Orbitrap fusion lumos high resolution mass spectrometer

Thermo Orbitrap Fusion Lumos high-resolution mass spectrometer, Easy-NLC 1200 NL liquid chromatography system, incubator, high speed centrifuge, ultramicro spectrophotometer (NanoDrop OneC), Multiskan Sky microplate reader were purchased from Thermo Fisher, USA; Electric thermostatic incubator was purchased from Shanghai Yiheng Technology Co., LTD. Concentrator Plus was purchased from Eppendorf Germany; Deionized water was prepared by the laboratory's own Mill-Q pure water system (Millipore, USA).

(1) Chromatographic conditions: Chromatograph: EASY nLC 1,200 chromatograph (ThermoFisher); Mobile phase: A (water/0.1%FA), B (80%ACN/0.1%FA); Analytical column: 75 μm × 15 cm, Inhouse packed C18 column, 1.9 μm; Flow rate: 0.3 μL/min. (2) Mass spectrometry conditions: Mass spectrometer: Orbitrap Fusion Lumos high-resolution mass spectrometer (ThermoFisher). Software setup parameters: spray voltage, 2.2 kV; parent ion scanning was detected by Orbitrap, mass scanning range: 350–1,500, resolution set to 60 K; daughter ions were detected by Orbitrap, resolution set to 50 K, HCD collision mode, energy 36%.

Two types of liquid chromatography mobile phases were first prepared. Phase A: an aqueous solution containing 0.1% formic acid (Fluka, USA) and 2% acetonitrile; Phase B: an aqueous solution containing 0.1% formic acid and 90% acetonitrile. Peptides were dissolved in phase A and separated using the EASY-nLC 1000 UPLC system (Thermo Fisher Scientific, USA) at a constant flow rate of 400 nL/min. The separation gradient was set to increase from 8 to 16% in phase B within 30 min, then increased to 30% within 25 min and 80% within 2 min, which was maintained for 3 min. The peptides were injected into the nanospray ionization source for ionization at a voltage of 2.0 kV. The precursor ions and the secondary fragments of the peptides were detected and analyzed using the Orbitrap Fusion Lumos high-resolution mass spectrometer (Thermo Fisher Scientific, USA). According to the data dependent acquisition (DDA) mode, the precursor ions with top 20 signal intensities after primary scan were fragmented with 32% fragmentation energy in the HCD collision cell. The secondary MS/MS scan then followed. The MS scan parameters are shown in Table 2.Table 2

MS scan parameters.

Parameter	Value
Range
Primary MS	350–1550 m/z
MS/MS	100 m/z (fixed starting point)
Resolution
Primary MS	60,000
MS/MS	15,000
Automatic gain control	50,000
Signal threshold	50,000 ions/s
Maximum injection time	70 ms
Dynamically exclude time	30 s