Peptidomic Identification of Cysteine-Rich Peptides from Plants
Peptidomic Identification of Cysteine-Rich Peptides from Plants
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DOI：
10.1007/978-1-4939-7537-2_26
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发表时间：
2018-01-01
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影响因子：
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通讯作者：
Tam, James P.
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文献类型：
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作者：
Hemu, Xinya;Serra, Aida;Tam, James P.
富含半胱氨酸的植物肽（Cystine-rich peptides，CRPs）是植物源肽的主要组成部分，具有高度的分子多样性。该方案描述了一种快速有效的肽组学方法来鉴定植物提取物中的全谱CRPs，并破译其分子多样性和生物加工机制。环肽类化合物C. ternatea被用作模型CRP来证明我们的方法。环肽以环状和线性形式天然存在，尽管线性形式（无环肽）通常以低得多的浓度存在。环肽和非环肽都需要在片段化和测序之前将其骨架线性化。本文提出了一种新的、实用的三步化学酶处理方法，用于线性化和区分两种形式：（1）N-末端乙酰化，预标记无环肽;（2）通过氮丙啶介导的S-烷基化将Cys转化为假Lys，以减少二硫键并增加肽的净电荷;和（3）通过在天然生物加工位点切割的新型天冬酰胺酰内肽酶butelase 2打开环状骨架。随后使用电子转移解离片段化通过液相色谱-质谱联用分析经处理的肽，并通过将MS/MS光谱与转录组数据库直接匹配来鉴定序列。
Plant cysteine-rich peptides (CRPs) constitute a majority of plant-derived peptides with high molecular diversity. This protocol describes a rapid and efficient peptidomic approach to identify a whole spectrum of CRPs in a plant extract and decipher their molecular diversity and bioprocessing mechanism. Cyclotides from C. ternatea are used as the model CRPs to demonstrate our methodology. Cyclotides exist naturally in both cyclic and linear forms, although the linear forms (acyclotide) are generally present at much lower concentrations. Both cyclotides and acyclotides require linearization of their backbone prior to -fragmentation and sequencing. A novel and practical three-step chemoenzymatic treatment was developed to linearize and distinguish both forms: (1) N-terminal acetylation that pre-labels the acyclotides; (2) conversion of Cys into pseudo-Lys through aziridine-mediated S-alkylation to reduce disulfide bonds and to increase the net charge of peptides; and (3) opening of cyclic backbones by the novel asparaginyl endopeptidase butelase 2 that cleaves at the native bioprocessing site. The treated peptides are subsequently analyzed by liquid -chromatography coupled to mass spectrometry using electron transfer dissociation fragmentation and sequences are identified by matching the MS/MS spectra directly with the transcriptomic database.