Easylc

Samples were separated briefly on a standard 10% SDS PAGE. Gel pieces, containing all the proteins in the sample, were cut out and digested using the Shevchenko protocol (Shevchenko et al., 2002 (link)). Digested peptides were analyzed using a QExactive mass spectrometer (Thermo Fisher Scientific) coupled with an Easy-LC (Thermo Fisher Scientific) and a nanospray ionization source. The peptides were loaded onto a trap (100micron, C18 100Å 5U) and desalted online before being separated using a reverse phase column (75micon, C18 200Å 3U). The gradient duration for separation of peptides was 60 minutes using 0.1% formic acid and 100% acetonitrile for solvents A and B, respectively. Data were acquired using a data dependent MS/MS method which had a full scan range of 300–1600Da and a resolution of 70,000. The MS/MS method’s resolution is 17,500 and an isolation width of 2 m/z with normalized collision energy of 27. The nanospray source was operated using 2.2 kV spray voltage and a heated transfer capillary temperature of 250°C.

Data were acquired using selected reaction monitoring (SRM) on a Proxeon EasyLC coupled to a Thermo Altis triple quadrupole mass spectrometer. Peptides were separated by reverse phase liquid chromatography using pulled tip columns created from 75 μm inner diameter fused silica capillary (New Objectives, Woburn, MA) in-house using a laser pulling device and packed with 3 μm ReproSil-Pur C18 beads (Dr. Maisch GmbH, Ammerbuch, Germany) to 30 cm. Trap columns were created from 150 μm inner diameter fused silica capillary fritted with Kasil on one end and packed with the same C18 beads to 3 cm. Solvent A was 0.1% formic acid in water (v/v), and solvent B was 0.1% formic acid in 80% acetonitrile (v/v). For each injection, approximately 1 μg total protein was loaded and eluted using a 90 min gradient from 5 to 40% B in 25 min, 40 to 60% B in 5 min, followed by a 15 min wash and then 15 min equilibration back to initial conditions. Total analytical run time was 45 min. Thermo RAW files were imported into Skyline^{11 (link)} (Skyline-daily version 4.1.1.18151) for processing and Total Area Fragment results were exported using a Custom Report.