Probing the Coordination of Metal Ions by Diethylenetriaminepentaacetic Acid-Conjugated Proteins with Electrospray Ionisation Mass Spectrometry

Diethylenetriaminepentaacetic acid (DTPA) was covalently linked to L-lysine, the synthetic peptide cyclic GMP protein kinase substrate (cGMP), horse heart myoglobin and the B72.3 monoclonal antibody (MAb) F_ab fragment via an acylation reaction using cyclic DTPA anhydride (cDTPA). The reaction of cDTPA with myoglobin at 10 and 100 fold molar ratios gave rise to the addition of one diethylenetriaminetetraacetic acid (DTTA) group per protein molecule and this was independent of the initial concentration of the protein. We have shown that the modification is readily observed by electrospray ionisation mass spectrometry (ESI-MS) and that the coordination of a range of metal ions by the DTTA group can also be easily detected in ESI mass spectra. In addition, hydrolysed DTPA alone is found to conjugate a range of metal cations in solution and the intensities of the [DTPA – (X – 1)H⁺ + M^X+] complexes (where X = the integer value of the charge on the metal ion and M = the metal cation) observed in ESI mass spectra correlate with known solution stabilities of these complexes. This clearly demonstrates the potential of ESI-MS to determine the number of metal ions that can be incorporated into a protein via a chelating ligand, which in turn has implications, for example, in generating radiolabelled proteins for use in immunotherapy and immunodiagnosis of carcinomas.