HPLC Fractionation and Affinity Enrichment

The protein sample was fractionated into factions by reverse-phase HPLC (high pH) using Agilent 300Extend C18 column (5 μm particles, 4.6 mm ID, 250 mm length). Briefly, peptides were first separated with a gradient of 2% to 60% acetonitrile in ammonium bicarbonate (10 mM, pH 10) over 80 min into 80 fractions. Then, the peptides were combined into 8 fractions and dried by vacuum centrifuging.

To enrich Ksu peptides, a NETN buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, 0.5% NP-40, pH 8.0) was used to dissolve the tryptic peptides. Then, the tryptic peptides were incubated with pre-washed antibody beads (PTM Biolabs) at 4 °C overnight with gentle shaking. The beads were washed four times with NETN buffer and twice with ddH₂O. The bound peptides were eluted from the beads with 0.1% trifluoroacetic acid (TFA). The eluted fractions were combined and vacuum-dried. The resulting peptides were cleaned with C₁₈ ZipTips (Millipore) according to the manufacturer’s instructions, followed by LC-MS/MS analysis.