Multinotch-MS3 for Protein Quantification

To increase the accuracy and confidence in measurements of protein abundance, a multinotch-MS3 method was employed for analyzing MS data [33 (link)]. Raw data were acquired by using RSLC Ultimate 3000 nano-UPLC (Dionex) coupled to Orbitrap Fusion MS (Thermo Fisher Scientific, MA). Two microliters from each fraction were resolved on an Acclaim PepMap C18 reverse phase column (2 microns, 75 μm i.d. × 50 cm) using a 0.1% formic/acetonitrile gradient at 300 nl/min. The mass spectrometer was set to collect one MS1 scan (Orbitrap; 120 K resolution; AGC target 2×10⁵; max IT 100 ms) followed by data-dependent, “Top Speed” (3 s) MS2 scans (collision induced dissociation; ion trap; NCD 35; AGC 5×10³; max IT 100 ms). For multinotch-MS3, the top ten precursors from each MS2 scan were fragmented by high-energy collisional dissociation (HCD) followed by Orbitrap analysis (NCE 55; 60,000 resolution; AGC 5×10⁴; max IT 120 ms, 100–500 m/z scan range).