MoP-18


CHARACTERISATION OF SYNTHETIC HUMAN CALGRANULIN C: OBSERVATION OF NONCOVALENT INTERACTIONS BY QUADRUPOLE ESI & ESI-TOF MASS SPECTROMETRY

L. P. Miranda, A. Jones,1 I. Chernushevich,2 K. Standing,3 P. F. Alewood,1 M. Raftery3 and C. L. Geczy3

1Centre for Drug Design & Development, The University Of Queensland, Brisbane, Qld 4072 Australia
2Department of Physics, The University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada
3Cytokine Research Unit, School of Pathology, The University Of New South Wales, Sydney NSW 2052 Australia


EF-hand calcium-binding proteins (CaBPs) play a central role in calcium-mediated signal transduction, a critical step in the regulation of many biological processes. A characteristic of this large family is a conserved EF-hand (helix-loophelix) structural motif in their calcium binding sites.1 The largest subfamily of EF-hand proteins is the S100 family which consists of abundant low molecular weight (10-14 kDa) acidic calcium and zinc-binding proteins with two EF-hands. A new member of the S100 family is human calgranulin C (hCAGC, P6, CAAF1, CGRP & S100A12), a 91 residue protein recently isolated from neutrophils (PMN).2-4 hCAGC may play a role in host-parasite defence and experiments to determine its functions) are currently underway.3,4 However, unlike a number of other S100 proteins only minute amounts of endogenous hCAGC are available. We have chemically synthesised hCAGC using highly optimised Boc chemistry to provide sufficient protein for comprehensive biochemical characterisation, functional analysis and the production of a polyclonal antibody.

Recent developments with electrospray ionisation mass spectromtetry (MS) allow the investigation of noncovalent protein-protein, protein-peptide and protein-metal interactions.5 To further this work, we report the characterisation of synthetic hCAGC using a combination of ESI-MS (shown below), tryptic digestion, SDS-PAGE, CD and NMR. The observation of homodimers of hCAGC ESI-QuadTOF-MS and Ca(II) & Zn(II) binding to the monomer of hCAGC by quadrupole ESI-MS will also be presented.

mass spectrum
  1. Kretsinger, R. H. and Kockolds, C. E., J. Biol. Chem., 248, 3313-3326 (1973).
  2. Guignard, F., Mauel, J. and Markert, M., Biochem J., 309, 395-401 (1995).
  3. Marti, T. and Gallin, M. Y., Biochem. Biophys. Res. Comm., 221, 454-458 (1996).
  4. Ilg, E.C., Troxler, H., Bürgisser, D.M., Kuster, T., Market, M., Guignard, F., Hunziker, P., Birchler, N. and Heizmann, C.W., Biochem. Biophys. Res. Comm., 225, 146-150 (1996).
  5. Fitzgerald, M.C., Chernushevich, I., Standing K.G., Whitman, C.P. and Kent, S.B.H., Proc. Nat. Acad. Sci., 93, 6851-6856 (1996).