Multinotch-MS3 for Accurate Peptide Quantification

In order to obtain superior quantitation accuracy, we employed multinotch-MS3 which minimizes the precursor ion interference that occurs from fragmentation of co-isolated peptides during MS analysis^{62 (link)}. Orbitrap Fusion (Thermo Fisher Scientific) and RSLC Ultimate 3000 nano-UPLC (Dionex) was used to acquire the data. 2 μL of the sample was resolved on a PepMap RSLC C18 column (75 μm i.d. × 50 cm; Thermo Fisher Scientific) at the flow-rate of 300 nl/min using 0.1% formic acid/acetonitrile gradient system (2–22% acetonitrile in 150 min;22–32% acetonitrile in 40 min; 20 min wash at 90% followed by 50 min re-equilibration) and directly spray onto the mass spectrometer using EasySpray source (Thermo Fisher Scientific). 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; NCE 35; AGC 5 × 10³; max IT 100 ms). For multinotch-MS3, top 10 precursors from each MS2 were fragmented by HCD followed by Orbitrap analysis (NCE 55; 60 K resolution; AGC 5 × 10⁴; max IT 120 ms, 100–500 m/z scan range). Since the TMT modification is used as a “fixed modification”, unlabeled peptides are not identified or used.