Symmetry c18 resin

Each fraction was further subjected to a TripleTOF 5600 mass spectrometer system (AB SCIEX, USA) equipped with a nanoACQuity UPLC system (Waters, USA) according to previously described [2 (link)]. Briefly, the peptide sample from each SCX fraction was loaded onto a nanoACQuity UPLC BEH130 column (Waters, USA) packed with Symmetry C18 resin (Waters, USA). A Triple TOF 5600 platform was used for peptide identification. The ion spray voltage was set at 2.5 kV, the curtain gas was set at 30 psi, the nebulizer gas was set at 15 psi, and the interface heater temperature was set at 150 °C, respectively. For TOF–MS scans, the resolving power (RP) was greater than or equal to 30,000 FWHM. 250 ms was required for survey scans for the information dependent acquisition (IDA) analysis. 30 products were collected if the ion scans were more than 120 counts per second and with a 2+ to 5+ charge state. The Q2 transmission window was set at 100 Da for 100%. A sweeping collision energy setting of 35 ± 5 eV coupled with iTRAQ adjusted rolling collision energy was applied to all precursor ions for collision-induced dissociation. The parent ion dynamic exclusion was set to half of the peak time, and then the precursor was refreshed off the exclusion list.

LC-MS/MS analysis was carried out using a nanoAcquity HPLC (Waters, Milford, MA) and a LTQ-Orbitrap XL mass spectrometer (Thermo Scientific). For each analysis, 5 μL of tryptic digest was loaded onto a 180 μm x 20 mm trap column packed with 5 μm Symmetry C18 resin (Waters) using solvent A (0.1% formic acid in Milli-Q H₂O [Millipore, Billerica, MA]) followed by separation on a 75 μm x 250 mm analytical column packed with 1.7 μm BEH130 C18 resin (Waters). Peptides were eluted with either a 4 or 8 hour chromatographic gradient using solvent A and solvent B (0.1% formic acid in acetonitrile). For the 4 hour gradient, peptides were eluted using 5–28% B over 170 minutes, 28–50% B over 50 minutes, 50–80% B over 10 minutes, constant 80% B for 10 minutes, and 80–5% B over 5 minutes. For the 8 hour gradient, peptides were eluted using 5–28% B over 350 minutes, 28–50% B over 150 minutes, 50–80% B over 20 minutes, constant 80% B for 10 minutes, and 80–5% B over 5 minutes. A 30 minute blank gradient was run in between each sample injection to minimize carryover. Full scans were carried out from 400–2000 m/z with 60,000 resolution. MS2 data were acquired in data-dependent mode of the top six most intense ions with dynamic exclusion enabled for 60 s, monoisotopic precursor selection enabled, and single charged ions rejected.