Department of Chemistry, University of Warwick, Coventry, United Kingdom.
Anal Chem. 2011 Dec 15;83(24):9507-15. doi: 10.1021/ac202267g. Epub 2011 Nov 18.
Calmodulin (CaM) is a highly conserved, ubiquitous, calcium-binding protein; it binds to and regulates many different protein targets, thereby functioning as a calcium sensor and signal transducer. CaM contains 9 methionine (Met), 1 histidine (His), 17 aspartic acid (Asp), and 23 glutamine acid (Glu) residues, all of which can potentially react with platinum compounds; thus, one-third of the CaM sequence is a possible binding target of platinum anticancer drugs, which represents a major challenge for identification of specific platinum modification sites. Here, top-down electron capture dissociation (ECD) was used to elucidate the transition metal-platinum(II) modification sites. By using a combination of top-down and bottom-up mass spectrometric (MS) approaches, 10 specific binding sites for mononuclear complexes, cisplatin and [Pt(dien)Cl]Cl, and dinuclear complex [{cis-PtCl(2)(NH(3))}(2)(μ-NH(2)(CH(2))(4)NH(2))] on CaM were identified. High resolution MS of cisplatin-modified CaM revealed that cisplatin mainly targets Met residues in solution at low molar ratios of cisplatin-CaM (2:1), by cross-linking Met residues. At a high molar ratio of cisplatin:CaM (8:1), up to 10 platinum(II) bind to Met, Asp, and Glu residues. [{cis-PtCl(2)(NH(3))}(2)(μ-NH(2)(CH(2))(4)NH(2))] forms mononuclear adducts with CaM. The alkanediamine linker between the two platinum centers dissociates due to a trans-labilization effect. [Pt(dien)Cl]Cl forms {Pt(dien)}(2+) adducts with CaM, and the preferential binding sites were identified as Met51, Met71, Met72, His107, Met109, Met124, Met144, Met145, Glu45 or Glu47, and Asp122 or Glu123. The binding of these complexes to CaM, particularly when binding involves loss of all four original ligands, is largely irreversible which could result in their failure to reach the target DNA or be responsible for unwanted side-effects during chemotherapy. Additionally, the cross-linking of cisplatin to CaM might lead to the loss of the biological function of CaM or CaM-Ca(2+) due to limiting the flexibility of the CaM or CaM-Ca(2+) complex to recognize target proteins or blocking the binding region of target proteins to CaM.
钙调蛋白（CaM）是一种高度保守、普遍存在的钙结合蛋白；它与许多不同的蛋白质靶标结合并调节它们的活性，从而充当钙传感器和信号转导器。CaM 含有 9 个蛋氨酸（Met）、1 个组氨酸（His）、17 个天冬氨酸（Asp）和 23 个谷氨酰胺（Glu）残基，所有这些残基都可能与铂化合物发生反应；因此，CaM 序列的三分之一可能是铂类抗癌药物的潜在结合靶标，这给鉴定特定的铂修饰位点带来了重大挑战。在这里，自上而下的电子俘获解离（ECD）被用于阐明过渡金属-铂（II）修饰位点。通过自上而下和自下而上的质谱（MS）方法的结合，在 CaM 上鉴定到了 10 个单核复合物、顺铂和[Pt（dien）Cl]Cl，以及双核配合物[{cis-PtCl2（NH3）}2（μ-NH2（CH2）4NH2）]的单核复合物、顺铂和[Pt（dien）Cl]Cl，以及双核配合物[{cis-PtCl2（NH3）}2（μ-NH2（CH2）4NH2）]的 10 个特定结合位点。顺铂修饰的 CaM 的高分辨率 MS 显示，顺铂主要通过交联 Met 残基，在低摩尔比（2:1）的顺铂-CaM 时靶向溶液中的 Met 残基。在高摩尔比（8:1）的顺铂：CaM 时，多达 10 个铂（II）与 Met、Asp 和 Glu 残基结合。[{cis-PtCl2（NH3）}2（μ-NH2（CH2）4NH2）]与 CaM 形成单核加合物。两个铂中心之间的链烷二胺连接物由于反式致活作用而解离。[Pt（dien）Cl]Cl 与 CaM 形成{Pt（dien）}2+加合物，并且鉴定到的优先结合位点为 Met51、Met71、Met72、His107、Met109、Met124、Met144、Met145、Glu45 或 Glu47，以及 Asp122 或 Glu123。这些配合物与 CaM 的结合，特别是当结合涉及失去所有四个原始配体时，在很大程度上是不可逆的，这可能导致它们无法到达靶 DNA 或导致化疗期间产生不必要的副作用。此外，顺铂与 CaM 的交联可能导致 CaM 或 CaM-Ca（2+）的生物功能丧失，因为这限制了 CaM 或 CaM-Ca（2+）复合物识别靶蛋白的灵活性，或阻止靶蛋白与 CaM 的结合区域。
