1. 广东药科大学药学院
2. 军事科学院军事医学研究院生命组学研究所
3. 蛋白质组学国家重点实验室
4. 北京蛋白质组研究中心
5. 国家蛋白质科学中心,北京
6. 北京工业大学生命科学与生物工程学院
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孟波, 李晓宇, 崔新玲, 等. 微量样本中N-糖组分析技术的发展与应用[J]. 分析测试学报, 2019,38(12):1416-1422.
Development and Application of an Analysis Technique for Micro-scale N-Glycome[J]. 2019,38(12):1416-1422.
针对糖组学分析面临的初始样本量需求较高的技术挑战,该研究发展了一种微量样本N 糖链制备技术(GPAT),通过在移液枪头(Tip)中分别装填C18和HILIC填料,实现了一站式原位蛋白消化、N-糖链释放和富集。与目前普遍采用的基于过滤辅助样品制备(FASP)技术的N 糖组分析策略相比,使用GPAT技术可以实现微量免疫球蛋白G(IgG)和人肝癌HepG2细胞提取蛋白的原位N 糖链的释放和富集,样本起始量减少90%,可以从10 μg IgG和10 μg的HepG2细胞蛋白中分别检测到20条和39条N-糖链。从6例健康人(3例男性,3例女性)尿液中提取的10 μg尿蛋白中检测到49条N-糖链。该方法实现了微量复杂蛋白样本N-糖链简便、快速的定量分析策略,为进一步的糖组学方法推广应用奠定了基础。
One of challenges for current glycomic analysis is that it often requires high quantity of initial sample,which is difficult to meet the needs of micro-sample analysis.In this study,a micro-sample N-glycan preparation technique(GPAT) was developed for the release,enrichment and analysis of N glycans in micro protein samples.By loading C18 and HILIC fillers separately in tips,a one stop platform was set up to perform protein digestion,N glycan release and enrichment in situ.Compared with other N-glycome analysis based on the filter aided sample preparation(FASP) strategy,N-glycome analysis with GPAT was able to detect 20 and 39 N glycans in 10 μg IgG and 10 μg HepG2 cell proteins,respectively,realizing almost the same identification effect with 90% decrease of the initial sample consumption.Otherwise,forty nine N glycans was also detected in 10 μg of urine protein extracted from the urine of 6 healthy people(3 males,3 females).Results show that GPAT realizes a simple and fast quantitative analysis for N-glycans in micro scale protein samples,and lays a foundation for further application of glycome researches.
N-糖链糖组学尿蛋白基质辅助激光解吸电离飞行时间串联质谱仪
N-glycanglycomeurinaryproteinMALDI-TOF MS
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