Quantitative Mass Spectrometry Analysis

MRM analyses were performed on a 6500 QTRAP mass spectrometer (SCIEX) equipped with an electrospray source, a 1290 Infinity UPLC system (Agilent Technologies, Santa Clara, CA), and a XBridge Peptide BEH300 C18 (3.5 μm, 2.1 mm × 150 mm) column (Waters, Milford, MA). Liquid chromatography was carried out at a flow rate of 400 µL/min, and the sample injection volume was 30 µL. The column was maintained at a temperature of 60 °C. Mobile phase A consisted of 0.1% formic acid (Sigma Aldrich) in water (Thermo Fisher Scientific), and mobile phase B consisted of 0.1% formic acid in acetonitrile (Thermo Fisher Scientific). The gradient with respect to %B was as follows: 0–1.5 min, 5%; 1.5–2 min, 5–15%; 2–5 min, 15%; 5–7.1 min, 15–20%; 7.1–8.1 min, 20–80%; 8.1–9.0 min, 80%; and 9.0–9.1 min, 80–5%. 9.1–16 min, 5%. The instrument parameters for 6500 QTRAP mass spectrometer were as follows: Ion spray voltage of 5500 V, curtain gas of 20 psi, collision gas set to “medium”, interface heater temperature of 400 °C, nebulizer gas (GS1) of 80 psi and ion source gas (GS2) of 80 psi, and unit resolution for both Q1 and Q3 quadrupoles as previously described in Ravipaty et al. 2017^{19 (link)}.