Mandal Amit Kumar, Ramasamy Mani Ramakrishnan Santhana, Sabareesh Varatharajan, Openshaw Matthew E, Krishnan Kozhalmannom S, Balaram Padmanabhan
Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India.
J Am Soc Mass Spectrom. 2007 Aug;18(8):1396-404. doi: 10.1016/j.jasms.2007.04.009. Epub 2007 Apr 24.
De novo mass spectrometric sequencing of two Conus peptides, Vi1359 and Vi1361, from the vermivorous cone snail Conus virgo, found off the southern Indian coast, is presented. The peptides, whose masses differ only by 2 Da, possess two disulfide bonds and an amidated C-terminus. Simple chemical modifications and enzymatic cleavage coupled with matrix assisted laser desorption ionization (MALDI) mass spectrometric analysis aided in establishing the sequences of Vi1359, ZCCITIPECCRI-NH(2), and Vi1361, ZCCPTMPECCRI-NH(2), which differ only at residues 4 and 6 (Z = pyroglutamic acid). The presence of the pyroglutamyl residue at the N-terminus was unambiguously identified by chemical hydrolysis of the cyclic amide, followed by esterification. The presence of Ile residues in both the peptides was confirmed from high-energy collision induced dissociation (CID) studies, using the observation of w(n)- and d(n)-ions as a diagnostic. Differential cysteine labeling, in conjunction with MALDI-MS/MS, permitted establishment of disulfide connectivity in both peptides as Cys2-Cys9 and Cys3-Cys10. The cysteine pattern clearly reveals that the peptides belong to the class of T-superfamily conotoxins, in particular the T-1 superfamily.
本文介绍了从印度南部海岸发现的食虫芋螺（Conus virgo）中两种芋螺肽Vi1359和Vi1361的从头质谱测序。这两种肽的质量仅相差2道尔顿，具有两个二硫键和一个酰胺化的C末端。简单的化学修饰和酶切，结合基质辅助激光解吸电离（MALDI）质谱分析，有助于确定Vi1359（ZCCITIPECCRI-NH₂）和Vi1361（ZCCPTMPECCRI-NH₂）的序列，它们仅在第4和第6位残基处不同（Z = 焦谷氨酸）。通过环酰胺的化学水解，然后酯化，明确鉴定了N末端焦谷氨酰残基的存在。使用w(n)-和d(n)-离子作为诊断依据，通过高能碰撞诱导解离（CID）研究证实了两种肽中异亮氨酸残基的存在。差分半胱氨酸标记结合MALDI-MS/MS，确定了两种肽中二硫键的连接方式为Cys2-Cys9和Cys3-Cys10。半胱氨酸模式清楚地表明，这些肽属于T-超家族芋螺毒素类别，特别是T-1超家族。
