LC-MS/MS-based in vitro kinase assays performed with recombinant maltose-binding protein-tagged P. falciparum calcium dependent protein kinase 4 using synthetic peptides as substrates
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ABSTRACT: In vitro kinase assays were performed with recombinant maltose-binding protein (MBP) tag-fused PfCDPK4 protein using synthetic peptides as substrates. Kinase reaction buffer contained CaCl2 or the calcium chelator EGTA. Triplicate reactions with either the CaCl2 buffer or the EGTA buffer were performed in parallel and the peptides were detected and quantified by LC-MS/MS. The suitability of a peptide as a substrate for PfCDPK4 was quantified as the depletion of the unmodified peptide as it was converted to phosphopeptide. Additionally, autophosphorylation of PfCDPK4 was assessed by performing the reaction in the absence of substrates followed by tryptic digest and LC-MS/MS of the resulting peptides to identify phosphorylation sites.
INSTRUMENT(S): Q Exactive HF
ORGANISM(S): Plasmodium Falciparum (ncbitaxon:5833)
SUBMITTER:
Kristian Swearingen
PROVIDER: MSV000087575 | MassIVE | Sat Jun 05 12:23:00 BST 2021
SECONDARY ACCESSION(S): PXD026499
REPOSITORIES: MassIVE
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