Hydrophilic Interaction Chromatography-Based High-Throughput Sample Preparation Method for N-Glycan Analysis from Total Human Plasma Glycoproteins

L. Renee Ruhaak†, Carolin Huhn‡, Willem-Jan Waterreus†, Arjen R. de Boer†, Christian Neusss‡, Cornelis H. Hokke†, Andr M. Deelder† and Manfred Wuhrer*†
Leiden University Medical Center, Biomolecular Mass Spectrometry Unit, Department of Parasitology, P.O. Box 9600, 2300RC Leiden, The Netherlands, and Aalen University, Faculty of Chemistry, Beethovenstrasse 1, 73430 Aalen, Germany
Anal. Chem., 2008, 80 (15), pp 6119–6126
DOI: 10.1021/ac800630x
Publication Date (Web): July 2, 2008
Copyright © 2008 American Chemical Society
†

Leiden University Medical Center.

,
‡

Aalen University.

,
* To whom correspondence should be addressed. E-mail: m.wuhrer@lumc.nl.

Abstract

Many diseases are associated with changes in the glycosylation of plasma proteins. To search for glycan biomarkers, large sample sets have to be investigated for which high-throughput sample preparation and analysis methods are required. We here describe a 96 well plate-based high-throughput procedure for the rapid preparation of 2-aminobenzoic acid (2-AA) labeled N-glycans from 10 μL of human plasma. During this procedure, N-glycans are released from glycoproteins and subsequently labeled with 2-AA without prior purification. A hydrophilic interaction chromatography (HILIC)-based solid phase extraction method is then applied to isolate the 2-AA labeled N-glycans, which can be analyzed by MALDI-TOF-MS, HPLC with fluorescence detection, and CE−MS. The relative standard deviation for the intrabatch repeatability and the interbatch repeatability of the sample preparation method remained below 7% and below 9%, respectively, for all peaks observed by HPLC. Similar results were obtained with MALDI-TOF-MS, where 47 N-glycans could be measured consistently. The 2-AA labeled N-glycans were additionally analyzed by a CE−ESI-Q-TOF-MS method, which featured high resolution and mass accuracy, allowing the unambiguous determination of the N-glycan compositions. Up to four times, 96 human plasma samples can be handled in parallel, which, together with the versatility of the 2-AA label, makes this procedure very attractive for glycomics analysis of larger sample cohorts.

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History

  • Published In Issue August 01, 2008
  • Article ASAPJuly 02, 2008
  • Received: March 28, 2008
    Accepted: June 3, 2008

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