Zhao Ting, King Fred L
C. Eugene Bennett Department of Chemistry, West Virginia University, Morgantown, West Virginia 26506-6045, USA.
J Am Soc Mass Spectrom. 2009 Jun;20(6):1141-7. doi: 10.1016/j.jasms.2009.02.013. Epub 2009 Feb 13.
Protein-cisplatin interactions lie at the heart of both the effectiveness of cisplatin as a therapeutic agent and side effects associated with cisplatin treatment. Because a greater understanding of the protein-cisplatin interactions at the molecular level can inform the design of cisplatin-like agents for future use, mass spectrometric determination of the binding site of cisplatin on a model protein, cytochrome c, was undertaken in this paper. The monoadduct cytochrome c-Pt(NH(3))(2)(H(2)O) is found to be the primary adduct produced by the cytochrome c-cisplatin interactions under native conditions. To locate the primary binding site of cisplatin, both free cytochrome c and the cytochrome c adducts underwent trypsin digestion, followed by Fourier transform mass spectrometry (FT-MS) to identify unique fragments in the adduct digest. Four such fragments were found in the adduct digest. Tandem mass spectrometry (MS/MS and MS(3) indicates that two fragments are Pt(NH(3))(2)(H(2)O) bound peptides (Gly56-Glu104 and Asn54-Glu104) with one water associated at the peptide bond Lys79-Met80, and the other two fragments are heme containing peptides (acety1-Gly1-Lys53 and acety1-Gly1-Lys55). The product-ion spectra of the four fragments reveal that Met65 is the primary binding site of cisplatin on cytochrome c.
蛋白质与顺铂的相互作用是顺铂作为治疗药物有效性及顺铂治疗相关副作用的核心所在。由于在分子水平上对蛋白质与顺铂相互作用有更深入的了解有助于设计未来使用的类顺铂药物，本文对顺铂在模型蛋白细胞色素c上的结合位点进行了质谱测定。在天然条件下，单加合物细胞色素c-Pt(NH(3))(2)(H(2)O)被发现是细胞色素c与顺铂相互作用产生的主要加合物。为了确定顺铂的主要结合位点，游离的细胞色素c和细胞色素c加合物都进行了胰蛋白酶消化，随后通过傅里叶变换质谱(FT-MS)来鉴定加合物消化产物中的独特片段。在加合物消化产物中发现了四个这样的片段。串联质谱(MS/MS和MS(3))表明，两个片段是与Pt(NH(3))(2)(H(2)O)结合的肽段(Gly56-Glu104和Asn54-Glu104)，在肽键Lys79-Met80处有一个水分子相连，另外两个片段是含血红素的肽段(acety1-Gly1-Lys53和acety1-Gly1-Lys55)。这四个片段的产物离子谱显示，Met65是顺铂在细胞色素c上的主要结合位点。
