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首页> 外文期刊>Journal of mass spectrometry: JMS >Probing peptide libraries from Conus achatinus using mass spectrometry and cDNA sequencing: identification of delta and omega-conotoxins
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Probing peptide libraries from Conus achatinus using mass spectrometry and cDNA sequencing: identification of delta and omega-conotoxins

机译：使用质谱和cDNA测序从骆马连续锥中探索肽库：δ和ω-芋螺毒素的鉴定

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摘要

The peptide library present in the venom of the piscivorous marine snail Conus achatinus has been probed using a combination of mass spectrometry and cDNA sequencing methods. Matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) analysis, before and following global reduction/alkylation of peptide mixtures, permits the rapid classification of individual components on the basis of the number of disulfide bonds. Mass fingerprinting and the reverse phase HPLC retention times permit a further deconvolution of the library in terms of peptide size and hydrophobicity. Sequencing of cDNA derived using O-superfamily specific primers yielded five complete conotoxin precursor sequences, ranging in polypeptide length from 75-87 residues containing six Cys residues at the C-terminus. Sequence analysis permits classification of the five putative mature peptides (Ac 6.1 to Ac 6.5) as delta, omega, and omega-like conotoxins. The presence of these predicted peptides in crude venom was established by direct matrix assisted laser desorption ionization tandem mass spectrometry (MALDI-MS/MS) sequencing following trypsin digestion of the peptide mixture after global reduction/alkylation. The determination of partial peptide sequences and comparison with the predicted sequences resulted in the identification of four of the five predicted conotoxins. The characterization of posttranslationally modified analogs, which are hydroxylated at proline or amidated at the C-terminus is also demonstrated. Crude venom analysis should prove powerful in studying both inter- and intra-species variation in peptide libraries.
机译：使用质谱和cDNA测序方法相结合，对食肉海洋蜗牛壳螺中的毒液中存在的肽库进行了探测。在肽混合物的整体还原/烷基化之前和之后，基质辅助激光解吸电离质谱（MALDI-MS）分析允许根据二硫键的数量对各个组分进行快速分类。质谱指纹图谱分析和反相HPLC保留时间允许在肽大小和疏水性方面对文库进一步解卷积。使用O超家族特异性引物对cDNA进行测序，产生了5个完整的芋螺毒素前体序列，多肽长度范围为75-87个残基，在C端含有6个Cys残基。序列分析允许将五个推定的成熟肽（Ac 6.1至Ac 6.5）分类为δ，ω和类似ω的毒素。在总体还原/烷基化后，胰蛋白酶消化肽混合物后，通过直接基质辅助激光解吸电离串联质谱法（MALDI-MS / MS）测序确定了这些预测的肽在粗毒液中的存在。确定部分肽序列并将其与预测序列进行比较，从而鉴定出五种预测的芋螺毒素中的四种。还证明了翻译后修饰的类似物的特性，该类似物在脯氨酸上被羟基化或在C端被酰胺化。粗毒液分析在研究肽库中物种间和物种内变异方面应被证明具有强大的作用。

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