Chip-based Reversed-phase Liquid Chromatography-Mass Spectrometry of Permethylated N-Linked Glycans: A Potential Methodology for Cancer-biomarker Discovery

被引:134
作者
Alley, William R., Jr. [1 ]
Madera, Milan [1 ]
Mechref, Yehia [1 ,2 ]
Novotny, Milos V. [1 ]
机构
[1] Indiana Univ, Natl Ctr Glyc & Glycoprote, Bloomington, IN 47405 USA
[2] Indiana Univ, MetaCyt Biochem Anal Ctr, Dept Chem, Bloomington, IN 47405 USA
基金
美国国家卫生研究院;
关键词
HYDROPHILIC-INTERACTION CHROMATOGRAPHY; LC-ESI-MS; ANION-EXCHANGE CHROMATOGRAPHY; GRAPHITIC CARBON; SERUM GLYCANS; PERFORMANCE; OLIGOSACCHARIDES; TIME; GLYCOPEPTIDES; GLYCOSYLATION;
D O I
10.1021/ac100131e
中图分类号
O65 [分析化学];
学科分类号
070302 ; 081704 ;
摘要
The study of protein glycosylation in biological fluids and tissues has substantial medical importance, as changes in glycan structures have now been associated with a number of diseases. Quantification of glycomic-profile changes is becoming increasingly important in the search for disease biomarkers. Here, we report a highly reproducible combination of a glycomic sample preparation/solid-phase derivatization of glycoprotein-derived N-linked glycans with their subsequent microchip-based separation and mass-spectrometric (MS) measurements. Following our previously described reductive beta-elimination for O-linked glycans with ammonia-borane complex to reduce N-linked structures, the N-linked alditol structures are effectively methylated in dimethylformamide medium to avoid artefacts in MS measurements. Reversed-phase microfluidic liquid chromatography (LC) of methylated N-linked oligosaccharide alditols resolved some closely related structures into regular retention increments, aiding in their structural assignments. Optimized LC gradients, together with nanospray MS, have been applied here in the quantitative measurements of N-linked glycans in blood serum, distinguishing breast cancer patients from control individuals.
引用
收藏
页码:5095 / 5106
页数:12
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