LC-MS/MS analysis

LC separation was done on a Waters Nano Acquity UHPLC (Waters Corporation) with a Proxeon nanospray source. Each SCX fraction (9 total) was reconstituted in 2 % acetonitrile /0.1 % trifluoroacetic acid and loaded onto a 100 μm × 25 mm Magic C18 100 Å 5U reverse phase trap. Peptides were eluted using a gradient of 0.1 formic acid (A) and 100 % acetonitrile (B) with a flow rate of 300 nL/min. A 60 min gradient was run with 5 to 35 B over 50 min, 35 to 80 B over 3 min, 80 B for 1 min, 80 to 5 B over 1 min, and finally held at 5 % B for 5 min.

Mass spectra were collected on an Orbitrap Q Exactive Plus mass spectrometer (Thermo Fisher Scientific). A dynamic exclusion of 15 s was used. MS spectra were acquired with a resolution of 70,000 and a target of 1 × 106 ions or a maximum injection time of 30 ms. MS/MS spectra were acquired with a resolution of 17,500 and a target of 5 × 104 ions or a maximum injection time of 50 ms, and a fixed first mass of 110 m/z. Peptide fragmentation was performed using higher-energy collision dissociation with a normalized collision energy value of 30. Unassigned charge states as well as +1 and ions > +5 were excluded from MS/MS fragmentation.