title: Quantification of bindings of organometallic ruthenium complexes to GSTπ by mass spectrometry.
abstract: 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 [(η(6)-arene)Ru(en)Cl](+) (arene=p-cymene (1), biphenyl (2) or 9,10-dihydrophenanthrene (3); en=ethylenediamine) bound to human glutathione-S-transferase π (GSTπ) at Cys15 and Cys48 within the G-site, and Cys102 and Met92 on the interface of the GSTπ 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 GSTπ. The differences in signal intensities of the heavy-labelled peptides produced by tryptic digestion of the ruthenated GSTπ complexes and the respective light-labelled peptides in the tryptic digest of equimolar GSTπ were used to calculate the binding stoichiometry at specific residues. The results indicated that the pre-complexation of GSTπ 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 GSTπ are similar to those against GSTπ in complexation with GSH, these results suggest that the inhibition of the ruthenium complexes on GSTπ 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 GSTπ, but also a general method for quantitative characterization of metal-protein interactions.
authors.affiliation: Beijing National Laboratory for Molecular Sciences, China; CAS Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Lin
authors.affiliation: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China.
authors.forename: Yongdong
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Beijing National Laboratory for Molecular Sciences, China; CAS Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Zheng
authors.affiliation: Beijing National Laboratory for Molecular Sciences, China; CAS Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China.
authors.forename: Kui
authors.initials: K
authors.lastname: Wu
authors.affiliation: Beijing National Laboratory for Molecular Sciences, China; CAS Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China.
authors.forename: Qun
authors.initials: Q
authors.lastname: Luo
authors.affiliation: Beijing National Laboratory for Molecular Sciences, China; CAS Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China.
authors.forename: Yao
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Beijing National Laboratory for Molecular Sciences, China; CAS Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China; Beijing Mass Spectrometry Centre, China.
authors.forename: Shaoxiang
authors.initials: S
authors.lastname: Xiong
authors.affiliation: Beijing National Laboratory for Molecular Sciences, China; CAS Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China; Beijing Mass Spectrometry Centre, China. Electronic address: fuyi.wang@iccas.ac.cn.
authors.forename: Fuyi
authors.initials: F
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D051549:Glutathione S-Transferase pi / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009942:Organometallic Compounds / Q000737:chemistry* / Q000494:pharmacology; D011485:Protein Binding; D017975:Ruthenium Compounds / Q000737:chemistry* / Q000494:pharmacology
chemical_list: D009942:Organometallic Compounds; D017975:Ruthenium Compounds; D051549:Glutathione S-Transferase pi
keywords: Binding stoichiometry; Glutathione-S-transferase; Mass spectrometry; Organometallic ruthenium complexes; Quantification