Optimized Workflow for Preparation of APTS-Labeled N-Glycans Allowing High-Throughput Analysis of Human Plasma Glycomes using 48-Channel Multiplexed CGE-LIF
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- 作者：L. Renee Ruhaak ; Ren Hennig ; Carolin Huhn ; Matthias Borowiak ; Radboud J. E. M. Dolhain ; Andr M. Deelder ; Erdmann Rapp ; Manfred Wuhrer
- 刊名：Journal of Proteome Research
- 出版年：2010
- 出版时间：December 3, 2010
- 年：2010
- 卷：9
- 期：12
- 页码：6655-6664
- 全文大小：503K
- 年卷期：v.9,no.12(December 3, 2010)
- ISSN：1535-3907
文摘
High-throughput methods for oligosaccharide analysis are required when searching for glycan-based biomarkers. Next to mass spectrometry-based methods, which allow fast and reproducible analysis of such compounds, further separation-based techniques are needed, which allow for quantitative analysis. Here, an optimized sample preparation method for N-glycan-profiling by multiplexed capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF) was developed, enabling high-throughput glycosylation analysis. First, glycans are released enzymatically from denatured plasma glycoproteins. Second, glycans are labeled with APTS using 2-picoline borane as a nontoxic and efficient reducing agent. Reaction conditions are optimized for a high labeling efficiency, short handling times, and only limited loss of sialic acids. Third, samples are subjected to hydrophilic interaction chromatography (HILIC) purification at the 96-well plate format. Subsequently, purified APTS-labeled N-glycans are analyzed by CGE-LIF using a 48-capillary DNA sequencer. The method was found to be robust and suitable for high-throughput glycan analysis. Even though the method comprises two overnight incubations, 96 samples can be analyzed with an overall labor allocation time of 2.5 h. The method was applied to serum samples from a pregnant woman, which were sampled during first, second, and third trimesters of pregnancy, as well as 6 weeks, 3 months, and 6 months postpartum. Alterations in the glycosylation patterns were observed with gestation and time after delivery.