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 MRP14 (S100A9)-and its isoform MRP14*-show two calcium-binding sites (EF hands) per protein chain. MRP8, MRP14*, and MRP14, isolated from human granulocytes or monocytes, are known to form noncovalently associated complexes; the exact stoichiometries of these complexes in the presence of calcium are still controversially discussed in the literature. The present electrospray ionization-mass spectrometry (ESI-MS) study shows that MRP8, MRP14*, and MRP14 exist as heterodimers MRP8/14* and MRP8/14, respectively, in the absence of calcium confirming both a recent nuclear magnetic resonance study and a biochemical study on this topic. Furthermore, this ESI-MS study confirms the previously published 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, respectively, in the presence of calcium. The number of Ca2+ ions bound to the individual tetramer is determined to be eight for nonphosphorylated fractions; this is in agreement with the previously reported MALDI study on these fractions. 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 groups in the tetramers. This study is, therefore, convincing proof of the reliability of MALDI-MS in studying noncovalent protein-protein interactions.
Strupat
K
K
Laboratoire de Spectrométrie de Masse Bio-Organique, Université Louis Pasteur, Strasbourg, France.
Rogniaux
H
H
Van Dorsselaer
A
A
Roth
J
J
Vogl
T
T
D000943:Antigens, Differentiation / Q000737:chemistry*; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry*; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D040501:Calgranulin A; D040502:Calgranulin B; D002138:Calibration; D006098:Granulocytes / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D010766:Phosphorylation; D011485:Protein Binding; D011489:Protein Denaturation; D009418:S100 Proteins / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000943:Antigens, Differentiation; D002135:Calcium-Binding Proteins; D040501:Calgranulin A; D040502:Calgranulin B; D009418:S100 Proteins; D002118:Calcium