High-Throughput and Site-Specific N-Glycosylation Analysis of Human Alpha-1-Acid Glycoprotein Offers a Great Potential for New Biomarker Discovery.
High-Throughput and Site-Specific N-Glycosylation Analysis of Human Alpha-1-Acid Glycoprotein Offers a Great Potential for New Biomarker Discovery.
复制标题
人α-1-酸性糖蛋白的高重复性和位点特异性N-糖基化分析为发现新的生物标志物提供了巨大的潜力。
DOI：
10.1074/mcp.ra120.002433
复制
发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Novokmet M
中科院分区：
文献类型：
--
作者：
Keser T;Tijardović M;Gornik I;Lukić E;Lauc G;Gornik O;Novokmet M
α-1-酸性糖蛋白（AGP）是血液中的急性期糖蛋白，主要在肝脏中合成，其生物学作用尚未完全了解。它由45%的碳水化合物组成，这些碳水化合物以五种N-连接复合聚糖的形式存在。AGP N-糖基化在许多不同的疾病中显示出变化，并且一些变化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糖基化主要在小型队列中进行分析，没有详细的位点特异性聚糖信息。在这里，我们开发了一种具有成本效益的方法，用于AGP的高通量和位点特异性N-糖基化LC-MS分析，该方法可应用于大型队列，有助于寻找新的疾病生物标志物，并能够更好地了解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，但在96孔板中通过从5 μl血浆中酸沉淀来富集AGP。胰蛋白酶消化后，AGP糖肽纯化使用亲水性相互作用色谱法为基础的固相萃取和反相液相色谱-电喷雾电离-MS分析。我们用我们的方法表明，第一次AGP N-聚糖档案是稳定的健康个体（14个人在三个时间点），这是一个要求，其诊断潜力的评估。此外，我们在一个人群中测试了我们的方法，包括在危重病中登记高血糖的个体（59例和49例对照），这代表了发展2型糖尿病的风险显着增加。糖尿病风险较高的个体在AGP的第二个糖基化位点上表现出增加的N-聚糖分支，在AGP的第三个和AGP 1的第四个糖基化位点上表现出较低的N-聚糖唾液酸化。虽然这应该在更大的前瞻性队列中得到证实，但它表明位点特异性AGP N-聚糖谱可以帮助区分有2型糖尿病风险的个体。提出了高通量详细AGP糖谱分析的经济有效的方法。位点特异性AGP N-聚糖谱可从5 μl血浆中获得。AGP N-聚糖谱在健康个体中稳定。AGP糖谱可以帮助识别有2型糖尿病风险的个体。第一次，开发了一种高通量详细AGP N-糖基化分析的成本效益方法，其中包括位点特异性糖基化信息。使用该方法，证明了AGP N-聚糖谱在健康个体中是稳定的。此外，在试点队列中使用该方法，发现位点特异性AGP N-聚糖谱可以帮助区分有2型糖尿病风险的个体。该方法为研究AGP的大生物标志物潜力提供了一种新的有价值的工具。
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. Cost-effective method for high-throughput detailed AGP glycoprofiling is presented. Site-specific AGP N-glycan profile can be obtained from 5 μl of blood plasma. AGP N-glycan profile is stable in a healthy individual. AGP glycoprofile could help identify individuals who are at risk of type 2 diabetes. For the first time, a cost-effective method for a high-throughput detailed AGP N-glycosylation profiling was developed, which includes site-specific glycosylation information. Using the method, it was demonstrated that AGP N-glycan profile is stable in a healthy individual. Furthermore, using the method on a pilot cohort, it was found that site-specific AGP N-glycan profile could help distinguish individuals who are at risk of type 2 diabetes. The method presents a new valuable tool for investigation of AGP’s large biomarker potential.