope
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Variable analysis
independent variables
- Nano-UPLC gradient (2 to 22% acetonitrile in 110 min; 22 to 40% acetonitrile in 25 min; 6-min wash at 90% acetonitrile; 25 min re-equilibration)
- Collision energy (CID: 35 NCE; HCD: 55 NCE)
- Mass spectrometry data (MS1 scans, MS2 scans, MS3 scans)
- Orbitrap Fusion mass spectrometer
- RSLC Ultimate 3000 nano-UPLC
- PepMap RSLC C18 column (75 μm internal diameter by 50 cm)
- Flowrate (300 nL/min)
- Solvent system (0.1% formic acid/acetonitrile)
- MS1 resolution (120,000), AGC target (2 × 10^5), max IT (50 ms)
- MS2 resolution (ion trap), AGC (5 × 10^3), max IT (100 ms)
- MS3 resolution (60,000), AGC (5 × 10^4), max IT (120 ms), scan range (100-500 m/z)
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