Determination of binding sites in carboplatin-bound cytochrome c using electrospray ionization mass spectrometry and tandem mass spectrometry

interaction of carboplatin with cytochrome c (Cyt. c) has been investigated by electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS). ESI-MS studies revealed that the ring-opened adducts of carboplatin with Cyt. c were formed in the stoichiometric ratio of 1: 1 and 2: 1 at pH 5.0 and 37 degrees C and in the stoichiometric ratio of 1: 1 only at pH 7.0 and 37 degrees C. It was also found that Cyt. c could be cleaved by carboplatin at pH 2.5 and 50 degrees C. The cleaved fragments of Cyt. c were determined by ESI-MS and MS/MS analysis to be Glu66 similar to Met80, Ac-Gly01 similar to Met65, Glu66 similar to Glu104, Ac-Gly01 similar to Met80 and Ile81 similar to Glu104. The carboplatin prefers to anchor to Met65 first, then to Met80. To further confirm the binding site of Met, AcMet-Gly was used as the model molecule to investigate its interaction with carboplatin and its hydrolysis reaction. On the basis of species detected during the reaction monitored by ESI-MS, a possible pathway of the cleavage reaction was proposed. Copyright (c) 2005 John Wiley & Sons, Ltd.