title: Rapid purification and high sensitivity analysis of heparan sulfate from cells and tissues: toward glycomics profiling.
abstract: 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 fluorescent label, permits structural analyses on RIP-purified HS at approximately 1,000-fold higher sensitivity than standard UV detection methods and approximately 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.
authors: Centre for Glycobiology, School of Biological Sciences, University of Liverpool, Liverpool L69 7ZB, United Kingdom.
authors: Scott E
authors: SE
authors: Guimond
authors: Tania M
authors: TM
authors: Puvirajesinghe
authors: Mark A
authors: MA
authors: Skidmore
authors: Ina
authors: I
authors: Kalus
authors: Thomas
authors: T
authors: Dierks
authors: Edwin A
authors: EA
authors: Yates
authors: Jeremy E
authors: JE
authors: Turnbull
mesh_terms: D016475:3T3 Cells; D000818:Animals; D001896:Boron Compounds / Q000737:chemistry; D005260:Female; D005346:Fibroblast Growth Factors / Q000378:metabolism / Q000494:pharmacology; D006497:Heparan Sulfate / Q000032:analysis* / Q000302:isolation & purification* / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D015238:Sulfotransferases / Q000235:genetics / Q000378:metabolism
chemical_list: D001896:Boron Compounds; D005346:Fibroblast Growth Factors; D006497:Heparan Sulfate; D015238:Sulfotransferases; C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; C486773:Sulf1 protein, mouse