Calcium-induced noncovalently linked tetramers of MRP8 and MRP14 are confirmed by electrospray ionization-mass analysis

Proteins of the S100- family such as MRP8 (S100A8) and MRT14 (S100A9)-and i ts isoform MRP14*-show two calcium-binding sites (EF hands) per protein cha in. MRP8, MRP14*, and MRP14, isolated from human granulocytes or monocytes, are known to form noncovalently associated complexes; the exact stoichiome tries of these complexes in the presence of calcium are still controversial ly discussed in the literature. The present electrospray ionization-mass sp ectrometry (ESI-MS) study shows that MRP8, MRP14*, and MRT14 exist as heter odimers MRP8/14* and MRP8/14, respectively, in the absence of calcium confi rming both a recent nuclear magnetic resonance study and a biochemical stud y on this topic. Furthermore, this ESI-MS study confirms the previously pub lished matrix-assisted laser desorption ionization (MALDI)-MS study, which states that the MRP8/14* and MRP8/14 heterodimeric complexes tetramerize to heterotetramers (MRP8/14*)(2), (MRP8/14*)(MRP8/14), and (MRP8/14)(2) respe ctively, in the presence of calcium. The number of Ca2+ ions bound to the i ndividual tetramer is determined to be eight for nonphosphorylated fraction s; this is in agreement with the previously reported MALDI study on these f ractions. About 1.2 Ca2+ ions more are bound to the phosphorylated form; it is speculated that the additional Ca2+ ions are bound to the phosphate gro ups in the tetramers. This study is, therefore, convincing proof of the rel iability of MALDI-MS in studying noncovalent protein-protein interactions. (C) 2000 American Society for Mass Spectrometry.