Sequest search software

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)}.

Stained protein bands resolved by SDS-PAGE gel electrophoresis under reducing conditions were cut out and subjected to in-gel trypsin digestion^{19 (link)}. Electrospray mass spectrometry was performed on the tryptic peptides using an LCQ Deca XP ion-trap mass spectrometer equipped with in-line microbore LC (Thermo-Finnigan) using a C8 capillary column (0.3 × 150 mm; Grace Vydac 208MS5.315) eluted at 4 µl/min. The LC mobile phase consisted of Buffer A (0.1% formic acid in MilliQ water) and Buffer B (0.1% formic acid in 3:1 acetonitrile:n-propanol, v/v). Sequest search software (ThermoFinnigan) was used for peptide identification using the NCBI protein database.