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 Ala43 CP10 was identified after electrospray ionization mass spectrometry using a nondenaturing solvent of 5-mM ammonium acetate (pH 6.5) and source temperature of 50 degrees C. With a low cone voltage (75 V), major ions were observed at approximately 2075, 2305, and 2613 Da, which were attributed to partially desolvated multiply charged noncovalently linked dimeric species (+10, +9, and +8 charge states, respectively). Deconvolution produced a broad peak centered around 20750 Da corresponding to the approximate mass of dimeric recombinant Ala43 CP10. Increasing the cone voltage, the collisionally activating the complex, gradually reduced the intensity of these dimeric ions, with a concomitant increase 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, indicating a thermally unstable noncovalent complex. Similar spectra were obtained using recombinant CP10. Specific noncovalent S100 dimeric complexes were confirmed by chemically cross-linking recombinant Ala43 CP10 or CP10 with bis (sulfosuccinimidyl) suberate, followed by SDS/PAGE. The dominant silver-stained and CP10-immunoreactive component migrated at 20,000 suggested that the complex represents the major isoform in solution.
Raftery
M J
MJ
Cytokine Research Unit, School of Pathology, University of New South Wales, Kensington, New South Wales, Australia. m.raftery@unsw.edu.au
Geczy
C L
CL
D000818:Animals; D003432:Cross-Linking Reagents; D003956:Dialysis; D013058:Mass Spectrometry; D051379:Mice; D011994:Recombinant Proteins / Q000032:analysis; D009418:S100 Proteins / Q000032:analysis*
D003432:Cross-Linking Reagents; D011994:Recombinant Proteins; D009418:S100 Proteins