Department of Pathology, Johns Hopkins University, Baltimore, Maryland 21231, United States.
Anal Chem. 2013 Apr 2;85(7):3606-13. doi: 10.1021/ac3033867. Epub 2013 Mar 18.
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.
唾液酸糖链分析对于理解唾液酸在正常生物过程以及疾病中的作用至关重要。然而，唾液酸的不稳定性通常使得通过质谱方法对这种单糖进行常规分析变得困难。为了克服这一困难，我们采用了衍生化方法，将已建立的乙酰肼方法扩展到苯胺方法，最终优化了对甲苯胺衍生化。这种新的定量糖型分析方法，采用 MALDI-TOF 在正离子模式下进行，首先通过比较胎球蛋白和血清中分离的 N-聚糖进行验证，然后利用轻、重对甲苯胺的共标记策略分析 SW1990 胰腺癌细胞中唾液酸途径代谢通量增加的影响。后者的结果表明，代谢通量与唾液酸转移酶表达更为人所熟知的影响相辅相成，可以显著调节特定糖链的唾液酸化，而其他糖链几乎不变。
