Velos orbitrap elite

MudPIT was carried out as described previously^{28 (link)} with the following modifications. Peptide mixtures were loaded onto a 250 μm i.d. capillary packed first with 3.5 cm of 5 μm Luna SCX, followed by 2.5 cm of 5 μm Aqua C18. The biphasic column was washed with buffer A for more than 20 column volumes. After desalting, the biphasic column was connected via a 2-μm filtered union (UpChurch Scientific) to a 100 μm i.d. column, which had been pulled to a 5 μm tip, then packed with 10 cm of 5 μm C18 RP particles (Aqua). The split three-phase column was placed in line with an Agilent 1200 quaternary HPLC pump (Palo Alto, CA) and a Velos Pro Orbitrap (VPO) mass spectrometer or a Velos Orbitrap Elite (VOE) mass spectrometer (Thermo Fisher Scientific). The 200 ng and 3 ug HeLa digests were analyzed using 10- and 12-step MudPIT, on VPO and VOE, respectively. The solvent solutions used were described above with the additional buffer C for salt bumps consisting of buffer A with 500 mM ammonium acetate.

All peptide samples were dried using a SpeedVac vacuum concentrator (Thermo Scientific Savant) and resuspended in 150 ml 5% acetonitrile, 0.1% formic acid (Buffer A). Peptides were loaded on a split-triple-phase fused-silica micro-capillary column (Florens and Washburn, 2006 (link)) and placed in-line with an Agilent 1260 series quaternary HPLC pump and Velos Pro Orbitrap or Velos Orbitrap Elite mass spectrometers (Thermo Fisher Scientific). A fully automated 12-step MudPIT chromatography run was carried out, as described in (Florens and Washburn, 2006 (link)). Each full MS scan (400–1600 m/z) was followed by 15 data-dependent MS/MS scans. MS1 scans were acquired in Orbitrap at 60,000 resolution, while ddMS2 were acquired in the ion trap with Rapid Scan settings. The number of the micro scans was set to 1 for both MS and MS/MS. MS1 AGC target was set to 1.00E+06, MS2 AGC targets at 1.00E+04, and MS2 max injection times at 150 ms. MS1 charge states were between 2–5. MS2 collision energy was set at 35%. Dynamic exclusion settings were: 2 repeat counts; 30 s repeat duration; exclusion list size of 500; and 90 s exclusion duration.

Protein (0.2−0.3 μg) diluted in water at 0.1 μg/μL was loaded on a Waters NanoAcquity LC equipped with a C2 reversed phase column (packed in-house, length 70 cm, 75 μm ID, 3 μm C2 particle). Intact proteins were separated using water/acetonitrile mobile phases with 0.1% formic acid over a gradient of 1 h (ramping acetonitrile gradient 10−50%) at a flow rate of 300 nL/min. Mass spectra of eluting proteins were collected on a Thermo VelosOrbitrap Elite. MS¹ spectra were acquired at 120000 resolution (at m/z 200) and were averaged over 8 microscans. ProMex^{35 (link)} was used to deconvolute the data and obtain the intact mass of the proteins. LCMS data were aggregated across retention time and binned into unit mass (sum intensities within 1 Da) to generate the intact mass plot (Supplementary Figure 5). Raw data and processing results are deposited in MassIVE (massive.ucsd.edu) with accession MSV000085233.