Biochemistry & Proteomics Group, Department of Chemistry & Biomolecular Science, Clarkson University, 8 Clarkson Ave., Box 5810, Potsdam, NY, 13699, USA.
Institute of Education and Research Santa Casa Belo Horizonte-Laboratory of Toxins, Rua Domingos Vieira 590, Belo Horizonte, Minas Gerais, 30150-240, Brazil.
J Am Soc Mass Spectrom. 2018 May;29(5):827-841. doi: 10.1007/s13361-018-1904-3. Epub 2018 Apr 16.
Native Phα1β is a peptide purified from the venom of the armed spider Phoneutria nigriventer that has been shown to have an extensive analgesic effect with fewer side effects than ω-conotoxin MVIIA. Recombinant Phα1β mimics the effects of the native Phα1β. Because of this, it has been suggested that Phα1β may have potential to be used as a therapeutic for controlling persistent pathological pain. The amino acid sequence of Phα1β is known; however, the exact structure and disulfide arrangement has yet to be determined. Determination of the disulfide linkages and exact structure could greatly assist in pharmacological analysis and determination of why this peptide is such an effective analgesic. Here, we used biochemical and mass spectrometry approaches to determine the disulfide linkages present in the recombinant Phα1β peptide. Using a combination of MALDI-MS, direct infusion ESI-MS, and nanoLC-MS/MS analysis of the undigested recombinant Phα1β peptide and digested with AspN, trypsin, or AspN/trypsin, we were able to identify and confirm all six disulfide linkages present in the peptide as Cys1-2, Cys3-4, Cys5-6, Cys7-8, Cys9-10, and Cys11-12. These results were also partially confirmed in the native Phα1β peptide. These experiments provide essential structural information about Phα1β and may assist in providing insight into the peptide's analgesic effect with very low side effects. Graphical Abstract ᅟ.
天然的 Phα1β 是一种从武装蜘蛛 Phoneutria nigriventer 的毒液中纯化出来的肽，已被证明具有广泛的镇痛作用，副作用比 ω-芋螺毒素 MVIIA 少。重组的 Phα1β 模拟天然 Phα1β 的作用。正因为如此，有人认为 Phα1β 可能有潜力被用作控制持续性病理性疼痛的治疗药物。Phα1β 的氨基酸序列是已知的；然而，其确切的结构和二硫键排列尚未确定。确定二硫键连接和确切的结构可以极大地帮助进行药理学分析，并确定为什么这种肽是如此有效的镇痛药。在这里，我们使用生化和质谱方法来确定重组 Phα1β 肽中存在的二硫键连接。通过 MALDI-MS、直接注入 ESI-MS 和未消化的重组 Phα1β 肽和用 AspN、胰蛋白酶或 AspN/胰蛋白酶消化的 nanoLC-MS/MS 分析的组合，我们能够鉴定和确认肽中存在的所有六个二硫键连接，即 Cys1-2、Cys3-4、Cys5-6、Cys7-8、Cys9-10 和 Cys11-12。这些结果在天然 Phα1β 肽中也得到了部分证实。这些实验提供了关于 Phα1β 的重要结构信息，可能有助于深入了解该肽的镇痛作用，且副作用极低。
