Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia.
Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia.
Mol Cell Proteomics. 2021;20:100044. doi: 10.1074/mcp.RA120.002433. Epub 2021 Jan 23.
Alpha-1-acid glycoprotein (AGP) is an acute phase glycoprotein in blood, which is primarily synthetized in the liver and whose biological role is not completely understood. It consists of 45% carbohydrates that are present in the form of five N-linked complex glycans. AGP N-glycosylation was shown to be changed in many different diseases, and some changes appear to be disease-specific; thus, it has a great diagnostic and prognostic potential. However, AGP glycosylation was mainly analyzed in small cohorts and without detailed site-specific glycan information. Here, we developed a cost-effective method for a high-throughput and site-specific N-glycosylation LC-MS analysis of AGP which can be applied on large cohorts, aid in search for novel disease biomarkers, and enable better understanding of AGP's role and function in health and disease. The method does not require isolation of AGP with antibodies and affinity chromatography, but AGP is enriched by acid precipitation from 5 μl of bloodplasma in a 96-well format. After trypsinization, AGP glycopeptides are purified using a hydrophilic interaction chromatography-based solid-phase extraction and analyzed by reversed-phase-liquid chromatography-electrospray ionization-MS. We used our method to show for the first time that AGP N-glycan profile is stable in healthy individuals (14 individuals in three time points), which is a requirement for evaluation of its diagnostic potential. Furthermore, we tested our method on a population including individuals with registered hyperglycemia in critical illness (59 cases and 49 controls), which represents a significantly increased risk of developing type 2 diabetes. Individuals at higher risk of diabetes presented increased N-glycan branching on AGP's second glycosylation site and lower sialylation of N-glycans on AGP's third and AGP1's fourth glycosylation site. Although this should be confirmed on a larger prospective cohort, it indicates that site-specific AGP N-glycan profile could help distinguish individuals who are at risk of type 2 diabetes.
α-1-酸性糖蛋白（AGP）是血液中的一种急性期糖蛋白，主要在肝脏中合成，其生物学功能尚不完全清楚。它由 45%的碳水化合物组成，这些碳水化合物以 5 种 N 连接的复合糖的形式存在。已经表明，AGP 的 N 糖基化在许多不同的疾病中发生改变，并且一些改变似乎是疾病特异性的；因此，它具有很大的诊断和预后潜力。然而，AGP 糖基化主要在小队列中进行分析，并且没有详细的位点特异性聚糖信息。在这里，我们开发了一种经济高效的方法，用于对 AGP 进行高通量和位点特异性的 N-糖基化 LC-MS 分析，该方法可应用于大样本量，有助于寻找新的疾病生物标志物，并更好地理解 AGP 在健康和疾病中的作用和功能。该方法不需要用抗体和亲和层析分离 AGP，而是通过在 96 孔格式中从 5 μl 血浆中用酸沉淀来富集 AGP。在胰蛋白酶消化后，AGP 糖肽通过亲水相互作用色谱基于固相萃取进行纯化，并通过反相液相色谱-电喷雾电离-MS 进行分析。我们首次使用该方法表明，AGP N-聚糖谱在健康个体中是稳定的（14 名个体在三个时间点），这是评估其诊断潜力的要求。此外，我们在包括危重病中血糖升高的个体的人群中测试了我们的方法（59 例病例和 49 例对照），这代表了发展为 2 型糖尿病的风险显著增加。患有糖尿病风险较高的个体在 AGP 的第二个糖基化位点上具有增加的 N-聚糖分支，并且在 AGP 的第三个和 AGP1 的第四个糖基化位点上的 N-聚糖的唾液酸化程度较低。尽管这需要在更大的前瞻性队列中得到证实，但它表明 AGP 的位点特异性 N-聚糖谱可能有助于区分患 2 型糖尿病的风险个体。
