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Abstract：
Hydrophilic interaction liquid chromatography, HILIC, is a relatively new HPLC mode. Compared with other HPLC modes, HILIC is a high resolution chromatographic mode with high peak capacity for separations of complex mixtures. Although the separation mechanism is still not completely clear, HILIC has been widely used for analysis of hydrophilic compounds which are difficult for reversed phase chromatography to retain and separate. In this study, we fabricated and investigated nanoHILIC columns in terms of separation efficiency, van Deemter curves and more importantly, we focused on long packed capillary columns, and studied their extreme resolution for protein digests. Using meter long nanoHILIC columns packed with 5 μm particles, we realized a high peak capacity of 130. Based on nanoLC-MS, we compared the resolution and protein identification capabilities of nanoHILIC and nanoRPLC. The results indicate both nanoHILIC and nanoRPLC can provide high resolution for protein sequencing but neither mode is significantly better than the other. Among the 99 digest peptides identified, 17 were uniquely identified by nanoHILIC-MS and 20 were uniquely identified by nanoRPLC-MS and 62 were identified by both methods. Although at this moment in time, nanoRPLC is the most popular microseparation tool in proteomics, the excellent complementarity of nanoHILIC and nanoRPLC suggests their combined use in achieving deep-coverage in MS-based proteomics.
摘要：
亲水相互作用液相色谱,HILIC,是一种相对较新的HPLC模式。与其他HPLC模式相比，HILIC是一种高分辨率色谱模式，具有分离复杂混合物的高峰值容量。虽然分离机理还不完全清楚,HILIC已广泛用于分析亲水性化合物,所述亲水性化合物对于反相色谱难以保留和分离。在这项研究中,我们制造并研究了纳米HILIC柱的分离效率，vanDeemter曲线，更重要的是，我们专注于长填充毛细管柱，并研究了他们对蛋白质消化的极端分辨率。使用装有5μm颗粒的米长nanoHILIC色谱柱，我们实现了130的高峰容量。基于nanoLC-MS,我们比较了nanoHILIC和nanoRPLC的分辨率和蛋白质识别能力。结果表明，nanoHILIC和nanoRPLC都可以为蛋白质测序提供高分辨率，但两种模式都不明显优于另一种。在鉴定的99个消化肽中，17个由nanoHILIC-MS唯一鉴定,20个由nanoRPLC-MS唯一鉴定,62个由两种方法鉴定。虽然此时此刻,nanoRPLC是蛋白质组学中最流行的微分离工具，nanoHILIC和nanoRPLC的优异的互补性表明它们在基于MS的蛋白质组学中的结合应用。
