Collagen molecular phenotype (mass spectrometry)

Dorsal skin collagen was defatted with chloroform/methanol (3:1 v/v). Collagen was solubilized by 3% acetic acid extraction at 4°C for 24 hrs or heat denaturation in SDS-PAGE sample buffer. The extracts were run on 6% SDS-PAGE and stained with Coomassie Blue. Bands corresponding to collagen α1(I) and α2(I) chains were cut out and processed for mass spectrometry using trypsin in-gel digestion, without reduction or alkylation. Collagen peptides were analyzed with LC-MS using an LTQ XL ion trap mass spectrometer (ThermoFisher) equipped with in-line liquid chromatography using a C4 5um capillary column (300um x 150mm; Higgins Analytical RS-15M3-W045) eluted at 4.5ul 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.