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High-pH reverse-phase liquid chromatography (RPLC) fractionation

JJ Jae Hun Jung
YJ Yong Woo Ji
HH Ho Sik Hwang
JO Jae Won Oh
HK Hyun Chang Kim
HL Hyung Keun Lee
KK Kwang Pyo Kim
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Peptide separation was carried out using high-pH RPLC fractionation based on peptide hydrophobicity. Samples were divided into six fractions using the Agilent 1260 series HPLC system (Agilent Technologies, Santa Clara, CA, USA). Briefly, an Accucore™ 150 C18 LC column (150 mm × 2.1 mm, 4 μm) was used for fractionation with the high-pH buffer A, B; 10 mM ammonium formate (pH = 10) as mobile phase A and 10 mM ammonium formate in 90% ACN (pH = 10) as mobile phase B. The gradient was as follows: 0–10 min, 5% B; 10–70 min, 35% B; 70–80 min, 70% B; 80–85 min, 70% B; 85–90 min, 5% B; 90–105 min, 5% B. The separated peptides were collected and dried in a speed-vac. Each fraction was desalted with a C18 spin column.

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