High-performance liquid chromatography–mass spectrometry and electron-capture dissociation tandem mass spectrometry of osteocalcin: Determination of γ-carboxyglutamic acid residues

Two mass spectrometry methods, high-performance liquid chromatography combined on-line with electrospray ionization mass spectrometry (HPLC–ESI-MS) and electron-capture (EC) dissociation tandem mass spectrometry (MS–MS), were applied for structural analysis of bovine and human osteocalcins. Osteocalcin contains γ-carboxyglutamic acid (Gla) residues, which bind metal ions, among its amino acids. Ethylenediaminetetraacetic acid (EDTA) was added to all samples in order to chelate bound metal ions. After elimination of interfering metal ions MS spectra became uncomplicated to interpret. EDTA is incompatible with ESI and it was removed from samples using either on-line HPLC or micropurification method. The number of Gla residues varies in osteocalcin. These subforms, which contain different amounts of Gla residues, were separated using the HPLC–ESI-MS method. In order to determine locations of Gla residues in human osteocalcin, which contained two Gla residues, dissociation MS–MS method was successfully applied.