Rapid purification and high sensitivity analysis of heparan sulfate from cells and tissues: toward glycomics profiling.
Rapid purification and high sensitivity analysis of heparan sulfate from cells and tissues: toward glycomics profiling.
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DOI：
10.1074/jbc.m109.032755
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发表时间：
2009-09-18
期刊：
影响因子：
--
通讯作者：
Turnbull JE
中科院分区：
文献类型：
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作者：
Guimond SE;Puvirajesinghe TM;Skidmore MA;Kalus I;Dierks T;Yates EA;Turnbull JE
糖胺聚糖和蛋白聚糖（pg）的研究一直受到传统方法分离和分析困难、灵敏度低和通量低的限制。本研究采用苯酚/胍/氯仿试剂从多种组织和细胞中定量纯化硫酸肝素（HS） PGs，建立了一种快速分离蛋白聚糖（RIP）的方法。我们进一步表明，当与BODIPYTM荧光标记一起应用时，这种通用纯化方法允许对rip纯化的HS进行结构分析，灵敏度比标准紫外检测方法高~ 1000倍，灵敏度比以前的荧光检测方法高~ 10 - 100倍。通过对小组织样本、多个小鼠器官和少量几千个培养细胞的HS结构组成进行快速分析，证明了RIP-BODIPY方法的实用性。该研究还首次对Sulf1敲除小鼠体内HS结构变化产生了新的见解，这与以往仅限于组织培养实验的观察结果有很大不同。RIP还用于纯化HS进行生物检测，例如成纤维细胞生长因子信号激活的细胞检测；这从2- o -硫转移酶敲除小鼠中获得数据，并揭示了杂合小鼠的HS在成纤维细胞生长因子激活方面的意外缺陷。这些数据表明，RIP将为开发HS和其他糖胺聚糖的糖组学分析策略奠定基础，以探索它们在复杂生物系统中的结构-功能关系。
Studies on glycosaminoglycans and proteoglycans (PGs) have been hampered by difficulties in isolation and analysis by traditional methods that are laborious and lack sensitivity and throughput. Here we demonstrate a simple method for rapid isolation of proteoglycans (RIP) employing phenol/guanidine/chloroform reagent to purify heparan sulfate (HS) PGs quantitatively from various tissues and cells. We further show that this generic purification methodology, when applied in concert with a BODIPYTM fluorescent label, permits structural analyses on RIP-purified HS at ∼1,000-fold higher sensitivity than standard UV detection methods and ∼10–100-fold higher sensitivity than previous fluorescence detection methods. The utility of RIP-BODIPY methodology was demonstrated by rapid profiling of HS structural composition from small tissue samples, multiple mouse organs, and as little as a few thousand cultured cells. It was also used to generate novel insights into in vivo structural changes in HS from Sulf1 knock-out mice for the first time that differed significantly from previous observations limited to tissue culture experiments. RIP was also applied to purify HS for bioassay testing, exemplified by cell assays of fibroblast growth factor signaling activation; this generated data from 2-O-sulfotransferase knock-out mice and revealed an unexpected deficiency in fibroblast growth factor activation by HS from heterozygous mice. These data demonstrate that RIP will underpin emerging efforts to develop glycomics profiling strategies for HS and other glycosaminoglycans to explore their structure-function relationships in complex biological systems.