3-Amino-1-phenyl-2-pyrazoline-5-ketone as a heterobifunctional chromogenic reagent to derivatize reducing glycans for subsequent online LC/MS analysis.
3-Amino-1-phenyl-2-pyrazoline-5-ketone as a heterobifunctional chromogenic reagent to derivatize reducing glycans for subsequent online LC/MS analysis.
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DOI：
10.1016/j.ab.2018.02.029
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发表时间：
2018-05
影响因子：
2.9
通讯作者：
Yu Lu;Chengjian Wang;Rendan Liu;W. Jin;Yanan Wen;Linjuan Huang;Zhongfu Wang
中科院分区：
文献类型：
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作者：
Yu Lu;Chengjian Wang;Rendan Liu;W. Jin;Yanan Wen;Linjuan Huang;Zhongfu Wang
通过液相色谱/质谱法对聚糖进行灵敏分析受到聚糖结构上缺乏显色或荧光基团以及其差的电离性质的显著阻碍。本文采用具有氨基和活性亚甲基的异双功能显色剂3-氨基-1-苯基-2-吡唑啉-5-酮（PAP），通过高效液相色谱/电喷雾质谱（HPLC/ESI-MS）对柱前标记的聚糖进行检测。在酸性介质中通过还原胺化反应得到了具有活性亚甲基的PAP衍生物，在碱性介质中通过活性亚甲基与聚糖的还原端缩合得到了具有活性氨基的PAP衍生物，并且PAP衍生物可以进一步进行功能化，通过聚糖微阵列制备。优化了两种反应模式的条件，研究了PAP衍生物的HPLC分离方法，并对生物样品中的几种还原性聚糖进行了PAP衍生物的ESI-MS和LC-MS分析。
Sensitive analysis of glycans by liquid chromatography/mass spectrometry is significantly hampered by the lack of chromogenic or fluorescent groups on the glycan structures, as well as, their poor ionization properties. In the present, a heterobifunctional chromogenic reagent 3-amino-1-phenyl-2-pyrazoline-5-ketone (PAP) bearing amino and active methylene groups, which readily reacts with reducing glycans, was used for detection of the pre-column-labeled glycans via high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS). The PAP derivatives with active methylene and amino groups were obtained via reductive amination in acidic medium and condensation of an active PAP methylene group with the reducing end of glycans in alkaline medium, respectively, and the PAP derivatives could be further functionalized, e.g., via glycan microarray preparation. The conditions for the two reaction modes were optimized, the HPLC separation method of PAP derivatives was investigated, and the PAP derivatives of some glycans derived from biological samples were obtained and analyzed by ESI-MS and LC-MS. Using this new reagent, reducing glycans can be selectively derivatized by different reaction mechanisms, having great importance for functional glycomics studies.