De novo sequencing and disulfide mapping of a bromotryptophan-containing conotoxin by Fourier transform ion cyclotron resonance mass spectrometry
De novo sequencing and disulfide mapping of a bromotryptophan-containing conotoxin by Fourier transform ion cyclotron resonance mass spectrometry
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DOI：
10.1021/ac0607764
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发表时间：
2006-12-01
影响因子：
7.4
通讯作者：
Marshall, Alan G.
中科院分区：
文献类型：
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作者：
Nair, Sudarslal Sadasivan;Nilsson, Carol L.;Marshall, Alan G.
t -1家族concontoxins属于t超家族，由10-17个氨基酸组成。它们具有共同的半胱氨酸框架和二硫连通性，并表现出不寻常的翻译后修饰，如色氨酸溴化、谷氨酸羧化和苏氨酸糖基化。我们分离并鉴定了一种新的多肽Mo1274，它含有11个氨基酸，显示出与t -1家族成员相同的半胱氨酸模式，- CC- CC和二硫键。完整序列GNWCCSARVCC （W表示溴色氨酸）来自MS-based de novo测序。FT-ICRMS/MS电子捕获解离（ECD）、红外多光子解离和碰撞诱导解离技术用于色氨酸溴化的检测和定位。溴在所有含有溴色氨酸的片段中具有独特的同位素分布。ECD破碎导致溴的损失，并恢复到正态同位素分布。用质谱法测定了Mo1274半胱氨酸对1-3和2-4之间的二硫连连性，并用三（2-羧基乙基）膦进行了化学衍生化，然后与n -乙基马来酰亚胺和碘乙酰胺进行了差异烷基化。天然和部分修饰肽的ECD光谱显示在需要二硫键存在的过程中溴的损失。
T-1-family conotoxins belong to the T-superfamily and are composed of 10-17 amino acids. They share a common cysteine framework and disulfide connectivity and exhibit unusual posttranslational modifications, such as tryptophan bromination, glutamic acid carboxylation, and threonine glycosylation. We have isolated and characterized a novel peptide, Mo1274, containing 11 amino acids, that shows the same cysteine pattern, - CC- CC, and disulfide linkage as those of the T-1-family members. The complete sequence, GNWCCSARVCC, in which W denotes bromotryptophan, was derived from MS-based de novo sequencing. The FT-ICRMS/MS techniques of electron capture dissociation (ECD), infrared multiphoton dissociation, and collision-induced dissociation served to detect and localize the tryptophan bromination. The bromine contributes a distinctive isotopic distribution in all fragments that contain bromotryptophan. ECD fragmentation results in the loss of bromine and return to the normal isotopic distribution. Disulfide connectivity of Mo1274, between cysteine pairs 1-3 and 2-4, was determined by mass spectrometry in combination with chemical derivatization employing tris(2-carboxyethyl)phosphine, followed by differential alkylation with N-ethylmaleimide and iodoacetamide. The ECD spectra of the native and partially modified peptide reveal a loss of bromine in a process that requires the presence of a disulfide bond.