Computational protocol: O-GlcNAcylation of SIRT1 enhances its deacetylase activity and promotes cytoprotection under stress

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Protocol publication

[…] The His-tagged SIRT1 and MBP-fused OGT were co-expressed in E. coli BL21(DE3) strain, and recombinant SIRT1 was purified by Ni-affinity chromatography. After electrophoresis using SDS-PAGE and staining with Bio-Safe Coomassie blue R250 Stain (Bio-Rad), the SIRT1 protein band was excised and manually digested in the gel with chymotrypsin (Promega). Sample fractions were analyzed by LC-MS/MS. Chromatography was performed using a LC-20AD nano HPLC (Shimadzu) at a flow rate of 400 nL/min. The column was a BEH C18 Column 75 μM × 100 mm (Waters). Peptides were eluted by a gradient from 2 to 35% solvent B (98% ACN, 0.1% formic acid) for 44 min followed by a short wash at 80% solvent B and then solvent A (2% ACN, 0.1% formic acid), before returning to starting conditions. Then the eluted peptide components were analyzed by using either an LTQ Orbitrap XL™ ETD mass spectrometer (Thermo Scientific) equipped with an ETD option or a TripleTOF 5600 (AB SCIEX, Concord, ON) equipped with Nanospray III source. For electrospray ionization (ESI) MS/MS analysis performed on the TripleTOF 5600, the nanospray voltage was typically 2500 V in the nano-LC (liquid chromatography) ESI MS/MS mode. For LTQ Orbitrap XL™ ETD mass spectrometer analysis, the ion source voltage is set to 1500 V. MS/MS data were searched using the pFind program for O-GlcNAcylated peptides, and the spectra were annotated using pLabel–. […]

Pipeline specifications

Software tools pFind, pLabel
Application MS-based untargeted proteomics
Organisms Homo sapiens, Mus musculus