Hydrophilic interaction chromatography-based high-throughput sample preparation method for N-glycan analysis from total human plasma glycoproteins
Hydrophilic interaction chromatography-based high-throughput sample preparation method for N-glycan analysis from total human plasma glycoproteins
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DOI：
10.1021/ac800630x
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发表时间：
2008-08-01
影响因子：
7.4
通讯作者：
Wuhrer, Manfred
中科院分区：
文献类型：
--
作者：
Ruhaak, L. Renee;Huhn, Carolin;Wuhrer, Manfred
许多疾病都与血浆蛋白糖基化的变化有关。为了寻找葡聚糖生物标记物，必须研究大样本集，为此需要高通量的样本制备和分析方法。本文介绍了一种以96孔板为基础的高通量快速制备2-氨基苯甲酸(2-AA)标记N-聚糖的方法，该方法从10亩L血浆中提取。在这个过程中，N-葡聚糖从糖蛋白中释放出来，然后用2-AA标记，而不需要事先进行纯化。采用基于亲水作用层析(HILIC)的固相萃取方法分离2-AA标记的N-葡聚糖，并用MALDI-TOF-MS、高效液相色谱荧光检测和CE-MS对其进行分析。样品制备方法的批内重复性和批间重复性的相对标准偏差分别保持在7%和9%以下。MALDI-TOF-MS也得到了类似的结果，其中47个N-糖链可以被一致地测量到。此外，还利用CE-ESI-Q-TOF-MS方法对2-AA标记的N-糖链进行了分析，该方法具有较高的分辨率和质量准确度，可以明确地确定N-糖链的组成。多达四次，96个人体血浆样本可以并行处理，再加上2-AA标记的多功能性，这使得这一过程对于更大样本队列的糖组分析非常有吸引力。
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.