Hydrophilic interaction chromatography-based high-throughput sample preparation method for N-glycan analysis from total human plasma glycoproteins

作者：Ruhaak L Renee; Huhn Carolin; Waterreus Willem Jan; de Boer Arjen R; Neusuess Christian; Hokke Cornelis H; Deelder Andre M; Wuhrer Manfred^*

来源：Analytical Chemistry, 2008, 80(15): 6119-6126.

DOI：10.1021/ac800630x

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 mu L 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.

• 出版日期2008-8-1