Antia Imeobong U, Yagnik Darshna R, Pantoja Munoz Leonardo, Shah Ajit J, Hills Frank A
Glycan Research Group, Department of Natural Sciences, Faculty of Science and Technology, Middlesex University, The Burroughs, London NW4 4BT, UK.
Anal Biochem. 2017 Aug 1;530:17-30. doi: 10.1016/j.ab.2017.04.019. Epub 2017 Apr 30.
Glycosaminoglycans are a heterogeneous family of linear polysaccharides comprised of repeating disaccharide subunits that mediate many effects at the cellular level. There is increasing evidence that the nature of these effects is determined by differences in disaccharide composition. However, the determination of GAG disaccharide composition in biological samples remains challenging and time-consuming. We have developed a method that uses derivatization and selected ion recording and RP-UPLCMS resulting in rapid separation and quantification of twelve heparin/heparin sulfate disaccharides from 5 μg GAG. Limits of detection and quantitation were 0.02-0.15 and 0.07-0.31 μg/ml respectively. We have applied this method to the novel analysis of disaccharide levels extracted from heparan sulfate and human cancer cell lines. Heparan sulfate disaccharides extracted from biological samples following actinase and heparinase incubation and derivatized using reductive amination with 2-aminoacridone. Derivatized disaccharides were analyzed used UPLC-MS with single ion monitoring. Eight HS disaccharide subunits were separated and quantified from HS and cell lines in eleven minutes per sample. In all samples the most abundant subunits present were the unsulfated ΔUA-GlcNAc, ΔUA-GlcNAc,6S and ΔUA,2S-GlcNS,6S. There was considerable variation in the proportions and concentrations of disaccharides between different cell lines. Further studies are needed to examine the significance of these differences.
糖胺聚糖是一类由重复二糖亚基组成的线性多糖异质家族，在细胞水平介导多种效应。越来越多的证据表明，这些效应的性质由二糖组成的差异决定。然而，生物样品中糖胺聚糖二糖组成的测定仍然具有挑战性且耗时。我们开发了一种方法，该方法使用衍生化、选择离子记录和反相超高效液相色谱-质谱联用技术，能够从5μg糖胺聚糖中快速分离并定量12种肝素/硫酸乙酰肝素二糖。检测限和定量限分别为0.02 - 0.15μg/ml和0.07 - 0.31μg/ml。我们已将该方法应用于从硫酸乙酰肝素和人癌细胞系中提取的二糖水平的新型分析。生物样品经放线酶和肝素酶孵育后提取的硫酸乙酰肝素二糖，使用2-氨基吖啶通过还原胺化进行衍生化。衍生化的二糖使用超高效液相色谱-质谱联用技术进行单离子监测分析。每个样品在11分钟内从硫酸乙酰肝素和细胞系中分离并定量8种硫酸乙酰肝素二糖亚基。在所有样品中，含量最丰富的亚基是未硫酸化的ΔUA-GlcNAc、ΔUA-GlcNAc,6S和ΔUA,2S-GlcNS,6S。不同细胞系中二糖的比例和浓度存在相当大的差异。需要进一步研究来检验这些差异的意义。
