Biogen Idec, Cambridge, MA 02142, USA.
Anal Bioanal Chem. 2012 Mar;402(9):2687-700. doi: 10.1007/s00216-012-5712-5. Epub 2012 Jan 26.
The development of a general method for the purification and quantitative glycomic analysis of human plasma samples to characterize global glycosylation changes shall be presented. The method involves multiple steps, including the depletion of plasma via multi-affinity chromatography to remove high abundant proteins, the enrichment of the lower abundant glycoproteins via multi-lectin affinity chromatography, the isotopic derivatization of released glycans, and quantitative analysis by MALDI-TOF MS. Isotopic derivatization of glycans is accomplished using the well-established chemistry of reductive amination to derivatize glycans with either a light analog ((12)C anthranilic acid) or a heavy analog ((13)C(7) anthranilic acid), which allows for the direct comparison of the alternately labeled glycans by MALDI-TOF MS. The method displays a tenfold linear dynamic range for both neutral and sialylated glycans with sub-picomolar sensitivity. Additionally, by using anthranilic acid, a very sensitive fluorophore, as the derivatization reagent, the glycans can be analyzed by chromatography with fluorescence detection. The utility of this methodology is highlighted by the many diseases and disorders that are known to either show or be the result of changes in glycosylation. A method that provides a generic approach for sample preparation and quantitative data will help to further advance the field of glycomics.
将介绍一种用于纯化和定量糖组分析人血浆样品以表征全局糖基化变化的通用方法。该方法涉及多个步骤，包括通过多亲和层析耗尽富含大量蛋白质的血浆，通过多凝集素亲和层析富集低丰度糖蛋白，释放糖的同位素衍生化，以及 MALDI-TOF MS 的定量分析。糖的同位素衍生化是通过还原胺化的成熟化学方法完成的，用轻模拟物（(12)C 邻氨基苯甲酸）或重模拟物（(13)C(7)邻氨基苯甲酸）衍生化糖，这允许 MALDI-TOF MS 直接比较交替标记的糖。该方法对中性和唾液酸化糖显示出十倍的线性动态范围，具有亚皮摩尔的灵敏度。此外，通过使用邻氨基苯甲酸作为衍生化试剂，一种非常灵敏的荧光团，糖可以通过具有荧光检测的色谱法进行分析。该方法的实用性通过许多已知表现出或导致糖基化变化的疾病和病症得到了强调。提供通用样品制备和定量数据方法的方法将有助于进一步推进糖组学领域的发展。
