Proteome discoverer search software

Adult zebrafish were euthanized with 0.4% tricaine and vertebral bone was dissected manually from two plod2 mutant and two control fish, residual soft tissue was removed by incubation in Accumax solution (Sigma-Aldrich) for 1 hour. Bone was demineralized in 0.1 M HCl at 4°C, washed, and solubilized by heat denaturation in SDS-PAGE sample buffer. The method of Laemmli was used with 6% gels for the denaturant extracts. Collagen α-chains were cut from SDS-PAGE and subjected to in-gel trypsin digestion. Electrospray MS was performed on tryptic peptides using an LTQ XL ion-trap mass spectrometer (Thermo Scientific) equipped with in-line liquid chromatography using a C4 5μm capillary column (300um × 150mm; Higgins Analytical RS-15M3-W045) 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). The LC sample stream was introduced into the mass spectrometer by electrospray ionization (ESI) with a spray voltage of 4kV. Proteome Discoverer search software (Thermo 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.

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.

Electrospray MS was performed with Lys-C peptides and individual collagenase HPLC fractions using an LTQ XL ion-trap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) equipped with in-line liquid chromatography using a C4 5-μm capillary column (300 μm × 150 mm; Higgins Analytical RS-15M3-W045, Mountain View, CA, USA) eluted at 4.5 μL minute. 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). The LC sample stream was introduced into the mass spectrometer by electrospray ionization (ESI) with a spray voltage of 4 kV. Proteome Discoverer search software (Thermo Fisher Scientific) was used for peptide identification using the NCBI protein database.

Femurs from male 12-week-old Col1a2^+/+;Lrp5^+/+, Col1a2^+/p.G610C;Lrp5^+/+, and Col1a2^+/p.G610C;Lrp5^+/p.A214V mice were recovered following euthanasia. Mid-shaft bone was decalcified overnight in 0.1M HCl at 4°C, minced and heat denatured at 90°C for 10 min in SDS sample buffer. Equal amounts of demineralized tissue were separated by 6% SDS-PAGE.
Individual α-chains were cut from the gel and subjected to in-gel trypsin digestion (30 (link), 31 (link)). Electrospray MS was performed on the tryptic peptides using an LTQ XL ion-trap mass spectrometer equipped with in-line liquid chromatography (LC) (Thermo Scientific, Waltham, MA) using a C4 5 μm capillary column (300 μm × 150 mm; Higgins Analytical RS-15M3-W045) eluted at 4.5 μl min. Proteome Discoverer search software (Thermo Scientific) was used for peptide identification using the NCBI protein database.