PDF(Pubmed)
Abstract：
Alpha-1-acid glycoprotein (AGP) is a heterogeneous glycoprotein fulfilling key roles in many biological processes, including transport of drugs and hormones and modulation of inflammatory and immune responses. The glycoform profile of AGP is known to change depending on (patho)physiological states such as inflammatory diseases or pregnancy. Besides complexity originating from five N-glycosylation sites, the heterogeneity of the AGP further expands to genetic variants. To allow in-depth characterization of this intriguing protein, we developed a method using anion exchange chromatography (AEX) coupled to mass spectrometry (MS) revealing the presence of over 400 proteoforms differing in their glycosylation or genetic variants. More precisely, we could determine that AGP mainly consists of highly sialylated higher antennary structures with on average 16 sialic acids and 0 or 1 fucose per protein. Interestingly, a slightly higher level of fucosylation was observed for AGP1 variants compared to that of AGP2. Proteoform assignment was supported by integrating data from complementary MS-based approaches, including AEX-MS of an exoglycosidase-treated sample and glycopeptide analysis after tryptic digestion. The developed analytical method was applied to characterize AGP from plasma of women during and after pregnancy, revealing differences in glycosylation profiles, specifically in the number of antennae, HexHexNAc units, and sialic acids.
摘要：
α-1-酸性糖蛋白（AGP）是一种在许多生物过程中发挥关键作用的异质糖蛋白，包括药物和激素的运输以及炎症和免疫反应的调节。已知AGP的糖型谱根据(病理)生理状态如炎性疾病或妊娠而改变。除了来自五个N-糖基化位点的复杂性，AGP的异质性进一步扩展到遗传变异。为了深入表征这种有趣的蛋白质，我们开发了一种使用阴离子交换色谱（AEX）结合质谱（MS）的方法，揭示了存在超过400种糖基化或遗传变体不同的蛋白质形式。更确切地说,我们可以确定AGP主要由高度唾液酸化的高触角结构组成，平均每个蛋白有16个唾液酸和0或1个岩藻糖。有趣的是,与AGP2相比,观察到AGP1变体的岩藻糖基化水平略高。通过整合来自互补的基于MS的方法的数据来支持Proteoform分配，包括外切糖苷酶处理的样品的AEX-MS和胰蛋白酶消化后的糖肽分析。所开发的分析方法用于表征妊娠期间和之后妇女血浆中的AGP，揭示糖基化谱的差异，特别是在天线的数量上，HexHexNAc单位,和唾液酸。
