title: MALDI-TOF high mass calibration up to 200 kDa using human recombinant 16 kDa protein histidine phosphatase aggregates.
abstract: 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.
  
  
  RESULTS
  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
  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.
authors.affiliation: Institute for Pharmaceutical and Medical Chemistry, University of Münster, Münster, Germany.
authors.forename: Katrin
authors.initials: K
authors.lastname: Ludwig
authors.forename: Schähdi
authors.initials: S
authors.lastname: Habbach
authors.forename: Josef
authors.initials: J
authors.lastname: Krieglstein
authors.forename: Susanne
authors.initials: S
authors.lastname: Klumpp
authors.forename: Simone
authors.initials: S
authors.lastname: König
mesh_terms: D002138:Calibration; D002850:Chromatography, Gel; D006801:Humans; D008970:Molecular Weight; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry*; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D011522:Protons; D011786:Quality Control; D011994:Recombinant Proteins / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D011522:Protons; D011994:Recombinant Proteins; C504019:PHPT1 protein, human; D010744:Phosphoric Monoester Hydrolases