Professor Wolf Lehmann, from Molecular Structure Analysis at the German Cancer Research Center (DKFZ) Heidelberg, Germany, conducted a study to characterize in detail the in vitro phosphorylation of the recombinant Plasmodium falciparum protein GAP45 by a kinase from the same organism.
The study is documented in Analytical Biochemistry [393 (1), 41-47 (2009)] in which Plasmodium falciparum glideosome-associated protein 45 (PfGAP45) was in vitro phosphorylated by P. falciparum calcium-dependent protein kinase (PfCDPK1) and digested using the four proteases trypsin, chymotrypsin, AspN and elastase. Subsequently, phosphopeptide enrichment using Ga(III) immobilized metal affinity chromatography (IMAC) was performed. The resulting fractions were analysed using ultra performance liquid chromatography–electrospray ionization–tandem mass spectrometry (UPLC–ESI–MS/MS).
“The first level of information was the identification of 9 phosphorylation sites. The next findings were that two of these sites were phosphorylated sequentially and two sites were phosphorylated in a mutually exclusive way. Finally, it was observed that both tryptic and chymotryptic protein cleavage is substantially inhibited, when an acidic residue (phosphoserine, phosphothreonine, aspartic acid or glutamic acid) is located within a ± 3 position relative to the cleavage site,” Dr Lehmann explained about the significant outcomes.
For Lehmann, the results show that for an estimation of a site-specific phosphorylation degree, a possible interference of phosphorylation with proteolytic cleavage has to be considered. “Further, we learned that combinatorial aspects of multisite phosphorylation can be extracted from LC-MS/MS data by their manual inspection, in particular when proteolytic digests based on different proteases are available,” he concluded.