Fox Jay W, Ma Li, Nelson Kristina, Sherman Nicholas E, Serrano Solange M T
Department of Microbiology, University of Virginia, P.O. Box 800734, Charlottesville VA 22908-0734, USA.
Toxicon. 2006 May;47(6):700-14. doi: 10.1016/j.toxicon.2006.01.022. Epub 2006 Mar 30.
In a sense, the field of snake venom proteomics has been under investigation since the very earliest biochemical studies where it was soon recognized that venoms are comprised of complex mixtures of bioactive molecules, most of which are proteins. Only with the re-emergence of 2D polyacrylamide gel electrophoresis (2D PAGE) and the recent developments in mass spectrometry for the identification/characterization of proteins coupled with venom gland transcriptomes has the field of snake venom proteomics began to flourish and provide exciting insights into the protein composition of venoms and subsequently their pathological activities. In this manuscript we will briefly discuss the state of snake venom proteomics followed by the presentation of several straightforward experiments designed to explore approaches to investigating venom proteomics. The first set of experiments used 1D gel electrophoresis (1D PAGE) of Crotalus atrox venom followed by slice-by-slice analysis of the proteins using liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS). In the second set of experiments, C. atrox and Bothrops jararaca venoms were subjected to in-solution digestion followed by Fourier transform ion cyclotron resonance (FTICR) LC/MS/MS. The peptide ion-maps of these venoms were compared along with the proteins identified. In addition, the results were compared to the results observed from the 1D PAGE approach. From these studies it is clear that sample de-complexation/fractionation before mass spectrometry is still the best approach for maximum proteome coverage. Furthermore, comparison of venom proteomes based on tryptic peptide identities between the proteomes is not particularly effective since there does not appear to be a sufficient number of such identical peptides, derived from related proteins, present in venoms. Finally, as has previously been recognized without either better databases of venom protein sequences or facile and rapid de novo sequencing technologies for mass spectrometry, snake venom proteome investigation will remain a laborious task.
从某种意义上说，自最早的生化研究以来，蛇毒蛋白质组学领域就一直在进行研究。在这些早期研究中，人们很快就认识到毒液是由生物活性分子的复杂混合物组成，其中大部分是蛋白质。直到二维聚丙烯酰胺凝胶电泳（2D PAGE）重新出现，以及最近用于蛋白质鉴定/表征的质谱技术与毒腺转录组学相结合，蛇毒蛋白质组学领域才开始蓬勃发展，并为毒液的蛋白质组成及其随后的病理活性提供了令人兴奋的见解。在本手稿中，我们将简要讨论蛇毒蛋白质组学的现状，然后介绍几个简单的实验，旨在探索研究毒液蛋白质组学的方法。第一组实验对西部菱斑响尾蛇毒液进行一维凝胶电泳（1D PAGE），然后使用液相色谱/质谱/质谱（LC/MS/MS）对蛋白质进行逐片分析。在第二组实验中，对西部菱斑响尾蛇和巴西矛头蝮毒液进行溶液内消化，然后进行傅里叶变换离子回旋共振（FTICR）LC/MS/MS。将这些毒液的肽离子图谱与鉴定出的蛋白质进行比较。此外，还将结果与一维凝胶电泳方法得到的结果进行比较。从这些研究中可以清楚地看出，质谱分析前的样品去复合/分级分离仍然是实现最大蛋白质组覆盖的最佳方法。此外，基于蛋白质组之间胰蛋白酶肽同一性的毒液蛋白质组比较并不是特别有效，因为毒液中似乎没有足够数量的源自相关蛋白质的此类相同肽。最后，正如之前所认识到的，如果没有更好的毒液蛋白质序列数据库，也没有用于质谱分析的简便快速的从头测序技术，蛇毒蛋白质组研究仍将是一项艰巨的任务。
