Trypsin-based method was used to digest protein. Before digestion, protein solution was first reduced for 1 h at 37 °C with 10 mM DTT. Proteins then were alkylated at room temperature in darkness with 20 mM iodoacetamide (IAA) for 45 min. Afterward, a two-step trypsin digestion was performed as previously described [16 (link)].
To enrich succinylated peptides, tryptic peptides dissolved in NETN buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, 0.5% NP-40, pH 8.0) were incubated overnight with prewashed antibody beads (PTM Biolabs) at 4 °C. After washing four times with NETN buffer and twice with ddH2O, the bound peptides were eluted from the beads with 0.1% TFA.
After cleaning with C18 ZipTips (Millipore), the enriched peptides were analyzed following the procedure as described, with minor modification [16 (link)]. In brief, the peptides dissolved in 0.1% formic acid (FA) were firstly separated using a reversed-phase analytical column (Acclaim PepMap RSLC, Thermo Scientific) at a constant flow rate of 700 nl/min on EASY-nLC 1000 UPLC system. Then, the resulting peptides were subjected to MS/MS by an Orbitrap Fusion mass spectrometer (ThermoFisher Scientific). Detection of intact peptides and ion fragments in the Orbitrap were carried out at a resolution of 60,000 and 15,000, with the NCE setting as 35. For MS scans, the m/z scan range was 350 to 1550.
To enrich succinylated peptides, tryptic peptides dissolved in NETN buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, 0.5% NP-40, pH 8.0) were incubated overnight with prewashed antibody beads (PTM Biolabs) at 4 °C. After washing four times with NETN buffer and twice with ddH2O, the bound peptides were eluted from the beads with 0.1% TFA.
After cleaning with C18 ZipTips (Millipore), the enriched peptides were analyzed following the procedure as described, with minor modification [16 (link)]. In brief, the peptides dissolved in 0.1% formic acid (FA) were firstly separated using a reversed-phase analytical column (Acclaim PepMap RSLC, Thermo Scientific) at a constant flow rate of 700 nl/min on EASY-nLC 1000 UPLC system. Then, the resulting peptides were subjected to MS/MS by an Orbitrap Fusion mass spectrometer (ThermoFisher Scientific). Detection of intact peptides and ion fragments in the Orbitrap were carried out at a resolution of 60,000 and 15,000, with the NCE setting as 35. For MS scans, the m/z scan range was 350 to 1550.
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