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首页> 外文期刊>Journal of mass spectrometry: JMS >Qualitative and quantitative characterization of the arsenic-binding behaviour of sulfur-containing peptides and proteins by the coupling of reversed phase liquid chromatography to electrospray ionization mass spectrometry
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Qualitative and quantitative characterization of the arsenic-binding behaviour of sulfur-containing peptides and proteins by the coupling of reversed phase liquid chromatography to electrospray ionization mass spectrometry

机译：反相液相色谱与电喷雾电离质谱联用对含硫肽和蛋白质的砷结合行为进行定性和定量表征

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摘要

Phenylarsenic-substituted cysteine-containing peptides and proteins were completely differentiated from their unbound original forms by the coupling of reversed phase liquid chromatography with electrospray ionization mass spectrometry. The analysis of biomolecules possessing structure-stabilizing disulfide bridges after reduction provides new insights into requirements concerning the accessibility of cysteine residues for reducing agents as well as for arsenic compounds in a spatial protein structure. Complementary binding studies performed using direct ESI-MS without chromatographic coupling in different solvent systems demonstrated that more than one binding site were activated for aprotinin and lysozyme in denaturing solvents because of a stronger defolding. From the intensities of the different charge states occurring in the mass spectra as well as from the LC elution behaviour, it can be deduced that the folding state of the arsenic-bound protein species resembles the native, oxidized conformation. In contrast, although the milk protein α-lactalbumin has several disulfide bridges, only one phenylarsenic moiety was bound under strongly denaturing conditions. Because of the charge state distribution in the ESI mass spectra, a conformational change to a molten globule structure is assumed. For the second considered milk protein ?-lactoglobulin, a noncovalent interaction with phenylarsine oxide was detected. In general, smaller apparent binding constants for the condensation reactions of the biomolecules with phenylarsine oxide leading to covalent arsenic-sulfur bindings were determined from direct injection ESI-MS measurements than from LC-ESI-MS coupling. The following order of binding affinities for one phenylarsenic group can be assumed from both ESI-MS and LC-ESI-MS: nonapeptide vasopressin > nonapeptide vasotocin > lysozyme > aprotinin > α-lactalbumin > thioredoxin. Kinetic investigations by LC-ESI-MS yielded a partial reaction order of 2 for vasopressin, Lys and α-lactalbumin and corresponding half-lives of 0.93, 2.56 and 123.5 min, respectively.
机译：通过反相液相色谱与电喷雾电离质谱联用，将苯砷取代的含半胱氨酸的肽和蛋白质与未结合的原始形式完全区分开。还原后具有结构稳定的二硫键的生物分子的分析提供了有关还原剂和空间蛋白质结构中砷化合物的半胱氨酸残基可及性要求的新见识。在不同溶剂系统中使用不进行色谱偶联的直接ESI-MS进行的互补结合研究表明，由于变性更强，在变性溶剂中，抑肽酶和溶菌酶的结合位点被激活了。从质谱图中出现的不同电荷状态的强度以及从LC洗脱行为可以推断出，砷结合蛋白物种的折叠状态类似于天然的氧化构象。相反，尽管乳蛋白α-乳清蛋白具有几个二硫键，但是在强变性条件下仅结合了一个苯基砷部分。由于ESI质谱图中的电荷状态分布，因此假定了熔融小球结构的构象变化。对于第二种考虑的乳蛋白β-乳球蛋白，检测到与苯ar氧化物的非共价相互作用。通常，通过直接进样ESI-MS测量比通过LC-ESI-MS偶合确定了生物分子与苯ar氧化物的缩合反应导致共价砷硫结合的较小表观结合常数。从一个ESI-MS和一个LC-ESI-MS可以假定对一个苯基砷基团的结合亲和力的顺序如下：九肽血管加压素>九肽血管毒素>溶菌酶>抑肽酶>α-乳清蛋白>硫氧还蛋白。通过LC-ESI-MS进行的动力学研究得出，加压素，Lys和α-乳清蛋白的部分反应阶数为2，相应的半衰期分别为0.93、2.56和123.5分钟。

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