Utilizing Ion-Pairing Hydrophilic Interaction Chromatography Solid Phase Extraction for Efficient Glycopeptide Enrichment in Glycoproteomics
Utilizing Ion-Pairing Hydrophilic Interaction Chromatography Solid Phase Extraction for Efficient Glycopeptide Enrichment in Glycoproteomics
复制标题
DOI：
10.1021/ac100530w
复制
发表时间：
2010-07-01
影响因子：
7.4
通讯作者：
Thaysen-Andersen, Morten
中科院分区：
文献类型：
--
作者：
Mysling, Simon;Palmisano, Giuseppe;Thaysen-Andersen, Morten
糖肽富集是实现糖蛋白组学中蛋白糖基化结构表征的先决条件。本文提出了一种基于微柱形式的两性离子亲水相互作用色谱固相萃取（ZIC-HILIC SPE）的糖肽富集方法。该方法涉及TFA离子配对（IP），以增加糖肽和非糖基化肽之间的亲水性差异。研究了三种流动相，即含有80%乙腈的2%甲酸（定义为IP2% (FA) ZIC-HILIC SPE）、0.1% TFA和1% TFA（定义为IP0.1% （TFA）和IP1% (TFA) ZIC-HILIC SPE）。研究中使用了越来越复杂的样品，即单糖蛋白的消化物，五糖蛋白混合物和耗尽血浆。通过使用MALDI-TOF质谱和RP-LC-ESI-MS/MS增强糖肽检测来评估，流动相中TFA的存在显著提高了所有复杂性的糖肽富集，例如，与IP2% （FA）条件相比，糖肽离子信号增加了3.7倍。非糖基化肽的大量消耗促进了糖肽检测的增强，使用一个SPE富集步骤几乎可以完全分离IgG糖肽，并且在IP2% (FA) ZIC-HILIC SPE保留血浆部分中观察到的非糖基化肽从711个减少到只有157个和97个，当在流动相中使用0.1%和1% TFA时。总之，本系统的研究表明，当使用ZIC-HILIC SPE时，含tfa的流动相在广泛的样品复杂度范围内显著提高了糖肽富集效率。
Glycopeptide enrichment is a prerequisite to enable structural characterization of protein glycosylation in glycoproteomics. Here we present an improved method for glycopeptide enrichment based on zwitter-ionic hydrophilic interaction chromatography solid phase extraction (ZIC-HILIC SPE) in a microcolumn format. The method involves TFA ion pairing (IP) to increase the hydrophilicity difference between glycopeptides and non-glycosylated peptides. Three mobile phases were investigated, i.e., 2% formic acid (defined as IP2% (FA) ZIC-HILIC SPE), 0.1% TFA and 1% TFA (defined as IP0.1% (TFA) and IP1% (TFA) ZIC-HILIC SPE) all containing 80% acetonitrile. Samples of increasing complexities, i.e., digests of single glycoproteins, a five-glycoprotein mixture, and depleted plasma, were used in the study. The presence of TFA in the mobile phase significantly improved the glycopeptide enrichment for all complexities, as evaluated by enhanced glycopeptide detection using MALDI-TOF MS and RP-LC-ESI-MS/MS, e.g., the glycopeptide ion signals were increased by up to 3.7-fold compared to IP2% (FA) conditions. The enhanced glycopeptide detection was promoted by a substantial depletion of nonglycosylated peptides, offering an almost complete isolation of IgG glycopeptides using a single SPE enrichment step and a reduction from 711 non-glycosylated peptides observed in the IP2% (FA) ZIC-HILIC SPE retained plasma fraction, to only 157 and 97 when 0.1% and 1% TFA was used in the mobile phase. In conclusion, this systematic study has shown that TFA-containing mobile phases increase glycopeptide enrichment efficiency considerably for a broad range of sample complexities when using ZIC-HILIC SPE.