Peptide Fractionation and Analysis by LC-MS/MS

LC-MS/MS analyses were performed using a nanoLC Ultra 1D plus (Eksigent Technologies, AB SCIEX, Foster City, CA) coupled to a SCIEX TripleTOF 5600 Mass Spectrometer System (RRID : SCR_018053) via a Nanospray III source. Tryptic peptides were solubilized using solvent A (2% acetonitrile [ACN] in water, 0.1% FA) and the concentration was determined using Thermo Fisher Qubit fluorimeter (RRID : SCR_018095), according to manufacturer’s instructions. Tryptic peptides (1 µg) were loaded on a C18 Acclaim PepMap™ 100 trapping column (Thermo Scientific, 100 µm I.D. × 2 cm, 5 µm particle diameter, 100 Å) using solvent A at 2 µL/min and, after desalting, switched online with an Acquity UPLC^® M-Class Peptide BEH C18 analytical Column (Waters, 75 µm × 15 cm, 1.7 µm, 130 Å). Peptides were fractionated at a flow rate of 250 nL/min in a 250 min gradient with increasing concentrations of ACN (2% to 90%). TripleTOF 5600 system was operated in positive ion mode as follows: ion spray voltage 2300 V, curtain gas (CUR) 35, interface heater temperature (IHT) 150°C, ion source gas 1 (GS1) of 25, and declustering potential (DP) of 100 V. Data were acquired in information-dependent acquisition (IDA) mode with Analyst^®TF 1.7 Software (SCIEX, USA; RRID : SCR_015785). IDA parameters were: survey scan in the mass range of 350–1250 m/z, accumulation time 250 ms, followed by MS2 spectrum accumulation for 100 ms (100–1800 m/z) in a cycle of 4.04 sec. MS/MS fragmentation criteria were: ions in the 350-1250 m/z range with a charge state of 2–5 and an abundance threshold greater than 90 counts. Dynamic exclusion was set to 15s. IDA rolling collision energy (CE) parameter script was used to control the CE.