Calcium-induced noncovalently linked tetramers of MRP8 and MRP14 detected by ultraviolet matrix-assisted laser desorption/ionization mass spectrometry

MRP8 and MRP14 are members of the S100 family of calcium-binding proteins w hich play an important role during calcium-induced activation of phagocytes . Both proteins form noncovalently associated complexes as a prerequisite f or biological functions. The exact stoichiometric composition of these comp lexes, however, has not been completely clarified yet. In the present study we show for the first time by ultraviolet matrix-assisted laser desorption / ionization mass spectrometry (UV-MALDI-MS) the calcium-induced formation of noncovalently associated (MRP8/MRP14)(2) tetramers. Furthermore, we coul d determine posttranslational modifications of MRP8 and MRP14 the stoichiom etric proportion of the two known MRP14 isoforms in the complexes as well a s the number of calcium ions bound to the single MRP8 and MRP14 monomers an d tetramers. MRP14 showed a higher affinity for calcium than MRP8. Upon com plex formation the calcium binding increased to maximal saturation of the k nown EF hands in the complexed forms. Calcium-induced stabilization of the MRP8/MRP14 complexes was confirmed by DSC studies. Our results extend scope and application of UV-MALDI-MS by allowing identification of noncovalent p rotein complexes, the identification of minor alterations of subunits in su ch complexes as well as the determination of bound calcium ions. (J Am Soc Mass Spectrom 1999, 10, 1124-1130) (C) 1999 American Society for Mass Spect rometry.