Rslc ultimate 3000 nano uplc

In order to obtain superior quantitation accuracy, we employed multinotch-MS3 (McAlister GC, ref below) which minimizes the reporter ion ratio distortion resulting from fragmentation of co-isolated peptides during MS analysis [23 (link)]. Orbitrap Fusion (Thermo Fisher Scientific) and RSLC Ultimate 3000 nano-UPLC (Dionex) was used to acquire the data. Two μl of the sample was resolved on a PepMap RSLC C18 column (75 μm i.d. × 50 cm; Thermo 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; 120K 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; 60K resolution; AGC 5 × 10⁴; max IT 120 ms, 100–500 m/z scan range).

To obtain superior quantitative accuracy, we employed multinotch-MS3 (McAlister GC), as described by McAlister et al. [23 (link)]. This technique minimizes the reporter ion ratio distortion resulting from fragmentation of co-isolated peptides during MS analysis. Orbitrap Fusion (Thermo Fisher Scientific) and RSLC Ultimate 3000 nano-UPLC (Dionex) were used to acquire the data. Two μL of the sample was resolved on a PepMap RSLC C18 column (75 μm i.d. × 50 cm; Thermo 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 sprayed onto the mass spectrometer using EasySpray source (Thermo Fisher Scientific). 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; 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).

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.

For data acquisition, an Orbitrap Fusion (ThermoFisher) and RSLC Ultimate 3000 nano-UPLC (Dionex) was used to obtain raw data. To increase accuracy and confidence in protein abundance measurements, a multinotch-MS3 method was employed for MS data analysis. Two microliters from each fraction were resolved in 2D on a nanocapillary reverse phase column (Acclaim PepMap C18, 2 micron, 75 μm i.d. × 50 cm, ThermoFisher) using a 0.1% formic/acetonitrile gradient at 300 nl/m (2–22% acetonitrile in 150 m, 22–32% acetonitrile in 40 m, 20 min wash at 90% followed by 50 min reequilibration) and directly sprayed onto Orbitrap Fusion with EasySpray (ThermoFisher; Spray voltage (positive ion) = 1900 V, Spray voltage (negative ion) = 600 V, method duration = 180 min, ion source type = NSI). The mass spectrometer was set to collect the MS1 scan (Orbitrap; 120 K resolution; AGC target 2 × 10⁵; max IT 100 ms), and then 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 10 precursor ions from each MS2 scan 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).