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 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.
Highlights
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
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 ϳ1,000-fold higher sensitivity than standard UV detection methods and ϳ10 –100-fold higher sensitivity than previous fluorescence detection methods
Summary
Materials—Chloroform, DNase I, RNase, neuraminidase, chondroitin ABC lyase, sodium hydroxide, sodium borohydride, sodium chloride, Tris acetate, sodium acetate, calcium acetate, PBS, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, SDS, and DMSO were purchased from SigmaAldrich (Dorset, UK). Labeled samples were reduced using sodium borohydride (1 M, aqueous; 10 l) and incubated for 30 min (room temperature) prior to flash-freezing in liquid nitrogen and lyophilization. The fluorescently labeled disaccharides were resuspended in DMSO:H2O (50:50 v/v, 1 ml) prior to loading onto a Propac PA-1 column as above, eluted using a linear gradient of 0 –1 M sodium chloride (in 150 mM NaOH) over 30 min at a flow rate of 2 ml/min on a Shimadzu HPLC system. 10,000 cells/ well were plated on a 96-well plate with culture medium (100 l, RPMI 1640 supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 units/ml penicillin-G, and 100 g/ml streptomycin sulfate without IL-3) and were incubated with 2 ng/ml IL-3 or 1 nM FGF-2 with and without heparin/HS at the concentrations indicated.
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