Department of Cell and Molecular Pharmacology, Medical University of South Carolina, 173 Ashley Avenue, Charleston, 29425 South Carolina, United States.
J Am Soc Mass Spectrom. 2020 Dec 2;31(12):2511-2520. doi: 10.1021/jasms.0c00213. Epub 2020 Aug 18.
Changes in the levels and compositions of N-glycans released from serum and plasma glycoproteins have been assessed in many diseases across many large clinical sample cohorts. Assays used for N-glycan profiling in these fluids currently require multiple processing steps and have limited throughput, thus diminishing their potential for use as standard clinical diagnostic assays. A novel slide-based N-glycan profiling method was evaluated for sensitivity and reproducibility using a pooled serum standard. Serum was spotted on to an amine-reactive slide, delipidated and desalted with a series of washes, sprayed with peptide N-glycosidase F and matrix, and analyzed by MALDI-FTICR or MALDI-Q-TOF mass spectrometry. Routinely, over 75 N-glycan species can be detected from one microliter of serum in less than 6.5 h. Additionally, endoglycosidase F3 was applied to this workflow to identify core-fucosylated N-glycans and displayed the adaptability of this method for the determination of structural information. This method was applied to a small pooled serum set from either obese or nonobese patients that had breast cancer or a benign lesion. This study confirms the reproducibility, sensitivity, and adaptability of a novel method for N-glycan profiling of serum and plasma for potential application to clinical diagnostics.
在许多大型临床样本队列中，许多疾病的血清和血浆糖蛋白释放的 N-糖链水平和组成发生了变化。目前，这些液体中用于 N-糖链分析的测定法需要多个处理步骤，并且通量有限，因此降低了它们作为标准临床诊断测定法的潜力。我们评估了一种新的基于载玻片的 N-糖链分析方法，使用混合血清标准品评估其灵敏度和重现性。将血清点样到胺反应载玻片上，用一系列洗涤液进行去脂和脱盐，用肽 N-糖苷酶 F 和基质喷雾，然后通过 MALDI-FTICR 或 MALDI-Q-TOF 质谱分析。通常，从一微升血清中可以检测到超过 75 种 N-糖链，耗时不到 6.5 小时。此外，还将内切糖苷酶 F3 应用于该工作流程，以鉴定核心岩藻糖基化的 N-聚糖，并显示该方法对于确定结构信息的适应性。该方法应用于来自肥胖或非肥胖患者的小混合血清组，这些患者患有乳腺癌或良性病变。这项研究证实了一种新型血清和血浆 N-糖链分析方法的重现性、灵敏度和适应性，该方法可能适用于临床诊断。
