Basic pH Reverse-Phase HPLC Fractionation

A 500 μg aliquot of pooled sample was fractionated using basic pH reverse-phase HPLC using previously established procedures [49 (link)]. Briefly, the sample was loaded onto a 4.6 mm × 250 mm Xbridge C18 column (Waters, 3.5 μm bead size) using an Agilent 1260 Infinity Bio-inert HPLC and separated over a 70 min linear gradient from 10 to 50% solvent B at a flow rate of 0.5 ml/min (Buffer A  =  10 mM ammonium formate, pH 10.0; Buffer B  =  90% ACN, 10 mM ammonium formate, pH 10.0). A total of 40 fractions were collected throughout the gradient. The early, middle and late eluting fractions were concatenated and combined into 10 final fractions. The combined fractions were concentrated in the SpeedVac and stored at -80°C until further analysis.