IDENTIFICATION OF NONCOVALENT DIMERIC COMPLEXES OF THE RECOMBINANT MURINE S100 PROTEIN CP10 BY ELECTROSPRAY-IONIZATION MASS-SPECTROMETRY AND CHEMICAL CROSS-LINKING

CP10 is a chemotactic S100 protein expressed by murine myeloid cells. A specific noncovalently linked dimeric complex of recombinant Ala(43) CP10 was identified after electrospray ionization mass spectrometry u sing a nondenaturing solvent of 5-mM ammonium acetate (pH 6.5) and sou rce temperature of 50 degrees C. With a low cone voltage (75 V), major ions were observed at similar to 2075, 2305, and 2613 Da, which were attributed to partially desolvated multiply charged noncovalently link ed dimeric species (+10, +9, and +8 charge states, respectively). Deco nvolution produced a broad peak centered around 20750 Da corresponding to the approximate mass of dimeric recombinant Ala(43) CP10. increasi ng the cone voltage, and collisionally activating the complex, gradual ly reduced the intensity of these dimeric ions, with a concomitant inc rease in higher and lower charge state monomeric ions. The intensities of these dimeric ions were greatly reduced in spectra recorded with a source temperature of 140 degrees C and cone voltage of 75 V, indicat ing a thermally unstable noncovalent complex. Similar spectra were obt ained using recombinant CP10. Specific noncovalent S100 dimeric comple xes were confirmed by chemically cross-linking recombinant Ala(43) CP1 0 or CP10 with bis (sulfosuccinimidyl) suberate, followed by SDS/PAGE. The dominant silver-stained and CP10-immunoreactive component migrate d at 20,000 suggesting that the complex represents the major isoform i n solution. (C) 1998 American Society for Mass Spectrometry.