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Elucidating the Molecular Composition of Cartilage by Proteomics

Hsueh, Ming Feng; Khabut, Areej LU ; Kjellström, Sven LU ; Önnerfjord, Patrik LU and Kraus, Virginia Byers (2016) In Journal of Proteome Research 15(2). p.374-388
Abstract

Articular cartilage consists of chondrocytes and two major components, a collagen-rich framework and highly abundant proteoglycans. Most prior studies defining the zonal distribution of cartilage have extracted proteins with guanidine-HCl. However, an unextracted collagen-rich residual is left after extraction. In addition, the high abundance of anionic polysaccharide molecules extracted from cartilage adversely affects the chromatographic separation. In this study, we established a method for removing chondrocytes from cartilage sections with minimal extracellular matrix protein loss. The addition of surfactant to guanidine-HCl extraction buffer improved protein solubility. Ultrafiltration removed interference from polysaccharides and... (More)

Articular cartilage consists of chondrocytes and two major components, a collagen-rich framework and highly abundant proteoglycans. Most prior studies defining the zonal distribution of cartilage have extracted proteins with guanidine-HCl. However, an unextracted collagen-rich residual is left after extraction. In addition, the high abundance of anionic polysaccharide molecules extracted from cartilage adversely affects the chromatographic separation. In this study, we established a method for removing chondrocytes from cartilage sections with minimal extracellular matrix protein loss. The addition of surfactant to guanidine-HCl extraction buffer improved protein solubility. Ultrafiltration removed interference from polysaccharides and salts. Almost four-times more collagen peptides were extracted by the in situ trypsin digestion method. However, as expected, proteoglycans were more abundant within the guanidine-HCl extraction. These different methods were used to extract cartilage sections from different cartilage layers (superficial, intermediate, and deep), joint types (knee and hip), and disease states (healthy and osteoarthritic), and the extractions were evaluated by quantitative and qualitative proteomic analyses. The results of this study led to the identifications of the potential biomarkers of osteoarthritis (OA), OA progression, and the joint specific biomarkers.

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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
cartilage, extracellular matrix proteins, guanidine-HCl extraction, in situ trypsin digestion, multiple reaction monitoring, proteomics
in
Journal of Proteome Research
volume
15
issue
2
pages
15 pages
publisher
The American Chemical Society
external identifiers
  • Scopus:84957628373
ISSN
1535-3893
DOI
10.1021/acs.jproteome.5b00946
language
English
LU publication?
yes
id
a86e5bba-2919-4fda-be8e-7d94e46b7b85
date added to LUP
2016-10-14 11:34:06
date last changed
2016-10-15 03:00:02
@misc{a86e5bba-2919-4fda-be8e-7d94e46b7b85,
  abstract     = {<p>Articular cartilage consists of chondrocytes and two major components, a collagen-rich framework and highly abundant proteoglycans. Most prior studies defining the zonal distribution of cartilage have extracted proteins with guanidine-HCl. However, an unextracted collagen-rich residual is left after extraction. In addition, the high abundance of anionic polysaccharide molecules extracted from cartilage adversely affects the chromatographic separation. In this study, we established a method for removing chondrocytes from cartilage sections with minimal extracellular matrix protein loss. The addition of surfactant to guanidine-HCl extraction buffer improved protein solubility. Ultrafiltration removed interference from polysaccharides and salts. Almost four-times more collagen peptides were extracted by the in situ trypsin digestion method. However, as expected, proteoglycans were more abundant within the guanidine-HCl extraction. These different methods were used to extract cartilage sections from different cartilage layers (superficial, intermediate, and deep), joint types (knee and hip), and disease states (healthy and osteoarthritic), and the extractions were evaluated by quantitative and qualitative proteomic analyses. The results of this study led to the identifications of the potential biomarkers of osteoarthritis (OA), OA progression, and the joint specific biomarkers.</p>},
  author       = {Hsueh, Ming Feng and Khabut, Areej and Kjellström, Sven and Önnerfjord, Patrik and Kraus, Virginia Byers},
  issn         = {1535-3893},
  keyword      = {cartilage,extracellular matrix proteins,guanidine-HCl extraction,in situ trypsin digestion,multiple reaction monitoring,proteomics},
  language     = {eng},
  month        = {02},
  number       = {2},
  pages        = {374--388},
  publisher    = {ARRAY(0xa9ad378)},
  series       = {Journal of Proteome Research},
  title        = {Elucidating the Molecular Composition of Cartilage by Proteomics},
  url          = {http://dx.doi.org/10.1021/acs.jproteome.5b00946},
  volume       = {15},
  year         = {2016},
}