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.
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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通过反相液相色谱与电喷雾电离质谱联用定性和定量表征含硫肽和蛋白质的砷结合行为
DOI：
10.1002/jms.3025
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发表时间：
2012
期刊：
影响因子：
--
通讯作者：
K. Mickein
中科院分区：
文献类型：
--
作者：
A.C. Schmidt;K. Mickein
通过反相液相色谱与电喷雾电离质谱的耦合，将含苯胂取代的半胱氨酸的肽和蛋白质与其未结合的原始形式完全区分开。对还原后具有结构稳定二硫键的生物分子的分析，为有关还原剂半胱氨酸残基可及性以及空间蛋白质结构中砷化合物的要求提供了新的见解。使用直接ESI-MS在不同溶剂系统中进行的互补结合研究表明，由于更强的去折叠，在变性溶剂中，抑肽酶和溶菌酶的结合位点不止一个被激活。根据质谱中出现的不同电荷状态的强度以及LC洗脱行为，可以推断砷结合蛋白质物质的折叠状态类似于天然氧化构象。相比之下，尽管乳蛋白α-乳白蛋白具有几个二硫键，但在强变性条件下仅结合一个苯砷部分。由于ESI质谱中的电荷态分布，假设构象变化为熔融球结构。对于第二个考虑的牛奶蛋白α-乳球蛋白，检测到与氧化苯胂的非共价相互作用。一般来说，直接进样ESI-MS测量比LC-ESI-MS耦合确定的生物分子与氧化苯胂的缩合反应导致共价砷-硫结合的表观结合常数较小。根据ESI-MS和LC-ESI-MS，可以假定对一个苯砷基团的结合亲和力的以下顺序：九肽加压素>九肽催产素>溶菌酶>抑肽酶> α-乳白蛋白>硫氧还蛋白。通过LC-ESI-MS进行动力学研究，得出加压素、赖氨酸和α-乳清蛋白的部分反应级数为2，相应的半衰期分别为0.93、2.56和123.5分钟。版权所有© 2012约翰威利父子有限公司.
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 ß‐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. Copyright © 2012 John Wiley & Sons, Ltd.
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蛋白质液相色谱
DOI：
--
发表时间：
1999
期刊：
影响因子：
--
作者：
M. Kastner
通讯作者：
M. Kastner
影响因子：
3.6
作者：
Kitchin, KT;Wallace, K
通讯作者：
Wallace, K
影响因子：
2.9
作者：
Getz, EB;Xiao, M;Selvin, PR
通讯作者：
Selvin, PR
影响因子：
1.9
作者：
Alexander Tolkach;U. Kulozik
通讯作者：
U. Kulozik
影响因子：
3.8
作者：
Carter, DE;Aposhian, HV;Gandolfi, AJ
通讯作者：
Gandolfi, AJ