Department of Biochemistry , Medical College of Wisconsin , Milwaukee , Wisconsin 53226 , United States.
Center for Biomedical Mass Spectrometry Research , Medical College of Wisconsin , Milwaukee , Wisconsin 53226 , United States.
J Proteome Res. 2019 Apr 5;18(4):1644-1656. doi: 10.1021/acs.jproteome.8b00916. Epub 2019 Feb 28.
Peptide cleanup is essential for the removal of contaminating substances that may be introduced during sample preparation steps in bottom-up proteomic workflows. Recent studies have described benefits of carboxylate-modified paramagnetic particles over traditional reversed-phase methods for detergent and polymer removal, but challenges with reproducibility have limited the widespread implementation of this approach among laboratories. To overcome these challenges, the current study systematically evaluated key experimental parameters regarding the use of carboxylate-modified paramagnetic particles and determined those that are critical for maximum performance and peptide recovery and those for which the protocol is tolerant to deviation. These results supported the development of a detailed, easy-to-use standard operating protocol, termed SP2, which can be applied to remove detergents and polymers from peptide samples while concentrating the sample in solvent that is directly compatible with typical LC-MS workflows. We demonstrate that SP2 can be applied to phosphopeptides and glycopeptides and that the approach is compatible with robotic liquid handling for automated sample processing. Altogether, the results of this study and accompanying detailed operating protocols for both manual and automated processing are expected to facilitate reproducible implementation of SP2 for various proteomics applications and will especially benefit core or shared resource facilities where unknown or unexpected contaminants may be particularly problematic.
肽段净化对于去除在自上而下的蛋白质组学工作流程的样品制备步骤中可能引入的污染物质是至关重要的。最近的研究描述了羧酸修饰的超顺磁颗粒相对于传统反相方法在去除去污剂和聚合物方面的优势，但由于重复性方面的挑战，限制了该方法在实验室中的广泛应用。为了克服这些挑战，本研究系统地评估了羧酸修饰的超顺磁颗粒使用的关键实验参数，并确定了那些对最大性能和肽段回收至关重要的参数，以及对协议偏差具有容忍性的参数。这些结果支持了详细、易于使用的标准操作协议（称为 SP2）的开发，该协议可用于在浓缩样品的同时从肽样品中去除去污剂和聚合物，所使用的溶剂与典型的 LC-MS 工作流程直接兼容。我们证明了 SP2 可用于磷酸肽和糖肽，并且该方法与机器人液体处理兼容，可用于自动化样品处理。总之，这项研究的结果以及手动和自动处理的详细操作协议预计将促进 SP2 在各种蛋白质组学应用中的可重复性实施，特别是将使核心或共享资源设施受益，因为在这些设施中，未知或意外的污染物可能是特别成问题的。
