Peptidomics of Three Bothrops Snake Venoms: Insights Into the Molecular Diversification of Proteomes and Peptidomes
Peptidomics of Three Bothrops Snake Venoms: Insights Into the Molecular Diversification of Proteomes and Peptidomes
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DOI：
10.1074/mcp.m112.019331
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发表时间：
2012-11-01
影响因子：
7
通讯作者：
Serrano, Solange M. T.
中科院分区：
文献类型：
--
作者：
Tashima, Alexandre K.;Zelanis, Andre;Serrano, Solange M. T.
蛇毒蛋白质组/肽组非常复杂，维持其在腺腔内的完整性对于毒素活性的表达至关重要。毒液蛋白质组学领域已经取得了相当大的进展，然而，由于缺乏用于分析 MS 数据的全面的毒液序列数据库，肽组学进展缓慢。因此，在许多情况下，必须通过 MS/MS 分析或 Edman 降解对毒液肽进行手动测序。这对于稀有蛇类至关重要，例如被视为濒临灭绝的白蛇 (BC) 和丰塞凯蛇 (BF)。在本研究中，我们对 BC、BF 和 B. jararaca (BJ) 的毒液肽组进行了全面分析，采用固相萃取和反相 HPLC 相结合来分级肽，然后进行纳米液相色谱-串联 MS (LC-MS/MS) 或直接输注电喷雾电离 - (ESI)-MS/MS 或 MALDI-MS/MS 分析。我们检测到毒液肽组的显着差异，并通过从头测序鉴定出长度为 7 至 39 个残基的肽。人工鉴定了 44 个独特序列，其中 30 个是新肽，包括 17 个缓激肽增强肽、3 个聚组氨酸聚甘氨酸肽以及有趣的 10 个 L-氨基酸氧化酶片段。一些新的缓激肽增强肽显示出显着的缓激肽增强活性。自动数据库搜索揭示了肽组中几种毒素的片段，主要来自 L-氨基酸氧化酶，并允许确定蛋白酶的肽键特异性和 P4-P4' 位点的氨基酸出现情况。我们还证明，由于毒液蛋白质组的不平衡以及随之而来的蛋白酶对毒液成分的活性，毒液冻干/再溶解过程大大增加了肽组的复杂性。蛋白酶抑制剂的使用清楚地显示了肽组表征的不同结果，并表明蛇毒的降解组学-肽组学分析对采样程序的条件高度敏感。分子与细胞蛋白质组学 11：10.1074/mcp.M112.019331, 1245-1262, 2012。
Snake venom proteomes/peptidomes are highly complex and maintenance of their integrity within the gland lumen is crucial for the expression of toxin activities. There has been considerable progress in the field of venom proteomics, however, peptidomics does not progress as fast, because of the lack of comprehensive venom sequence databases for analysis of MS data. Therefore, in many cases venom peptides have to be sequenced manually by MS/MS analysis or Edman degradation. This is critical for rare snake species, as is the case of Bothrops cotiara (BC) and B. fonsecai (BF), which are regarded as near threatened with extinction. In this study we conducted a comprehensive analysis of the venom peptidomes of BC, BF, and B. jararaca (BJ) using a combination of solid-phase extraction and reversed-phase HPLC to fractionate the peptides, followed by nano-liquid chromatography-tandem MS (LC-MS/MS) or direct infusion electrospray ionization-(ESI)-MS/MS or MALDI-MS/MS analyses. We detected marked differences in the venom peptidomes and identified peptides ranging from 7 to 39 residues in length by de novo sequencing. Forty-four unique sequences were manually identified, out of which 30 are new peptides, including 17 bradykinin-potentiating peptides, three poly-histidine-poly-glycine peptides and interestingly, 10 L-amino acid oxidase fragments. Some of the new bradykinin-potentiating peptides display significant bradykinin potentiating activity. Automated database search revealed fragments from several toxins in the peptidomes, mainly from L-amino acid oxidase, and allowed the determination of the peptide bond specificity of proteinases and amino acid occurrences for the P4-P4' sites. We also demonstrate that the venom lyophilization/resolubilization process greatly increases the complexity of the peptidome because of the imbalance caused to the venom proteome and the consequent activity of proteinases on venom components. The use of proteinase inhibitors clearly showed different outcomes in the peptidome characterization and suggested that degradomic-peptidomic analysis of snake venoms is highly sensitive to the conditions of sampling procedures. Molecular & Cellular Proteomics 11: 10.1074/mcp.M112.019331, 1245-1262, 2012.