Quantifying Protein-Specific N-Glycome Profiles by Focused Protein and Immunoprecipitation Glycomics
Quantifying Protein-Specific N-Glycome Profiles by Focused Protein and Immunoprecipitation Glycomics
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DOI：
10.1021/acs.jproteome.9b00232
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发表时间：
2019-08-01
影响因子：
4.4
通讯作者：
Sakamoto, Naoya
中科院分区：
文献类型：
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作者：
Kobayashi, Takashi;Ogawa, Koji;Sakamoto, Naoya
据报道，血清n -聚糖是许多疾病和病症的潜在诊断和治疗生物标志物，如炎症、纤维化和癌症进展。我们之前描述了凝胶分离血清蛋白的聚焦蛋白糖化分析（FPG）。通过这种方法，我们寻找了非酒精性脂肪性肝炎（NASH）的新型聚糖生物标志物，并成功鉴定出一些n -聚糖在NASH患者中与非酒精性脂肪肝患者相比显著升高。其中，α -1抗胰蛋白酶的三甲基化单聚焦三天线聚糖（A3F）变化最为动态。为了快速鉴定聚焦蛋白上的n -聚糖，我们构建了一种称为免疫沉淀糖组学（IPG）的简化方法，用亲和珠免疫沉淀靶蛋白，然后用MALDI-TOF ms进行糖组学分析。以α -1抗胰蛋白酶和铜蓝蛋白为靶蛋白，我们比较了FPG和IPG测定的n -聚糖值。两种方法对各n -糖聚糖的定量值具有统计学显著的相关性，表明简化的IPG方法可以实现对目标蛋白的高通量和定量n -糖组学。因此，结合FPG和IPG的分析策略可以适用于适当疾病领域的一般生物标志物发现和验证。
Serum N-glycans have been reported to be potential diagnostic and therapeutic biomarkers for many diseases and conditions, such as inflammation, fibrosis, and cancer progression. We previously described the focused protein glycomic analysis (FPG) from gel-separated serum proteins. With this methodology, we sought novel glycan biomarkers for nonalcoholic steatohepatitis (NASH) and successfully identified some N-glycans that were significantly elevated in NASH patients compared to nonalcoholic fatty liver patients. Among them, trisialylated monofucosylated triantennary glycan (A3F) of alpha-1 antitrypsin showed the most dynamic change. For rapid identification of N-glycans on the focused proteins, we constructed a simplified method called immunoprecipitation glycomics (IPG), where the target proteins were immunoprecipitated with affinity beads and subsequently subjected to glycomic analysis by MALDI-TOF MS. Focusing on alpha-1 antitrypsin and ceruloplasmin as the target proteins, we compared the values of N-glycans determined by FPG and IPG. The quantified values of each N-glycan by these two methods showed a statistically significant correlation, indicating that high throughput and quantitative N-glycomics of targeted proteins can be achieved by the simplified IPG method. Thus, an analytical strategy combining FPG and IPG can be adapted to general biomarker discovery and validation in appropriate disease areas.