Abi 4800 maldi tof tof analyzer

NADase, SLO, or NADase plus SLO, each at concentrations of 12 μM in PBS, 1 mM DTT, pH 7.4, was cross-linked with 0.005% glutaraldehyde for 10 min at room temperature. Reactions were stopped with Laemmli buffer and analyzed by SDS-PAGE. For MALDI-TOF mass spectrometry analysis, the reaction volume was 500 μl; reactions were stopped after 10 min with addition of Tris to 100 mM and then concentrated and buffer exchanged into 20 mM Tris, pH 6.8, using a spin concentrator (molecular weight cutoff [MWCO], 10,000; Pierce). The samples were then diluted 1:10 in 0.1% trifluoroacetic acid (TFA) and spotted with α-cyano-4-hydroxycinnamic acid (CHCA) on an Opti-TOF 384-well insert (AB Sciex). Masses were determined using standard protocols (51 (link)) on an ABI 4800 MALDI-TOF/TOF analyzer (Applied Biosystems).

Following the treatment with celastrol-PEG4-alkyne, the cellular proteins were subjected to Click chemistry biotinylation and proteomic identification as previously described (Yang et al., 2015 (link)). In brief, RAW264.7 cells were treated with 10 μM celastrol-PEG4-alkyne for 24 hr and lysed with RIPA buffer while iodoacetamide was supplemented to block free cysteine residues in the proteins. The cellular proteins were biotinylated with biotin-azide under Click chemistry conditions (sodium ascorbate, CuSO₄, and triazole ligand) to allow ‘azide-alkyne cycloaddition’ to occur. Biotin-labeled proteins were purified by affinity isolation on streptavidin-coated superparamagnetic beads and identified on ABI4800 MALDI TOF/TOF Analyzer (Applied Biosystems, Foster City, CA) through technical support for proteomics from Center of Genomic Sciences, University of Hong Kong.