In line liquid chromatography

For mass spectrometry, we prepared type I collagen from Crtap^−/− and wildtype tibiae. We defatted bone with chloroform/methanol (3:1 v/v) and demineralized it in 0.5 M EDTA, 0.05 M Tris-HCl, pH 7.5, all steps at 4°C. We finely minced the bone samples and solubilized collagen by heat denaturation (90°C) in SDS-PAGE sample buffer. Collagen α-chains were cut from SDS-PAGE gels and subjected to in-gel trypsin digestion. We performed electrospray MS on the tryptic peptides using an LCQ Deca XP ion-trap mass spectrometer equipped with in-line liquid chromatography (LC) (ThermoFinnigan) using a C8 capillary column (300 μm × 150 mm; Grace Vydac 208 MS5.315) eluted at 4.5 μl min. Sequest search software (ThermoFinnigan) was used for peptide identification using the NCBI protein database.
We quantified pyridinoline cross-links (HP and LP) by HPLC after hydrolyzing demineralized bone in 6 N HCl as described^{45 (link)}.

Preparation of type I and type II collagens from adult bone and neonatal growth plates was performed as described in Weiss et al³⁸ . Briefly, bone was demineralized in 0.1 M HCl at 4°C, washed, and solubilized by heat denaturation in SDS-PAGE sample buffer. For the growth plate, proteoglycans were removed with 4 M guanidine HCl, 0.05 M Tris-HCl, pH 7.5 with protease inhibitors (5 mM 1,10-phenanthroline and 2 mM PMSF) for 24 hours at 4°C and the residue was washed thoroughly. Collagens were solubilized with pepsin (1:20, pepsin/dry tissue) in 3% acetic acid for 24 hours at 4°C, and were run on 6% SDS-PAGE gels. After in-gel trypsin digestion, electrospray MS was performed using an LTQ XL ion-trap mass spectrometer equipped with in-line liquid chromatography (Thermo Fisher Scientific) using a C4 5 μm capillary column (300μm x 150mm; Higgins Analytical RS-15M3-W045) eluted at 4.5 μl/min. Proteome Discoverer search software (Thermo Fisher Scientific) was used for peptide identification using the NCBI protein database. Proline and lysine modifications were examined manually by scrolling or averaging the full scan over several minutes so that all of the post-translational variations of a given peptide appeared together in the full scan.