title: Hydrophilic interaction chromatography-based high-throughput sample preparation method for N-glycan analysis from total human plasma glycoproteins.
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 microL 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.
authors.affiliation: Leiden University Medical Center, Biomolecular Mass Spectrometry Unit, Department of Parasitology, P.O. Box 9600, 2300RC Leiden, The Netherlands.
authors.forename: L Renee
authors.initials: LR
authors.lastname: Ruhaak
authors.forename: Carolin
authors.initials: C
authors.lastname: Huhn
authors.forename: Willem-Jan
authors.initials: WJ
authors.lastname: Waterreus
authors.forename: Arjen R
authors.initials: AR
authors.lastname: de Boer
authors.forename: Christian
authors.initials: C
authors.lastname: Neusüss
authors.forename: Cornelis H
authors.initials: CH
authors.lastname: Hokke
authors.forename: André M
authors.initials: AM
authors.lastname: Deelder
authors.forename: Manfred
authors.initials: M
authors.lastname: Wuhrer
mesh_terms: D002845:Chromatography / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D019411:Clinical Laboratory Techniques; D006023:Glycoproteins / Q000097:blood*; D006031:Glycosylation; D006801:Humans; D013058:Mass Spectrometry; D011134:Polysaccharides / Q000032:analysis*; D052616:Solid Phase Extraction
chemical_list: D006023:Glycoproteins; D011134:Polysaccharides