Optimized extraction of glycosaminoglycans from normal and osteoarthritic cartilage for glycomics profiling.
Journal: 2007/March - Glycobiology
ISSN: 0959-6658
Abstract:
Articular cartilage is a highly specialized smooth connective tissue whose proper functioning depends on the maintenance of an extracellular matrix consisting of an integrated assembly of collagens, glycoproteins, proteoglycans (PG), and glycosaminoglycans. Isomeric chondroitin sulfate glycoforms differing in position and degree of sulfation and uronic acid epimerization play specific and distinct functional roles during development and disease onset. This work introduces a novel glycosaminoglycan extraction method for the quantification of mixtures of chondroitin sulfate oligosaccharides from intact cartilage tissue for mass spectral analysis. Glycosaminoglycans were extracted from intact cartilage samples using a combination of ethanol precipitation and enzymatic release followed by reversed-phase and strong anion exchange solid-phase extraction steps. Extracted chondroitin sulfate glycosaminoglycans were partially depolymerized using chondroitinases, labeled with 2-anthranilic acid-d(4) (2-AA) and subjected to size exclusion chromatography with online electrospray ionization mass spectrometric detection in the negative ion mode. The method presented herein enabled simultaneous determination of sulfate position and uronic acid epimerization in juvenile bovine and adult human cartilage samples. The method was applied to a series of 13 adult human cartilage explants. Standard deviation of the mean for the measurements was 1.6 on average. Coefficients of variation were approximately 4% for all compositions of 40% or greater. These results show that the new method has sufficient accuracy to allow determination of topographical distribution of glycoforms in connective tissue.
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Glycobiology 17(1): 25-35

Optimized Extraction of Glycosaminoglycans from Normal and Osteoarthritic Cartilage for Glycomics Profiling

Department of Biochemistry, Boston University School of Medicine, 670 Albany St., Boston, Massachusetts 02118.
Department of Orthopedic Surgery, Brigham and Women’s Hospital and Harvard Medical School, Boston Massachusetts 02115
To whom correspondence should be addressed: Department of Biochemistry, Boston University School of Medicine, MS Resource, 670 Albany St., Boston, MA 02118. Telephone: (617)-638-6762. Fax: (617)-638-6760. Email:ude.ub@aiazj

Abstract

Articular cartilage is a highly specialized smooth connective tissue whose proper functioning depends on the maintenance of an extracellular matrix, consisting of an integrated assembly of collagens, glycoproteins, proteoglycans and glycosaminoglycans. Isomeric chondroitin sulfate glycoforms differing in position and degree of sulfation and uronic acid epimerization play specific and distinct functional roles during development and disease onset. This work introduces a novel glycosaminoglycan extraction method for the quantification of mixtures of chondroitin sulfate oligosaccharides from intact cartilage tissue for mass spectral analysis. Glycosaminoglycans were extracted from intact cartilage samples using a combination of ethanol precipitation and enzymatic release followed by reversed phase and strong anion exchange solid phase extraction steps. Extracted chondroitin sulfate glycosaminoglycans were partially depolymerized using chondroitinases, labeled with 2-anthranilic acid- d4 and subjected to size exclusion chromatography with on-line electrospray ionization mass spectrometric detection in the negative ion mode. The method presented herein enabled simultaneous determination of sulfate position and uronic acid epimerization in juvenile bovine and adult human cartilage samples. The method was applied to series of 13 adult human cartilage explants. Standard deviation of the mean for the measurements was 1.6 on average. Coefficients of variation were approximately 4% for all compositions of 40% or greater. These results show that the new method has sufficient accuracy to allow determination of topographical distribution of glycoforms in connective tissue.

Keywords: cartilage, glycomics, glycosaminoglycan, mass spectrometry
Abstract

Abbreviations

AMAC2-aminoacridone
CE-LIFcapillary electrophoresis-laser induced fluorescence
CIDcollision-induced dissociation
CSchondroitin sulfate
CSAchondroitin sulfate type A
CSBchondroitin sulfate type B
CSCchondroitin sulfate type C
dpdegree of polymerization
ESIelectrospray ionization
GAGglycosaminoglycan
LC/MS/MSliquid chromatography-tandem mass spectrometry
OAosteoarthritis
PGproteoglycan
SECsize-exclusion chromatography
SLRPsmall leucine-rich proteoglycan
MSmass spectrometry
2-AA2-anthranilic acid
Abbreviations
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