Mass Spectrometric Analysis of Sialylated Glycans with Use of Solid-Phase Labeling of Sialic Acids

The
analysis of sialylated glycans is critical for understanding the role
of sialic acid in normal biological processes as well as in disease.
However, the labile nature of sialic acid typically renders routine
analysis of this monosaccharide by mass spectrometric methods difficult.
To overcome this difficulty we pursued derivatization methodologies,
extending established acetohydrazide approaches to aniline-based methods,
and finally to optimized p-toluidine derivatization.
This new quantitative glycoform profiling method with use of MALDI-TOF
in positive ion mode was validated by first comparing N-glycans isolated from fetuin and serum and was then exploited to
analyze the effects of increased metabolic flux through the sialic
acid pathway in SW1990 pancreatic cancer cells by using a colabeling
strategy with light and heavy toluidine. The latter results established
that metabolic flux, in a complementary manner to the more well-known
impact of sialyltransferase expression, can critically modulate the
sialylation of specific glycans while leaving others virtually unchanged.

唾液酸化糖链（sialylated glycans）的分析对于解析唾液酸（sialic acid）在正常生理过程及疾病发生中的关键作用至关重要。然而，唾液酸的不稳定性通常使得常规质谱分析法难以对该单糖开展有效分析。为攻克这一技术难题，本研究开发了系列衍生化方法：将已成熟的乙酰肼衍生方法拓展至基于苯胺的衍生策略，最终优化得到对甲苯胺衍生化方法。本研究以正离子模式下的基质辅助激光解吸电离飞行时间质谱（MALDI-TOF）为检测平台，对该新型定量糖型分析方法进行了验证：首先对比分析了从胎球蛋白（fetuin）与血清中分离得到的N-糖链（N-glycans）；随后采用轻、重对甲苯胺双标记策略，探究了SW1990胰腺癌细胞（SW1990 pancreatic cancer cells）中唾液酸代谢通路代谢流量升高对糖基化修饰的影响。后续实验结果证实，相较于学界更为熟知的唾液酸转移酶（sialyltransferase）表达的调控作用，代谢流量可通过互补机制显著调控特定糖链的唾液酸化修饰水平，而对其余糖链的修饰状态几乎无影响。