Quantifying Collagen Post-Translational Modifications

The purified collagen samples were treated with trypsin as described above and subjected to electrospray LC–MS using an ultrahigh resolution (UHR) quadrupole time-of-flight (QTOF) mass spectrometer (maXis II, Bruker Daltonics, Bremen, Germany) with a Shimadzu Prominence UFLC-XR system (Shimadzu, Kyoto, Japan). The samples were applied to an Ascentis Express C18 HPLC column (5 μm particle size, 2.1 mm × 150 mm; Supelco) with a binary gradient of 0.1% formic acid and acetonitrile as described previously.^{19 (link)} The MS scan was obtained over the m/z range of 50–2500 with a frequency of 2 Hz in positive ion mode. The relative extent of Lys hydroxylation and Hyl glycosylation (Lys, Hyl, G-Hyl, and GG-Hyl) at the specific sites in type I collagen was calculated as reported previously.^{19 (link),21 (link)}