Mass Spectrometry Protocol for TMT Analysis

Mass spectrometry data were acquired using an Orbitrap Lumos coupled with a Proxeon EASY-nLC 1000 LC pump (Thermo Fisher Scientific, San Jose, CA). Peptides were separated on a 75 mm inner diameter microcapillary column packed with 0.5 cm of Magic C4 resin (5 mm, 100 A°, Michrom Bioresources) followed by approximately 20 cm of GP118 resin (1.8 mm, 120 A°, Sepax Technologies). Peptides were separated using a 3 hr gradient of 6 to 30% acetonitrile in 0.125% formic acid at a flow rate of 300 nl/min. Each analysis used an MS3-based TMT method (McAlister et al., 2012 (link), Ting et al., 2011 (link)). The scan sequence began with an MS1 spectrum (Orbitrap analysis, resolution 120,000, 350-1400 Th, AGC target 5 x 10⁵, maximum injection time 100 ms). ‘Rapid’ was selected for MS2 analysis, which consisted of CID (quadrupole ion trap analysis, AGC 1.8 x 10⁴, NCE 35, maximum injection time 120 ms). For MS3 analysis, precursors were fragmented by HCD prior to Orbitrap analysis (NCE 55, max AGC 2 x 10⁵, maximum injection time 150 ms, isolation specificity 0.7 Th, resolution 50,000).