Quantification of bindings of organometallic ruthenium complexes to GSTπ by mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) has been widely used to identify binding sites of metal complexes to proteins. However, the MS quantification of the metal-protein coordination remains a challenge. We have recently demonstrated by ESI-MS analysis that organometallic ruthenium complexes [(eta(6)-arene)Ru(en)Cl](+) (arene = p-cymene (1), biphenyl (2) or 9,10-dihydrophenanthrene (3); en = ethylenediamine) bound to human glutathione-S-transferase pi (GST pi) at Cys15 and Cys48 within the G-site, and Cys102 and Met92 on the interface of the GST pi dimer, showing inhibitory potency against the enzyme (J. Inorg. Biochem., 128 (2013) 77-84). Herein, we developed a mass spectrometric method to quantify the binding stoichiometry of the three complexes to GST pi. The differences in signal intensities of the heavy-labelled peptides produced by tryptic digestion of the ruthenated GST pi complexes and the respective light-labelled peptides in the tryptic digest of equimolar GST pi were used to calculate the binding stoichiometry at specific residues. The results indicated that the pre-complexation of GST pi with its substrate GSH significantly reduced the bindings of the ruthenium complexes at Met92 and Cys102, but had little impact on the bindings at Cys15 and Cys48. As the inhibitory activities of the ruthenium complexes against GST pi are similar to those against GST pi in complexation with GSH, these results suggest that the inhibition of the ruthenium complexes on GST pi is attributed to the ruthenation at Cys15 and Cys48. The present work provides not only insights into the understanding on the inhibitory mechanism of ruthenium complexes GST pi, but also a general method for quantitative characterization of metal-protein interactions. (C) 2015 Elsevier Inc. All rights reserved.