The tryptic peptides were then fractionated into several fractions by high-pH reverse-phase HPLC using Agilent 300 Extend C18 columns (5 μm particles, 4.6 mm ID, 250 mm length). Briefly speaking, peptides were first separated into 60 fractions with a gradient of 8%–32% acetonitrile (ACN, pH 9.0) for over 60 min. Afterwards, the peptide fractions were combined into 4 fractions and dried by vacuum centrifuging.
Enrichment was implemented by immunoprecipitation in accordance with previous studies [28 (link), 29 (link)]. Briefly, to enrich lysine-succinylation modified peptides, tryptic peptides were dissolved in NETN buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, 0.5% NP-40, and pH 8.0) and then incubated overnight with prewashed antisuccinyl lysine antibody agarose beads (catlog no. PTM402; PTM Bio, Hangzhou, China) at 4°C with gentle shaking. Finally, the bound peptides were eluted from the beads with 0.1% trifluoroacetic acid (TFA), combined, and vacuum-dried. Before LC-MS/MS analysis, the obtained peptides were desalted with C18 ZipTips (Millipore) according to the manufacturer's instructions.
Enrichment was implemented by immunoprecipitation in accordance with previous studies [28 (link), 29 (link)]. Briefly, to enrich lysine-succinylation modified peptides, tryptic peptides were dissolved in NETN buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, 0.5% NP-40, and pH 8.0) and then incubated overnight with prewashed antisuccinyl lysine antibody agarose beads (catlog no. PTM402; PTM Bio, Hangzhou, China) at 4°C with gentle shaking. Finally, the bound peptides were eluted from the beads with 0.1% trifluoroacetic acid (TFA), combined, and vacuum-dried. Before LC-MS/MS analysis, the obtained peptides were desalted with C18 ZipTips (Millipore) according to the manufacturer's instructions.
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