Eksigent nanolc 425

Individual samples were analyzed in randomized order. The peptide mixtures in 2% ACN in 0.5% FA were loaded for 10 min at 2 μL/min on Acclaim PepMap 100 C18 (5 μm, 0.1 × 20 mm; Thermo Fisher Scientific) trap column. The separation was performed on an in-house-packed 25-cm fused-silica column (75-μm inner diameter) with ProntoSIL 120 Å 3 μm C18 AQ beads (Bischoff Analysentechnik GmbH) in a trap-elute mode, using the Eksigent nano-LC 425 (Sciex) on-line connected to 5600+ TripleTOF (Sciex). A linear gradient was set to 5–35% ACN in 0.1% FA over 120 min and 35–50% ACN in 0.1% FA over 10 min at a flow rate of 200 nL/min. For data-dependent acquisition (DDA) mode, the top 30 precursors with accumulation time 300 ms and mass range 400–1,250 in high-sensitivity mode were fragmented (MS/MS accumulation time 150 ms and mass range 170–1,500 Da) in each cycle (exclusion time 13 s). For Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS), 35 variable windows calculated with a SWATH Variable window calculator (Sciex) were monitored with 150 ms accumulation time in MS and 100 ms accumulation time in MS/MS with mass ranges of 400–1,250 Da and 170–2,000 Da, respectively, and a cycle time of 3.5 s.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed on a TripleTOF 6600 (Sciex) coupled to an Eksigent nanoLC 425 (Sciex) operating in microLC flow mode. Twelve microliters of each sample was injected onto a ChromXP C18CL column (3 μm, 120 Å, 150 mm × 0.3 mm; Sciex). Gradient elution was performed with mobile phase A (0.1% formic acid in water; Fisher) and mobile phase B (0.1% formic acid in ACN; Fisher) at a flow rate of 5 μl/min. A program of 3% mobile phase B to 35% mobile phase B in 5 min, 35% mobile phase B to 80% mobile phase B in 1 min, and 80% mobile phase B for 2 min was used to elute peptides. The eluate was ionized with a dual spray source. The mass spectrometer was operated in positive ion mode with a full MS1 scan experiment in a mass range of 400 to 1,250 m/z with a scan time of 250 ms followed by MS/MS experiments in the mass range of 100 to 1,500 m/z with a scan time of 50 ms. The top 30 precursor ions were selected for fragmentation.