Schmidt Anne-Christine, Koppelt Jenny, Neustadt Madlen, Otto Matthias
TU Bergakademie Freiberg, Faculty of Chemistry and Physics, Institute of Analytical Chemistry, Leipziger Strasse 29, D-09596 Freiberg, Germany.
Rapid Commun Mass Spectrom. 2007;21(2):153-63. doi: 10.1002/rcm.2823.
Trivalent and pentavalent arsenic were incubated with sulfur-containing amino acid, peptide and protein solutions both as organic compounds (phenylarsine oxide, phenylarsonic acid, dimethylarsinic acid, monomethylarsonic acid) and as inorganic compounds (arsenite, As(III), and arsenate, As(V)). After incubation of phenylarsine oxide solutions with cysteine and glutathione the mass spectra showed a covalent bond between arsenic and sulfur, which was stable at both acidic and neutral pH values. The mass spectra were dominated by monovalent ions at m/z 272 for cysteine samples and at m/z 458 for glutathione samples. Based on these masses the ionic structures could be ascribed to either fragment ions of the covalent arsenic-sulfur complexes or to other arsenic-bonding sites presumably at the amino group. Interestingly, under the same conditions no interactions of inorganic arsenite or arsenate could be measured. In the presence of added Cu(2+) ions all mass signals caused by a reaction of phenylarsine oxide with glutathione disappeared. In these mass spectra only the oxidised form of glutathione (GSSG) was found because of the redox activity of Cu(II). For the model protein lysozyme, no interactions with arsenic could be detected, whereas definite Cu- and Zn-lysozyme complexes with a stoichiometry of 1:1 and 2:1 for Zn(2+) ions and Cu(2+) ions, respectively, were observed. In contrast, for thioredoxin a bonding of As that depended on the concentration of the disulfide-reducing agent tris(2-carboxyethyl) phosphine was demonstrated. For three different phenylarsonic acids and for dimethylarsinic acid that all contain pentavalent arsenic, complexes with glutathione appeared in the mass spectra, which can be attributed to non-covalent interactions or to a covalent bond caused by an additive reaction. The optimisation of the experimental conditions necessary for the mass spectrometric analysis of the interactions of the arsenic species with peptides and proteins is described and the obtained mass spectra that provide information on the kinds of bonds are discussed.
将三价和五价砷与含硫氨基酸、肽和蛋白质溶液一起孵育，这些溶液既包括有机化合物（氧化苯胂、苯胂酸、二甲基胂酸、一甲基胂酸），也包括无机化合物（亚砷酸盐、As(III)和砷酸盐、As(V)）。将氧化苯胂溶液与半胱氨酸和谷胱甘肽孵育后，质谱显示砷和硫之间形成了共价键，该共价键在酸性和中性pH值下均稳定。对于半胱氨酸样品，质谱以m/z 272的单价离子为主；对于谷胱甘肽样品，质谱以m/z 458的单价离子为主。基于这些质量，离子结构可归因于共价砷-硫配合物的碎片离子，或可能归因于氨基处的其他砷键合位点。有趣的是，在相同条件下未检测到无机亚砷酸盐或砷酸盐的相互作用。在添加Cu(2+)离子的情况下，氧化苯胂与谷胱甘肽反应产生的所有质谱信号均消失。在这些质谱中，由于Cu(II)的氧化还原活性，仅发现了谷胱甘肽的氧化形式（GSSG）。对于模型蛋白溶菌酶，未检测到与砷的相互作用，而分别观察到了化学计量比为1:1和2:1的Zn(2+)离子和Cu(2+)离子与溶菌酶形成的确定配合物。相比之下，对于硫氧还蛋白，证明了砷的结合取决于二硫键还原剂三(2-羧乙基)膦的浓度。对于三种不同的苯胂酸和二甲基胂酸（均含有五价砷），质谱中出现了与谷胱甘肽的配合物，这可归因于非共价相互作用或加成反应导致的共价键。描述了对砷物种与肽和蛋白质相互作用进行质谱分析所需的实验条件的优化，并讨论了所获得的提供键类型信息的质谱。
