Application of a strong anion exchange material in electrostatic repulsion-hydrophilic interaction chromatography for selective enrichment of glycopeptides
Application of a strong anion exchange material in electrostatic repulsion-hydrophilic interaction chromatography for selective enrichment of glycopeptides
复制标题
强阴离子交换材料在静电斥力-亲水相互作用色谱中选择性富集糖肽的应用
DOI：
10.1016/j.chroma.2013.05.037
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发表时间：
2013-07-19
影响因子：
4.1
通讯作者：
Liang, Xinmiao
中科院分区：
文献类型：
--
作者：
Cao, Liwei;Yu, Long;Liang, Xinmiao
糖蛋白参与各种细胞活动，包括细胞间和细胞外信号传导。然而，在基于质谱的分析中，糖肽信号被共洗脱的非糖基化肽显著抑制。为了详细阐明糖蛋白的生物学功能，从非糖基化肽中选择性富集糖肽是至关重要的。在本研究中，SAX材料，XCharge SAX，被用于ERLIC模式的柱中，目的是特异性富集糖肽。初步优化了富集条件，并随后评估了XCharge SAX的选择性、糖基化异质性覆盖率和检测灵敏度。在选择性评估中，使用XCharge SAX从肽混合物（人血清免疫球蛋白G（IgG）和人血清白蛋白酶）和人血清胰蛋白酶消化物中有效分离糖肽。在糖基化异质性覆盖率的评估中，用XCharge SAX富集后鉴定了辣根过氧化物酶的5个糖位点和11个糖肽。在检测灵敏度评估中，用XCharge SAX富集后鉴定出4个数量级内的糖肽。此外，在上样和洗脱缓冲液中使用挥发性溶剂，因此ERLIC组分不需要洗脱。我们的结果共同支持XCharge SAX作为用于全局糖基化位点分析的合适色谱材料的效用。(C)2013爱思唯尔有限公司版权所有。
Glycoproteins are involved in various cellular activities, including inter- and extracellular signaling. However, glycopeptide signals are significantly suppressed by coeluting non-glycosylated peptides in mass spectrometry-based analysis. For detailed elucidation of the biological functions of glycoproteins, selective enrichment of glycopeptides from non-glycosylated peptides is crucial. In the present study, a SAX material, XCharge SAX, was used in a column in the ERLIC mode with the aim of specifically enriching glycopeptides. Enrichment conditions were initially optimized, and selectivity, glycosylation heterogeneity coverage and detection sensitivity of XCharge SAX were subsequently assessed. In the selectivity assessment, glycopeptides were effectively isolated from a peptide mixture (human serum immunoglobulin G (IgG) and human serum albumin digests) and a tryptic digest of human serum using XCharge SAX. In the evaluation of glycosylation heterogeneity coverage, five glycosites and eleven glyco peptides from horseradish peroxidase were identified after enrichment with XCharge SAX. In detection sensitivity assessment, glycopeptides within four orders of magnitude were identified after enrichment with XCharge SAX. In addition, volatile solvents were used in the loading and eluting buffers so that desalting was not necessary for ERLIC fractions. Our results collectively support the utility of XCharge SAX as a suitable chromatographic material for global glycosylation site analysis. (C) 2013 Elsevier B.V. All rights reserved.