Microgradient separation technique for purification and fractionation of permethylated N-glycans before mass spectrometric analyses
Microgradient separation technique for purification and fractionation of permethylated N-glycans before mass spectrometric analyses
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DOI：
10.1002/jssc.201701339
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发表时间：
2018-05-01
影响因子：
3.1
通讯作者：
Novotny, Milos, V
中科院分区：
文献类型：
--
作者：
Rehulka, Pavel;Zahradnikova, Martina;Novotny, Milos, V
分析患者血清或其他临床样品中酶促释放的n -聚糖可能为人类疾病的诊断和预后提供重要信息。这些生物分子的过甲基化同时增加了它们的疏水性，并在随后的质谱分析中大大提高了它们的检测参数。整个过程，从聚糖裂解到最终的质谱测定，包括几个步骤，包括提取，衍生化和纯化。在这些步骤中，可能偶然引入的某些聚合污染物会妨碍最终的测量。为了理解和应对这些干扰，进一步对这些聚糖进行分馏或预浓缩，我们在这里介绍了一种有效的微梯度色谱技术，该技术采用连接到气密微注射器的小型反相微柱，提供移动相梯度。将glyca馏分装入微柱后，建议进行三个洗脱步骤：(1)去除极性污染物；(2)利用电喷雾电离质谱法或基质辅助激光解吸/电离质谱法回收过甲基化聚糖；(3)去除较大的聚合物污染物，使柱前再生。我们进一步证明，捕获的第二部分可以有效地预浓缩并进一步分离，以实现高达6300 Da的衍生化n -聚糖的基质辅助激光解吸/电离质谱检测。四天线n -聚糖的增强检测能力对疾病生物标志物的发现越来越感兴趣。
Analysis of N-glycans released enzymatically from patients' sera or other clinical samples may provide diagnostically and prognostically important information on human disease. Permethylation of these biomolecules simultaneously increases their hydrophobicity and substantially improves their detection parameters in the following mass spectrometric analyses. The overall procedure, from the glycan cleavage to the final mass spectrometric determinations, includes several steps involving extraction, derivatization, and purification. During these steps, certain polymeric contaminants that may have been coincidentally introduced could hamper the final measurements. T To understand and counter these interferences and further fractionate or preconcentrate these glycans, we introduce here an effective microgradient chromatographi technique that employs a small reversed-phase microcolumn connected to a gas-tight microsyringe delivering a mobile-phase gradient. After loading the glyca fraction onto the microcolumn, three elution steps are recommended: (1) remove polar contaminants; (2) recover permethylated glycans for either liquid chromatograph with electrospray ionization mass spectrometry or matrix-assisted laser desorption/ionizatio mass spectrometry; and (3) remove larger polymeric contaminants and regenerate the precolumn. We further demonstrate that the trapped second fraction can be beneficially preconcentrated and further separated to achieve matrix-assisted laser desorption/ionization mass spectrometric detection of the derivatized N-glycans up to 6300 Da. The enhanced detection capabilities for tetra-antennary N-glycans are of increasing interest in disease biomarker discovery.