Liquid chromatography coupled with multi-stage mass spectrometry
(LC-MS3)
An Ultimate 3000 RSLC nano UHPLC was used for online fractionation,
equipped with a 300 μm ID x 5 mm Acclaim PepMap μ-Precolumn (Thermo) and
a 75 μm ID x 50 cm 2.1 μm particle Acclaim PepMap RSLC analytical column
(Thermo). Loading solvent was 0.1% formic acid. Analytical solvent
contained HPLC grade H2O, acetonitrile, and formic acid.
Samples were loaded at 5 ml/min for 5 min in loading solvent before
beginning the analytical gradient. Formic acid concentration was
maintained at 0.1% during the analytical gradient while concentration
of acetonitrile gradually increased. All separations were carried out at
55 °C. Mass spectrometry data were acquired using Orbitrap Lumos mass
spectrometer (Thermo). TMT-based analysis used a MultiNotch MS3-based
method (McAlister et al., 2014). MS1 scans surveyed 380-1500 Th, with
resolution of 120,000, automatic gain control (AGC) target of 2 x
105, and maximum injection time of 50 ms. Ions that
had the counts of 5 x 103 counts and above triggered
MS2 analysis, with Quadrupole isolation at an isolation width of 0.7 Th,
normalised collision energy (NCE) set to 35% for CID fragmentation,
1.5x104 AGC target, and 120 ms maximum injection time.
Top 6 MS2 ions were selected for HCD fragmentation (NCE 65%) in MS3.
MS3 resolution was 60,000, with an AGC target of 1 x
105 and a maximum accumulation time of 150 ms. The
entire MS/MS/MS cycle had a target time of 3 sec. Dynamic exclusion was
set to +/- 10 ppm for 70 sec.