Tryptic peptides were analyzed by nLC-MS/MS using a NanoAcquity UPLC (Waters’ Corporation, Milford, MA) interfaced to a Velos Pro linear ion trap mass spectrometer (Thermo Fisher Scientific, Waltham, MA). For each analysis, a 1 μL volume of protein digest was injected onto a C18 column (PicoChip column packed with Reprosil C18 3μm 120 Å chromatography media in a 10.5 cm long, 75 μm ID column with a 15 μm tip, New Objective, Inc., Woburn, MA) and then eluted off to the mass spectrometer using a 37-minute linear gradient of 3–35% ACN/0.1% FA at a flow rate of 300 nL/min.
The Velos Pro was operated in positive ionization mode with a spray voltage of 1.95 kV and capillary temperature of 275 °C. The acquisition consisted of cycles of one full-scan MS1 (AGC of 3×104, 75 ms maximum ion accumulation time, and m/z range of 375–1800) followed by eight MS/MS spectra recorded sequentially for the most abundant ions in the ion trap (minimum signal required 1000 counts, 1×104 AGC target, 100 ms maximum injection time, isolation width 2 m/z, normalized collision energy 35, and activation time 10 ms). Dynamic exclusion (30 s) was enabled to minimize the redundant selection of peptides previously selected for MS/MS.
The Velos Pro was operated in positive ionization mode with a spray voltage of 1.95 kV and capillary temperature of 275 °C. The acquisition consisted of cycles of one full-scan MS1 (AGC of 3×104, 75 ms maximum ion accumulation time, and m/z range of 375–1800) followed by eight MS/MS spectra recorded sequentially for the most abundant ions in the ion trap (minimum signal required 1000 counts, 1×104 AGC target, 100 ms maximum injection time, isolation width 2 m/z, normalized collision energy 35, and activation time 10 ms). Dynamic exclusion (30 s) was enabled to minimize the redundant selection of peptides previously selected for MS/MS.