In particular, genotype-specific differences in the extraction of collagens XII and XIV and tenascins C and X were identified; interestingly, increased expression of several of these genes has recently been implicated in susceptibility and/or progression of murine osteoarthritis.
The extraction of collagens from tissue, due to its ability to form cross-linked fibrils, is very difficult and requires special conditions, i.e., acetic acid, high temperature, or pepsin digestion [ 24].
α-Chymotrypsin enables digestion and thereby extraction of denatured collagen while sparing triple helical collagen [ 18, 19].
A further difference in the extraction of a collagen-interacting protein was observed in Comp DelD469 cartilage extractions, where there was a significant decrease in the detection of tenascin X compared with other genotypes.
Interestingly, and somewhat in contrast to type XIV collagen, there was decreased extraction of type XII collagen from Matn3 V194D and Comp T585M cartilages based on LC-MS/MS analysis of buffer 2 samples.
From response surface plots, acetic-acid concentration, temperature and time exhibited interactive effects on the extraction of acid-soluble collagen.
Correlation analysis of the mathematical-regression model indicated that quadratic polynomial model could be employed to optimize the extraction of acid-soluble collagen.
Under these optimal conditions, the experimental values agreed with the predicted values, using analysis of variance, indicating a high goodness of fit of the model used and the success of response surface methodology for modeling extraction of acid-soluble collagen from grass carp skin.
However, in contrast to Western blot analysis which detected no differences in the extraction of type IX collagen from Comp DelD469 buffer 1 samples, LC-MS/MS revealed a small decrease from similar extractions (WT spectra = 2 vs DelD469 spectra = 0; P = 0.019).
