Collagen Cross-Link and Mass Spectral Analyses.

Pyridinoline cross-links (hydroxylysylpyridinoline [HP] and lysylpyridinoline [LP]) were quantified by high performance liquid chromatography (HPLC) after hydrolyzing saline washed tendons in 6N HCl as described (59). Briefly, samples were dried and redissolved in 1% (vol/vol) n-heptafluorobutyric acid and run on an Agilent 1260 HPLC (Agilent Technologies) using a C18 RP-HPLC (Brownlee Aquapore RP-300 7u, 250 × 4.6 mm; PerkinElmer) running at 1 mL/min. Samples were eluted with a linear gradient of 17 to 21% acetonitrile in a running buffer of 0.01M n-heptafluorobutyric acid in water. CNBr peptides were run on 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) using the method of Laemmli. CB peptide bands were cut from SDS/PAGE gels and digested with trypsin in-gel (60). Peptides were analyzed by electrospray liquid chromatography–mass spectrometry (LC/MS) using an LTQ XL ion trap mass spectrometer (Thermo Scientific) equipped with inline LC using a C4 5-mm capillary column (300 μm × 150 mm; Higgins Analytical; RS-15M3-W045) eluted at 4.5 μL/min. The LC mobile phase consisted of buffer A (0.1% formic acid in Milli-Q water; Millipore) and buffer B (0.1% formic acid in 3:1 acetonitrile:n-propanol vol/vol). An electrospray ionization source introduced the LC sample stream into the mass spectrometer with a spray voltage of 4 kV. Proteome Discoverer search software (Thermo Scientific) was used for peptide identification using the National Center for Biotechnology Information protein database (https://www.ncbi.nlm.nih.gov/protein). Large collagenous peptides not found by Sequest had to be identified manually by calculating the possible tandem MS (MS/MS) ions and matching these to the actual MS/MS. Hydroxyproline and hydroxylysine calculations were done manually by scrolling or averaging the full scan over several minutes so that all of the posttranslational variations of a given peptide appeared together in the full scan.