Leiden University Scholarly Publications

Persistent URL of this record https://hdl.handle.net/1887/109155

In Collections

This item can be found in the following collections:

Ruhaak, L.R.; Hennig, R.; Huhn, C.; Borowiak, M.; Dolhain, R.J.E.M.; Deelder, A.M.; ... ; Wuhrer, M. (2010)

Optimized Workflow for Preparation of APTS-Labeled N-Glycans Allowing High-Throughput Analysis of Human Plasma Glycomes using 48-Channel Multiplexed CGE-LIF

Article / Letter to editor
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...Show moreHigh 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 deliveryShow less
glycan profiling high throughput N-glycan analysis CGE-LIF APTS HILIC-SPE plasma hydrophilic interaction chromatography sample preparation method dna-sequencing equipment mass-spectrometry antiinflammatory activity reductive amination glycosylation hplc identification glycoproteins
All authors
Ruhaak, L.R.; Hennig, R.; Huhn, C.; Borowiak, M.; Dolhain, R.J.E.M.; Deelder, A.M.; Rapp, E.; Wuhrer, M.
Date
2010-12-31
Journal
Journal of Proteome Research
Volume
9
Issue
12
Pages
6655 - 6664
DOI
doi:10.1021/pr100802f