Mass spectrometry (MS)
Individual fractions from the strong cation exchange (SCX)
chromatography were desalted using ZipTips (ZTC18S096, EMD Millipore),
dried down and resuspended in 0.1% formic acid (Catalog no 94138,
Honeywell). Fractions were analyzed by LC-MS on an Orbitrap Fusion Lumos
(ThermoFisher) equipped with an Easy NanoLC1200 HPLC (ThermoFisher).
Peptides were separated on a 75 μm × 15 cm Acclaim PepMap100 separating
column (Thermo Scientific) downstream of a 2 cm guard column (Thermo
Scientific). Buffer A was 0.1% formic acid in water. Buffer B was 0.1%
formic acid in 80% acetonitrile. Peptides were separated on a two-hour
gradient from 0% B to 35% B. Peptides were collisionally fragmented
using HCD mode. Precursor ions were measured in the Orbitrap with a
resolution of 120,000. Fragment ions were measured in the Orbitrap with
a resolution of 50,000. The spray voltage was set at 2.2 kV. Orbitrap
MS1 spectra (AGC 1×106) were acquired from 400-1800
m/z followed by data-dependent HCD MS/MS (collision energy 42%,
isolation window of 0.7 Da) for a three second cycle time. Charge state
screening was enabled to reject unassigned and singly charged ions. A
dynamic exclusion time of 60 seconds was used to discriminate against
previously selected ions.