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 which play an important role during calcium-induced activation of phagocytes. Both proteins form noncovalently associated complexes as a prerequisite for biological functions. The exact stoichiometric composition of these complexes, 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 could determine posttranslational modifications of MRP8 and MRP14, the stoichiometric proportion of the two known MRP14 isoforms in the complexes as well as the number of calcium ions bound to the single MRP8 and MRP14 monomers and tetramers. MRP14 showed a higher affinity for calcium than MRP8. Upon complex formation the calcium binding increased to maximal saturation of the known 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 protein complexes, the identification of minor alterations of subunits in such complexes as well as the determination of bound calcium ions.
Vogl
T
T
Institute of Experimental Dermatology, University of Münster, Germany. vogl@uni-muenster.de
Roth
J
J
Sorg
C
C
Hillenkamp
F
F
Strupat
K
K
D000943:Antigens, Differentiation / Q000032:analysis*; D002118:Calcium / Q000737:chemistry*; D002135:Calcium-Binding Proteins / Q000032:analysis*; D040501:Calgranulin A; D040502:Calgranulin B; D002152:Calorimetry, Differential Scanning; D006098:Granulocytes / Q000737:chemistry; D006801:Humans; D066298:In Vitro Techniques; D007536:Isomerism; D011499:Protein Processing, Post-Translational; D009418:S100 Proteins / Q000032:analysis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature
D000943:Antigens, Differentiation; D002135:Calcium-Binding Proteins; D040501:Calgranulin A; D040502:Calgranulin B; D009418:S100 Proteins; D002118:Calcium