Murphy Kathryn M, Balsor Justin, Beshara Simon, Siu Caitlin, Pinto Joshua G A
McMaster Integrative Neuroscience Discovery and Study Program, McMaster University, Canada; Psychology, Neuroscience & Behavior, McMaster University, Canada.
McMaster Integrative Neuroscience Discovery and Study Program, McMaster University, Canada.
J Neurosci Methods. 2014 Sep 30;235:35-40. doi: 10.1016/j.jneumeth.2014.05.036. Epub 2014 Jul 2.
Synaptoneurosomes have become an important tool for studying synaptic proteins. The filtered synaptoneurosomes preparation originally developed by Hollingsworth et al. (1985) is widely used and is an easy method to prepare synaptoneurosomes. The hand processing steps in that preparation, however, are labor intensive and have become a bottleneck for current proteomic studies using synaptoneurosomes. For this reason, we developed new steps for tissue homogenization and filtration that transform the preparation of synaptoneurosomes to a high-throughput, semi-automated process.
We implemented a standardized protocol with easy to follow steps for homogenizing multiple samples simultaneously using a FastPrep tissue homogenizer (MP Biomedicals, LLC) and then filtering all of the samples in centrifugal filter units (EMD Millipore, Corp).
The new steps dramatically reduce the time to prepare synaptoneurosomes from hours to minutes, increase sample recovery, and nearly double enrichment for synaptic proteins. These steps are also compatible with biosafety requirements for working with pathogen infected brain tissue.
The new high-throughput semi-automated steps to prepare synaptoneurosomes are timely technical advances for studies of low abundance synaptic proteins in valuable tissue samples.
突触神经小体已成为研究突触蛋白的重要工具。霍林斯沃思等人（1985年）最初开发的经过滤的突触神经小体制备方法被广泛使用，是一种制备突触神经小体的简便方法。然而，该制备方法中的手工操作步骤劳动强度大，已成为当前使用突触神经小体进行蛋白质组学研究的瓶颈。因此，我们开发了新的组织匀浆和过滤步骤，将突触神经小体的制备转变为高通量、半自动过程。
我们实施了一个标准化方案，使用FastPrep组织匀浆器（MP Biomedicals有限责任公司）同时对多个样品进行匀浆，步骤易于遵循，然后在离心过滤装置（EMD Millipore公司）中对所有样品进行过滤。
新步骤显著缩短了制备突触神经小体的时间，从数小时缩短至数分钟，提高了样品回收率，使突触蛋白的富集量几乎增加了一倍。这些步骤也符合处理病原体感染脑组织的生物安全要求。
制备突触神经小体的新高通量半自动步骤是对珍贵组织样本中低丰度突触蛋白进行研究的及时技术进步。
