Jensen Pia Hønnerup, Mysling Simon, Højrup Peter, Jensen Ole Nørregaard
Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark.
Methods Mol Biol. 2013;951:131-44. doi: 10.1007/978-1-62703-146-2_10.
Glycoproteins, and in particular glycopeptides, are highly hydrophilic and are often not retained by reversed phase (RP) chromatography. The separation principle of normal phase (NP) is based on hydrophilic interactions, which in many aspects is complementary to RP separations. Hydrophilic interaction liquid chromatography (HILIC) is a fairly new variation of the NP separations used in the 1970s, the major difference being the use of aqueous solvents. HILIC provides a versatile tool for enrichment of glycopeptides before mass spectrometric (MS) analysis, particularly when used for solid phase extraction (SPE), or in combination with other chromatographic resins or ion-pairing reagents. HILIC SPE can be used for glyco-profiling, i.e., for determining the glycan heterogeneity at one specific glycosylation site, for enrichment of glycopeptides from a complex mixture of peptides, as well as for pre-fractionation of complex samples at the protein or peptide level. In this chapter we present a straightforward HILIC SPE enrichment technique and then combine C18 RP and HILIC enrichment for analysis of glycopeptides. Finally, we demonstrate HILIC enrichment using trifluoroacetic acid as an ion-pairing reagent for the enrichment of glycopeptides prior to mass spectrometry analysis.
糖蛋白，尤其是糖肽，具有高度亲水性，通常不能被反相（RP）色谱保留。正相（NP）的分离原理基于亲水相互作用，这在许多方面与RP分离互补。亲水作用液相色谱（HILIC）是20世纪70年代使用的NP分离的一种相当新的变体，主要区别在于使用水性溶剂。HILIC为质谱（MS）分析前糖肽的富集提供了一种通用工具，特别是用于固相萃取（SPE）时，或与其他色谱树脂或离子对试剂结合使用时。HILIC SPE可用于糖谱分析，即确定一个特定糖基化位点的聚糖异质性，从复杂的肽混合物中富集糖肽，以及在蛋白质或肽水平上对复杂样品进行预分级分离。在本章中，我们介绍一种直接的HILIC SPE富集技术，然后结合C18 RP和HILIC富集来分析糖肽。最后，我们展示了使用三氟乙酸作为离子对试剂的HILIC富集，用于在质谱分析前富集糖肽。
