公开(公告)号：WO2016069764A1

公开(公告)日：2016-05-06

申请号：PCT/US2015/057848

申请日：2015-10-28

Applicant: WATERS TECHNOLOGIES CORPORATION

Inventor： LAUBER, Matthew, A. ,  BROUSMICHE, Darryl, W. ,  KOZA, Stephan, M.

IPC: G01N21/76

CPC classification number: G01N33/58 ,  G01N33/582 ,  G01N33/6842 ,  G01N2333/924 ,  G01N2333/98 ,  G01N2440/38

Abstract: Methods of analyzing glycosylated biomolecules include the steps of producing a deglycosylation mixture of biomolecules deglycosylated by natural or synthetic enzymatic or chemical techniques; providing a reagent solution having a labeling reagent in a polar aprotic, non-nucleophilic organic solvent; and mixing the deglycosylation mixture with the reagent solution in an excess of labeling reagent to produce derivatized glycosylamines. The method steps can be carried out purposefully without depletion of protein matter. A quenching solution can be added to the reaction mixture so that the pH of the reaction mixture is shifted to above 10. The yield of derivatized glycosylamines can be in an amount of about 80 to about 100 mole percent of the reaction mixture with minimal overlabeling, less than 0.2 mole percent. The derivizated glycosylamines can be separated from the reaction mixture and detected by chromatographic detection, fluorescence detection, mass spectrometry ("MS"), or Ultra Violet ("UV") detection and/or a combination thereof.

Abstract translation: 分析糖基化生物分子的方法包括通过天然或合成酶或化学技术产生去糖基化的生物分子的去糖基化混合物的步骤; 在极性非质子非亲核有机溶剂中提供具有标记试剂的试剂溶液; 并将去糖基化混合物与试剂溶液在过量的标记试剂中混合以产生衍生的糖胺。 方法步骤可以有目的地进行，而不会消耗蛋白质物质。 可以将淬灭溶液加入到反应混合物中，使得反应混合物的pH转移到10以上。衍生化的糖胺的产率可以是反应混合物的约80至约100摩尔％，同时具有最小的重叠， 小于0.2摩尔％。 衍生的糖胺可以从反应混合物中分离，并通过色谱检测，荧光检测，质谱（“MS”）或超紫（“UV”）检测和/或其组合进行检测。