title: Comprehensive identification of the binding sites of cisplatin in hen egg white lysozyme.
abstract: Platinum drugs have become one of the most important kinds of chemotherapy agents, and the interactions of these drugs with proteins play very important roles in their side effects and drug resistance. However, it is still a challenge to determine the binding sites of platinum drugs in proteins with multiple disulfide bonds and stable three-dimensional structures using mass spectrometry. Here, the interaction between cisplatin and hen egg white lysozyme (HEWL), a multi-disulfide-bond-containing protein with a stable three-dimensional structure, was investigated using Fourier transform ion cyclotron resonance mass spectrometry. Typical disulfide bond reduction with dithiothreitol/tris(2-carboxyethyl)phosphine before trypsin digestion destroyed the binding of cisplatin to HEWL, and no platination sites were found. Efficient trypsin digestion methods for HEWL-cisplatin adducts were developed to avoid the loss of platinum binding to protein. At 55 °C, platinated HEWL was digested directly by trypsin in 6 h, and multiple platinated peptides were observed. In 60% acetonitrile, the digestion time of platinated HEWL was shortened to 2 h, and most of the platinated peptides were observed. In addition, the reduction of the disulfide bonds of HEWL greatly accelerated the reaction between HEWL and cisplatin, and the potential binding sites of cisplatin in reduced HEWL could be easily recognized. On the basis of the above-mentioned methods, multiple binding sites of cisplatin in HEWL were first identified by mass spectrometry.
authors.affiliation: Changchun Center of Mass Spectrometry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun, 130022, China.
authors.forename: Ningbo
authors.initials: N
authors.lastname: Zhang
authors.forename: Yonggang
authors.initials: Y
authors.lastname: Du
authors.forename: Meng
authors.initials: M
authors.lastname: Cui
authors.forename: Zhiqiang
authors.initials: Z
authors.lastname: Liu
authors.forename: Shuying
authors.initials: S
authors.lastname: Liu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry*; D001665:Binding Sites; D002645:Chickens; D002945:Cisplatin / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry; D005583:Fourier Analysis; D007477:Ions; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D012997:Solvents / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature; D014357:Trypsin / Q000737:chemistry
chemical_list: D000970:Antineoplastic Agents; D004220:Disulfides; D007477:Ions; D010455:Peptides; D012997:Solvents; C421864:hen egg lysozyme; D009113:Muramidase; D014357:Trypsin; D002945:Cisplatin