Rapid Purification and High Sensitivity Analysis of Heparan Sulfate from Cells and Tissues: TOWARD GLYCOMICS PROFILING
Résumé
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 BODIPY TM 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 knockout 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-sulfo-transferase knockout mice and revealed an unexpected deficiency in fibroblast growth factor activation by HS from het-erozygous 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.
Domaines
Biochimie [q-bio.BM]
Origine : Publication financée par une institution
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