For mass spectrometry analysis, bacterially purified GST‐PDK1 was used for in vitro oxidative assays in the presence/absence of copper, and the resulting products were subjected for mass spectrometry analysis. The band containing PDK1 was reduced with 10 mm DTT for 30 min, alkylated with 55 mm iodoacetamide for 45 min, and in‐gel‐digested with trypsin enzymes. The resulting peptides were extracted from the gel and analyzed by microcapillary reversed‐phase (C18) LC‐MS/MS), using a high resolution QExactive HF Orbitrap (Thermo Fisher Scientific) in positive ion DDA mode (Top 8) via higher energy collisional dissociation (HCD) coupled to a Proxeon EASY‐nLc II nano‐HPLC.[57 (link)
] MS/MS data were searched against the Uniprot Human protein database (version 20151209 containing 21 024 entries) using Mascot 2.5.1 (Matrix Science) and data analysis was performed using the Scaffold 4.4.8 software (Proteome Software). Peptides and modified peptides were accepted if they passed a 1% FDR threshold.
] MS/MS data were searched against the Uniprot Human protein database (version 20151209 containing 21 024 entries) using Mascot 2.5.1 (Matrix Science) and data analysis was performed using the Scaffold 4.4.8 software (Proteome Software). Peptides and modified peptides were accepted if they passed a 1% FDR threshold.
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