Cross-validation of cyanogen bromide-peptide ratios to measure the proportion of type II collagen in pepsin digests of equine articular cartilage, meniscus, and cartilage repair tissue.
Abstract: Collagen type I and type II were purified from equine flexor tendon and articular cartilage, respectively. Equal amounts of these collagens were cleaved with cyanogen bromide, and 11 mixtures containing increasing proportions of type II collagen were separated in seven identical sodium dodecyl sulfate-polyacrylamide gels. The density of bands was measured in wet gels and the peak areas were used to form six ratios of peptide bands that had polynomial relationships with the known proportions of type I and type II collagen in the mixtures. Calibration curves for determining the proportion of type II collagen in the mixtures were constructed using ratios and combinations of ratios of peak areas. Cross-validation was used to identify calibration curves with the smallest squared prediction error or squared average prediction error for all combinations of ratios. Ratios of peak areas of each one of the seven gels were treated, in turn, as the "unknown," and a prediction was carried out using these unknowns and the ratios from the other six gels. Two ratios had the smallest squared average prediction error and calibration curves were computed for these ratios with all seven gels. These curves were used to estimate the proportion of type II collagen in the pepsin-soluble and the pepsin-resistant fractions of articular cartilage inner and outer meniscus, and cartilage repair tissue. Cross-validation enabled selection of the cyanogen bromide-peptide ratios for calibration curves that resulted in the most accurate estimation of the proportion of type II collagen in pepsin digests of tissues.
Publication Date: 1994-01-01 PubMed ID: 8135352DOI: 10.1006/abio.1994.1025Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
- Research Support
- U.S. Gov't
- P.H.S.
Summary
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This research shows how the proportion of type II collagen in cartilage tissues can be quantified by using cyanogen bromide to break down collagen into peptides, which were then analyzed. This method was applied to equine tissues and validated for accuracy and reliability.
Research Overview
In their study, the researchers purified collagen type I and type II from equine tendon and articular cartilage, respectively. They divided the collagens into equal parts and cleaved them using cyanogen bromide, creating eleven mixtures with varying amounts of type II collagen.
Methodology and Findings
- Seven identical Sodium Dodecyl Sulfate-Polyacrylamide gels were used to separate the mixtures.
- The wet gels’ band density was measured, and peak areas of peptide bands were identified, forming ratios that presented a polynomial relationship with the known proportions of type I and type II collagen in the mixtures.
- They built calibration curves using ratios and combinations of peak area ratios, helping to ascertain the proportion of type II collagen in the mixtures.
- The scientists used cross-validation to decide the calibration curves with the least squared prediction error or squared average prediction error across all peptide band ratios.
- The peak area ratios of each gel were taken as the “unknown”, while predictions were done using these unknowns and the ratios from the remaining six gels, allowing the researchers to identify two ratios with the smallest squared average prediction error.
- Their new found ratios were then applied to determine the distribution of type II collagen in pepsin-soluble and pepsin-resistant fractions of articular cartilage, inner and outer meniscus, and cartilage repair tissue.
Conclusion
The investigation showed that the cross-validation process led to the selection of cyanogen bromide-peptide ratios as part of calibration curves that generated the most precise estimation of the percentage of type II collagen in pepsin digests of tissues. This study contributes to knowledge about how we analyse and understand the complex protein makeup of tissues such as cartilage and tendons.
Cite This Article
APA
Todhunter RJ, Wootton JA, Altman N, Lust G, Minor RR.
(1994).
Cross-validation of cyanogen bromide-peptide ratios to measure the proportion of type II collagen in pepsin digests of equine articular cartilage, meniscus, and cartilage repair tissue.
Anal Biochem, 216(1), 195-204.
https://doi.org/10.1006/abio.1994.1025 Publication
Researcher Affiliations
- James A. Baker Institute for Animal Health, Cornell University, Ithaca, New York 14853.
MeSH Terms
- Animals
- Calibration
- Cartilage, Articular / chemistry
- Collagen / analysis
- Cyanogen Bromide
- Horses
- Pepsin A / pharmacology
Grant Funding
- 07344-01A1 / PHS HHS
- AR 20793 / NIAMS NIH HHS
Citations
This article has been cited 2 times.- Kuijer R, Surtel DA, Van Der Linden AJ, Bulstra SK, Passier RC. A novel method to examine the phenotype of chondrocytes.. J Mater Sci Mater Med 1998 Dec;9(12):749-54.
- Simonin MA, Gegout-Pottie P, Minn A, Gillet P, Netter P, Terlain B. Proteoglycan and collagen biochemical variations during fluoroquinolone-induced chondrotoxicity in mice.. Antimicrob Agents Chemother 1999 Dec;43(12):2915-21.
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