Kalb Suzanne R, Goodnough Michael C, Malizio Carl J, Pirkle James L, Barr John R
National Center for Environmental Health/Agency for Toxic Substances and Disease Registry, Centers for Disease Control and Prevention, 4770 Buford Highway, NE Atlanta, Georgia 30341-3724, USA.
Anal Chem. 2005 Oct 1;77(19):6140-6. doi: 10.1021/ac0511748.
Botulinum neurotoxin (BoNT) causes the disease botulism, which can be lethal if untreated. Rapid determination of exposure to BoNT is an important public health goal. Previous work in our laboratory focused on the development of Endopep-MS, a mass spectrometry-based endopeptidase method for detecting and differentiating BoNT in buffer. This method can rapidly determine the presence of BoNT in a sample and differentiate the toxin type of BoNT present but does not yield additional information about the subtype. We now describe here the application of Endopep-MS to detect BoNT A in a spiked milk sample. This work also describes subtype identification achieved through mass spectrometric analysis of the protein toxin itself and does not require the presence of DNA from the toxin-producing bacteria. Tryptic digests of A1 and A2 subtypes of BoNT were analyzed by mass spectrometry, and peptides unique to either the A1 or A2 subtype were subjected to tandem mass spectrometry analysis to confirm their identities. Finally, subtype identification through mass spectrometric analysis was performed on BoNT A isolated from spiked milk. In its entirety, this method would allow for analysis of BoNT with toxin type identification in a few hours and subtype identification within 24 h.
肉毒杆菌神经毒素（BoNT）可引发肉毒中毒，若不治疗可能致命。快速测定是否接触BoNT是一项重要的公共卫生目标。我们实验室之前的工作重点是开发Endopep-MS，这是一种基于质谱的内肽酶方法，用于在缓冲液中检测和区分BoNT。该方法可以快速确定样品中是否存在BoNT，并区分所存在的BoNT毒素类型，但无法提供有关亚型的更多信息。我们在此描述Endopep-MS在加标牛奶样品中检测BoNT A的应用。这项工作还描述了通过对蛋白质毒素本身进行质谱分析实现的亚型鉴定，并且不需要存在来自产毒细菌的DNA。对BoNT的A1和A2亚型的胰蛋白酶消化产物进行质谱分析，对A1或A2亚型特有的肽段进行串联质谱分析以确认其身份。最后，对从加标牛奶中分离出的BoNT A进行质谱分析以鉴定亚型。总体而言，该方法可在数小时内完成毒素类型鉴定的BoNT分析，并在24小时内完成亚型鉴定。
