CHARACTERIZING TEREBRIDAE TOXINS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 在Orbitrap上用LC-MS法测定了石斑鱼(Terebridae Gutatta)、银鱼(Agyosia)、毛虾(Maunti)和福尔摩沙(Formosa)粗毒中存在的多肽。只有杜仲胶和银鱼毒液有足够的物质可以离线分离。用反相高效液相色谱分离这两种毒素，并用MALDI TOF质谱仪对在214 nm处有较强紫外吸收的部分进行分析，以确定这些部分中潜在毒素的二硫键的准确质量和数目。紫外可见光谱分析表明，绿背天牛毒液几乎没有显示任何物质，而杜仲胶天牛毒液有5个主要峰。不幸的是，使用标准的还原方案，所有的瓜塔多肽都会沉淀下来。通过增加有机溶剂(乙腈和甲醇)的用量，优化了还原方案。使用高有机溶剂时，毒素在还原时仍留在溶液中。鉴定出质量在3700~5030 Da之间的毒素。大多数毒素(5)含有6个二硫键，1个毒素含有8个二硫键。到目前为止，我们还不能获得这些毒素的碎片数据，因为我们还没有确定使毒素保持在溶液中的条件，并允许使用反相高效液相色谱法进行分析。我们目前正在制定方案，将这些疏水毒素保留在溶液中，同时保留在反相高效液相色谱柱上。这种方法的发展对于分析狼蛛毒液也是必要的，因为它在还原时也会从水溶液中沉淀出来。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The peptides present in the crude venom of Terebridae gutatta, argyosia, maunti and formosa were measured by LC-MS on the Orbitrap. Only the gutatta and argyosia venom had enough material to allow off-line separation. These two venoms were separated by RP-HPLC and the fractions exhibiting strong UV absorption at 214 nm were analyzed with MALDI TOF mass spectrometry to determine the accurate mass and number of disulfide bonds of potential toxins in these fractions. The Terebridae aergyosia venom showed hardly any material using UV detection, but Terebridae gutatta showed 5 major peaks. Unfortunately using a standard reduction protocol all of the gutatta peptides precipitated. The reduction protocol was optimized by increasing the amount of organic solvent (acetonitrile and methanol). Using high organic solvent the toxins remained in solution upon reduction. Toxins with masses ranging from 3700 to 5030 Da were identified. The majority of toxins (5) contained 6 disulfide bonds and one toxin contained 8 disulfide bonds. To date, we are not able to obtain fragmentation data of these toxins as we have not yet determined conditions that keep the toxins in solution and allow analysis with RP HPLC. We are currently working out protocols that keep these hydrophobic toxins in solution and at the same time retained on RP-HPLC columns. This method development is also necessary for analyzing tarantula venoms that also precipitate out of aqueous solution upon reduction.