Potriquet Jeremy, Laohaviroj Marut, Bethony Jeffrey M, Mulvenna Jason
Genetics & Computational Biology Department, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, 4060, Australia.
Department of Pathology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand.
PLoS One. 2017 Jul 27;12(7):e0175967. doi: 10.1371/journal.pone.0175967. eCollection 2017.
To facilitate high-throughput proteomic analyses we have developed a modified FASP protocol which improves the rate at which protein samples can be processed prior to mass spectrometry. Adapting the original FASP protocol to a 96-well format necessitates extended spin times for buffer exchange due to the low centrifugation speeds tolerated by these devices. However, by using 96-well plates with a more robust polyethersulfone molecular weight cutoff membrane, instead of the cellulose membranes typically used in these devices, we could use isopropanol as a wetting agent, decreasing spin times required for buffer exchange from an hour to 30 minutes. In a typical work flow used in our laboratory this equates to a reduction of 3 hours per plate, providing processing times similar to FASP for the processing of up to 96 samples per plate. To test whether our modified protocol produced similar results to FASP and other FASP-like protocols we compared the performance of our modified protocol to the original FASP and the more recently described eFASP and MStern-blot. We show that all FASP-like methods, including our modified protocol, display similar performance in terms of proteins identified and reproducibility. Our results show that our modified FASP protocol is an efficient method for the high-throughput processing of protein samples for mass spectral analysis.
为促进高通量蛋白质组学分析，我们开发了一种改良的FASP方案，该方案提高了蛋白质样品在质谱分析前的处理速度。由于96孔板设备所能耐受的离心速度较低，将原始FASP方案应用于96孔板形式时，缓冲液交换所需的离心时间会延长。然而，通过使用具有更强健的聚醚砜截留分子量膜的96孔板，而非这些设备通常使用的纤维素膜，我们可以使用异丙醇作为湿润剂，将缓冲液交换所需的离心时间从1小时减少至30分钟。在我们实验室的典型工作流程中，这相当于每块板减少3小时，对于每块板最多96个样品的处理，其提供的处理时间与FASP相当。为测试我们的改良方案是否能产生与FASP及其他类似FASP的方案相似的结果，我们将改良方案的性能与原始FASP、最近描述的eFASP及MStern-blot进行了比较。我们表明，所有类似FASP的方法，包括我们的改良方案，在鉴定出的蛋白质数量和重现性方面都表现出相似的性能。我们的结果表明，我们的改良FASP方案是一种用于蛋白质样品高通量处理以进行质谱分析 的有效方法。
