MALDI-TOF High Mass Calibration up to 200 kDa Using Human Recombinant 16 kDa Protein Histidine Phosphatase Aggregates

Background: Protein histidine phosphatase (PHP) is an enzyme which removes phosphate groups from histidine residues. It was described for vertebrates in the year 2002. The recombinant human 16 kDa protein forms multimeric complexes in physiological buffer and in the gas phase. High-mass calibration in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has remained a problem due to the lack of suitable standards. Large proteins can hardly be freed of their substructural microheterogeneity by classical purification procedures so that their use as calibrants is limited. A small adduct-forming protein of validated quality is a valuable alternative for that purpose. Methodology/Principal Findings: Three major PHP clusters of ,113, 209 and .600 kDa were observed in gel filtration analysis. Re-chromatography of the monomer peak showed the same cluster distribution. The tendency to associate was detected also in MALDI-TOF MS measuring regular adducts up to 200 kDa. Conclusions/Significance: PHP forms multimers consisting of up to more than 35 protein molecules. In MALDI-TOF MS it generates adduct ions every 16 kDa. The protein can be produced with high quality so that its use as calibration compound for high mass ranges above 100 kDa, where standards are difficult to obtain, is feasible

Influence of protein histidine phosphatase overexpression and downregulation on human umbilical vein endothelial cell viability.

Protein histidine phosphatase (PHP) discovered previously has been shown to be expressed in mammalian tissues. Particularly, in blood vessel walls PHP was markedly expressed. Therefore, we made an attempt to find out whether PHP plays a significant role in endothelial cells. We demonstrated by Western Blot and immunofluorescence analysis that PHP was present in human umbilical vein endothelial cells (HUVECs). Overexpression of PHP by using the vector pIRES2-AcGFP1-PHP induced apoptosis in HUVECs. To exclude that an increase of the cellular protein content only could unspecifically cause cell damage the inactive H53A mutant of PHP was also overexpressed. However, overexpression of the inactive PHP mutant did not lead to apoptosis. Downregulation of PHP by RNAi-technique did not affect cell viability. In conclusion, HUVECs are damaged by overexpression but not by downregulation of PHP suggesting a pronounced impact of the enzyme on the cells when its activity is increased

Protein kinase CK2 phosphorylates BAD at threonine-117

Reversible phosphorylation of the 22 kDa BAD protein is crucial for cell survival. Five phosphorylation sites, all serines, had been identified. Here we report on number six. It is threonine-117 phosphorylated by the constitutively active kinase, CK2. Phosphoamino acid analysis and phospho-specific antibodies confirmed Thr117 as additional phosphorylation site. Immunoprecipitation furthermore revealed that BAD is phosphorylated at Thr117 in cultured cortical neurons. PP1, PP2A and PP2C dephosphorylated BAD at Thr117, but PP2B did not. The discovery of the constitutively active CK2 phosphorylating BAD is shedding an unexpected light in the otherwise strictly signal-regulated phosphorylation events on BAD