title: Contaminants in commercial preparations of 'purified' small leucine-rich proteoglycans may distort mechanistic studies.
abstract: The present study reports the perplexing results that came about because of seriously impure commercially available reagents. Commercial reagents and chemicals are routinely ordered by scientists and expected to have been rigorously assessed for their purity. Unfortunately, we found this assumption to be risky. Extensive work was carried out within our laboratory using commercially sourced preparations of the small leucine-rich proteoglycans (SLRPs), decorin and biglycan, to investigate their influence on nerve cell growth. Unusual results compelled us to analyse the composition and purity of both preparations of these proteoglycans (PGs) using both mass spectrometry (MS) and Western blotting, with and without various enzymatic deglycosylations. Commercial 'decorin' and 'biglycan' were found to contain a mixture of PGs including not only both decorin and biglycan but also fibromodulin and aggrecan. The unexpected effects of 'decorin' and 'biglycan' on nerve cell growth could be explained by these impurities. Decorin and biglycan contain either chondroitin or dermatan sulfate glycosaminoglycan (GAG) chains whereas fibromodulin only contains keratan sulfate and the large (>2500 kDa), highly glycosylated aggrecan contains both keratan and chondroitin sulfate. The different structure, molecular weight and composition of these impurities significantly affected our work and any conclusions that could be made. These findings beg the question as to whether scientists need to verify the purity of each commercially obtained reagent used in their experiments. The implications of these findings are vast, since the effects of these impurities may already have led to inaccurate conclusions and reports in the literature with concomitant loss of researchers' funds and time.
authors.affiliation: Robert Jones & Agnes Hunt (RJAH) Orthopaedic Hospital NHS Foundation Trust & ISTM (Keele University), Oswestry, SY10 7AG, U.K.
authors.forename: Sharon J
authors.initials: SJ
authors.lastname: Brown
authors.affiliation: Robert Jones & Agnes Hunt (RJAH) Orthopaedic Hospital NHS Foundation Trust & ISTM (Keele University), Oswestry, SY10 7AG, U.K.
authors.forename: Heidi R
authors.initials: HR
authors.lastname: Fuller
authors.affiliation: Robert Jones & Agnes Hunt (RJAH) Orthopaedic Hospital NHS Foundation Trust & ISTM (Keele University), Oswestry, SY10 7AG, U.K.
authors.forename: Philip
authors.initials: P
authors.lastname: Jones
authors.affiliation: Cardiff School of Biosciences, The Sir Martin Evans Building, Museum Avenue, Cardiff, CF10 3AX, U.K.
authors.forename: Bruce
authors.initials: B
authors.lastname: Caterson
authors.affiliation: BSRC Mass Spectrometry and Proteomics Facility, Biomedical Sciences Research Complex, University of St Andrews, North Haugh, St Andrews, Fife, KY16 9ST, U.K.
authors.forename: Sally L
authors.initials: SL
authors.lastname: Shirran
authors.affiliation: BSRC Mass Spectrometry and Proteomics Facility, Biomedical Sciences Research Complex, University of St Andrews, North Haugh, St Andrews, Fife, KY16 9ST, U.K.
authors.forename: Catherine H
authors.initials: CH
authors.lastname: Botting
authors.affiliation: Robert Jones & Agnes Hunt (RJAH) Orthopaedic Hospital NHS Foundation Trust & ISTM (Keele University), Oswestry, SY10 7AG, U.K. sally.roberts@rjah.nhs.uk.
authors.forename: Sally
authors.initials: S
authors.lastname: Roberts
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016477:Artifacts; D058578:Biglycan / Q000032:analysis* / Q000494:pharmacology; D015153:Blotting, Western; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D002645:Chickens; D058575:Decorin / Q000032:analysis* / Q000494:pharmacology; D007202:Indicators and Reagents / Q000032:analysis; D013058:Mass Spectrometry; D009474:Neurons / Q000166:cytology / Q000187:drug effects; D016415:Sequence Alignment
chemical_list: D058578:Biglycan; D058575:Decorin; D007202:Indicators and Reagents
keywords: biglycan; commercial preparations; decorin; glycosaminoglycan; mass spectrometry; small leucine-rich proteoglycans (SLRPs)