Glycopeptide enrichment for MALDI-TOF mass spectrometry analysis by hydrophilic interaction liquid chromatography solid phase extraction (HILIC SPE).
Glycopeptide enrichment for MALDI-TOF mass spectrometry analysis by hydrophilic interaction liquid chromatography solid phase extraction (HILIC SPE).
复制标题
DOI：
10.1007/978-1-62703-146-2_10
复制
发表时间：
2013-01-01
期刊：
影响因子：
--
通讯作者：
Jensen, Ole Norregaard
中科院分区：
文献类型：
--
作者：
Jensen, Pia Honnerup;Mysling, Simon;Jensen, Ole Norregaard
糖蛋白，特别是糖肽，是高度亲水性的，并且通常不被反相（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分离的一个相当新的变化，主要区别是使用水性溶剂。HILIC为质谱（MS）分析前的糖肽富集提供了一种通用工具，特别是用于固相萃取（SPE）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，用于测定一个特定糖基化位点处的聚糖异质性，用于从肽的复杂混合物中富集糖肽，以及用于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，我们提出了一个简单的HILIC固相萃取富集技术，然后结合联合收割机C18 RP和HILIC富集的糖肽分析。最后，我们证明了HILIC富集使用三氟乙酸作为离子配对试剂富集糖肽之前，质谱分析。
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.