Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands.
Nat Protoc. 2012 Nov;7(11):2041-55. doi: 10.1038/nprot.2012.124. Epub 2012 Oct 25.
Multidimensional liquid chromatography (LC) combined with mass spectrometry (MS) has become a standard technique in proteomics to reduce sample complexity and to tackle the dynamic range in protein abundance. Fractionation is necessary to obtain a comprehensive analysis of complex biological samples such as tissue and mammalian cell lines. However, extensive fractionation comes at the expense of sample loss, presenting a bottleneck in the analysis of limited amounts of material. In this protocol, we describe a two-dimensional chromatographic strategy based on a combination of hydrophilic interaction liquid chromatography (HILIC; with a zwitterionic packing material, ZIC-cHILIC) and reversed-phase chromatography, which allows proteomic analyses with minimal sample loss. Experimental aspects related to obtaining maximum recovery are discussed, including how to optimally prepare samples for this system. Examples involving protein lysates originating from cultured cell lines and cells sorted by flow cytometry are used to show the power, sensitivity and versatility of the technique. Once the ZIC-cHILIC fractionation system has been optimized and standardized, this protocol requires ∼5-6 d, including sample preparation and fraction analysis.
多维液相色谱（LC）与质谱（MS）联用已成为蛋白质组学中减少样品复杂性和解决蛋白质丰度动态范围的标准技术。为了对组织和哺乳动物细胞系等复杂生物样品进行全面分析，需要进行分级分离。然而，广泛的分级分离以样品损失为代价，这在分析有限量的材料时成为了一个瓶颈。在本方案中，我们描述了一种基于亲水相互作用液相色谱（HILIC；采用两性离子填充材料 ZIC-cHILIC）和反相色谱相结合的二维色谱策略，可实现最小样品损失的蛋白质组学分析。讨论了与获得最大回收率相关的实验方面，包括如何为该系统优化样品制备。使用源自培养细胞系的蛋白质裂解物和流式细胞术分选的细胞的实例来说明该技术的强大功能、灵敏度和多功能性。一旦优化和标准化了 ZIC-cHILIC 分级分离系统，该方案大约需要 5-6 天，包括样品制备和分级分析。
