Protein Separation and Mass Analysis

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.