Nano-LC mass spectrometry

High pH reversed-phase fractions were further fractionated using an Ultimate 3000 nano high performance LC system in line with an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific). Peptides in 1% (vol/vol) formic acid were injected onto an Acclaim PepMap C18 nano-trap column (Thermo Scientific). After washing with 0.5% (vol/vol) acetonitrile, 0.1% (vol/vol) formic acid peptides were resolved on a 250-mm × 75-μm Acclaim PepMap C18 reversed-phase analytical column (Thermo Scientific) over a 150-minute organic gradient, using 7 gradient segments (1%-6% solvent B over 1 minute, 6%-15% B over 58 minutes, 15%-32% B over 58 minutes, 32%-40% B over 5 minutes, 40%-90% B for 1 minute, held at 90% B for 6 minutes, and then reduced to 1% B for 1 minute) with a flow rate of 300 nL per minute. Solvent A was 0.1% formic acid, and solvent B was aqueous 80% acetonitrile in 0.1% formic acid. Peptides were ionized by nanoelectrospray ionization at 2.0 kV using a stainless-steel emitter with an internal diameter of 30 μm (Thermo Scientific) and a capillary temperature of 275°C.

All spectra were acquired using an Orbitrap Fusion Tribrid mass spectrometer controlled by Xcalibur 2.0 software (Thermo Scientific) and operated in data-dependent acquisition mode using a synchronous precursor selection–MS3 workflow. Fourier transform mass analyzers (FTMS)1 spectra were collected at a resolution of 120 000, with an automatic gain control (AGC) target of 200 000 and a maximum injection time of 50 milliseconds. Precursors were filtered with an intensity threshold of 5000, according to charge state (to include charge states 2-7) and with monoisotopic precursor selection. Previously interrogated precursors were excluded using a dynamic window (60 seconds ± 10 ppm). The MS2 precursors were isolated with a quadrupole mass filter set to a width of 1.2 m/z. Ion-trap tandem mass spectrometry (ITMS2) spectra were collected with an AGC target of 10 000, the maximum injection time of 70 milliseconds, and collision-induced dissociation collision energy of 35%.

For FTMS3 analysis, the Orbitrap was operated at 50 000 resolution with an AGC target of 50 000 and a maximum injection time of 105 milliseconds. Precursors were fragmented by high-energy collision dissociation at a normalized collision energy of 60% to ensure maximal TMT reporter ion yield. Synchronous precursor selection was enabled to include up to 5 MS2 fragment ions in the FTMS3 scan.