Albrecht Simone, Mittermayr Stefan, Smith Josh, Martín Silvia Millán, Doherty Margaret, Bones Jonathan
Characterisation and Comparability Laboratory, NIBRT - The National Institute for Bioprocessing Research and Training, Dublin, Ireland.
Department of Life Sciences, School of Science, Institute of Technology Sligo, Sligo, Ireland.
Proteomics. 2017 Jan;17(1-2). doi: 10.1002/pmic.201600304.
Quantitative glycomics represents an actively expanding research field ranging from the discovery of disease-associated glycan alterations to the quantitative characterization of N-glycans on therapeutic proteins. Commonly used analytical platforms for comparative relative quantitation of complex glycan samples include MALDI-TOF-MS or chromatographic glycan profiling with subsequent data alignment and statistical evaluation. Limitations of such approaches include run-to-run technical variation and the potential introduction of subjectivity during data processing. Here, we introduce an offline 2D LC-MS workflow for the fractionation and relative quantitation of twoplex isotopically labeled N-linked oligosaccharides using neutral C and C aniline (Δmass = 6 Da). Additional linkage-specific derivatization of sialic acids using 4-(4,6-dimethoxy-1,3,5-trizain-2-yl)-4-methylmorpholinium chloride offered simultaneous and advanced in-depth structural characterization. The potential of the method was demonstrated for the differential analysis of structurally defined N-glycans released from serum proteins of patients diagnosed with various stages of colorectal cancer. The described twoplex C / C aniline 2D LC-MS platform is ideally suited for differential glycomic analysis of structurally complex N-glycan pools due to combination and analysis of samples in a single LC-MS injection and the associated minimization in technical variation.
定量糖组学是一个正在积极拓展的研究领域，其范围涵盖从发现与疾病相关的聚糖改变到对治疗性蛋白质上的N-聚糖进行定量表征。用于复杂聚糖样品相对定量比较的常用分析平台包括基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）或色谱聚糖谱分析，随后进行数据比对和统计评估。这些方法的局限性包括批次间的技术差异以及数据处理过程中可能引入的主观性。在此，我们介绍一种离线二维液相色谱-质谱（2D LC-MS）工作流程，用于使用中性碳-13（¹³C）和碳-14（¹⁴C）苯胺（质量差 = 6 Da）对双标记同位素标记的N-连接寡糖进行分级分离和相对定量。使用4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基吗啉鎓氯对唾液酸进行额外的连接特异性衍生化，可同时进行深入的结构表征。该方法的潜力在对诊断为不同阶段结直肠癌患者血清蛋白释放的结构明确的N-聚糖进行差异分析中得到了证明。所描述的双标记¹³C/¹⁴C苯胺二维液相色谱-质谱平台非常适合对结构复杂的N-聚糖库进行差异糖组学分析，因为它可以在一次液相色谱-质谱进样中对样品进行合并和分析，并最大限度地减少技术差异。
