Gel Shift Assay and LC-MS/MS for PdhD Modification Analysis

The reaction (100 μL) contained 50 mM sodium phosphate (pH 7.0), 5 mM disodium ATP, 1 mM MgCl₂, 1 mM lipoic acid, 20 μM apo-PdhD and 4 μM Mhp-LplJ. After incubation at 37°C for 3 h, the proteins in the reaction system were loaded on an 8% native polyacrylamide gel and separated by electrophoresis.

Nano-LC-LTQ-Orbitrap XL MS/MS was performed to detect lipoate modification of PdhD. Chymotrypsin and trypsin-digested peptides were separated by a C₁₈ reversed-phase column (filled with 3 μm ReproSil-Pur C₁₈-AQ from Dr. Maisch GmbH) and loaded by a C₁₈ reversed-phase column (filled with 5 μm ReproSil-Pur C₁₈-AQ from Dr. Maisch GmbH) onto the nanoLC-LTQ-Orbitrap XL system (Thermo). Data were analyzed by Proteome Discoverer (version 1.4.0.288, Thermo Fischer Scientific). The MS2 spectra were searched in the PdhD sequence plus Contaminants (cRAP) database using the SEQUEST search engine. Lipoylation of lysine and oxidation of methionine were set as variable modifications. The matching of searched peptide and MS spectra was filtered by Percolator calculation.