Gao Wenjie, Li Henghui, Liu Yanyan, Liu Yuhong, Feng Xiaojun, Liu Bi-Feng, Liu Xin
Britton Chance Center for Biomedical Photonics at Wuhan National Laboratory for Optoelectronics - Hubei Bioinformatics & Molecular Imaging Key Laboratory, Systems Biology Theme, Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China.
Department of Rheumatology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
Talanta. 2016 Dec 1;161:554-559. doi: 10.1016/j.talanta.2016.09.004. Epub 2016 Sep 4.
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has become an important technology for glycan analysis due to its ease of operation, short analysis time and impurity tolerance. However, the low ionization efficiency of N-glycans led to the difficulty in analyzing glycans of low abundance in complex biological samples due to the lack of basic site for protonation. Therefore, highly sensitive method for the glycans analysis is in urgent demand. Here we report a new strategy to introduce a permanent charge at the reducing end of N-linked glycans by a one pot reaction, where glycosylamines that were obtained by rapid deglycosylation within 5min were labeled with N-succinimidyloxycarbonylmethyl tris (2,4,6- trimethoxyphenyl) phosphonium bromide (TMPP-Ac-OSu). With TMPP-Ac labeling, more than 50 fold enhancement in the sensitivity of method was achieved for neutral glycans from ribonuclease B (RNase B) in comparison to their native counterparts. In combination with methylamidation of sialic acid residues, this novel developed strategy could also be used for sialylated glycans analysis from sialoglycoproteins and complex serum sample. As a result, more than 50 glycans were detected with only 25nL human serum sample.
基质辅助激光解吸/电离质谱（MALDI-MS）因其操作简便、分析时间短和对杂质耐受性强，已成为聚糖分析的一项重要技术。然而，N-聚糖的低电离效率导致在复杂生物样品中分析低丰度聚糖存在困难，因为缺乏质子化的碱性位点。因此，迫切需要一种高灵敏度的聚糖分析方法。在此，我们报道了一种通过一锅法反应在N-连接聚糖的还原端引入永久电荷的新策略，即在5分钟内通过快速去糖基化获得的糖基胺用N-琥珀酰亚胺氧基羰基甲基三（2,4,6-三甲氧基苯基）溴化鏻（TMPP-Ac-OSu）进行标记。通过TMPP-Ac标记，与天然核糖核酸酶B（RNase B）的中性聚糖相比，该方法对其灵敏度提高了50多倍。结合唾液酸残基的甲基酰胺化，这种新开发的策略也可用于分析唾液酸化糖蛋白和复杂血清样品中的唾液酸化聚糖。结果，仅用25nL人血清样品就检测到了50多种聚糖。
