Quantification of Proline and Hydroxyproline

All assays were prepared as previously described Levin et al. (2017) (link). Briefly, assays contained 20 mM Tris-HCl pH 7.5, 100 mM KCl, 0.8 mM NADH, 3 µM P5CR, 0.2 mM Hyp, and 0.3 µM HypD. HypD was first activated under conditions described for EPR spectroscopic assays. All assays were carried out in triplicate and were initiated by adding Hyp into reaction mixtures, which were then incubated for 21 hr at 22°C. Upon removal from the anaerobic chamber, reactions were quenched with a 2 × volume of methanol and protein precipitates were removed by centrifugation (15,200 g, 10 min). Supernatants were further diluted 30-fold with water for Pro detection and 7.5-fold for Hyp detection by LC-MS/MS using previously published methods (Levin et al., 2017 (link)). Briefly, LC-MS/MS analysis of Pro and Hyp were performed on an Agilent 6410 Triple Quadrupole LC-MS instrument (Agilent Technologies) using a Luna SCX column (5 μm, 100 Å, 50 × 2.0 mm, Phenomenex). Precursor and product ions of m/z 116.1 and m/z 70.1 were monitored for proline detection whereas m/z 132.1 and m/z 86.1 were monitored for hydroxyproline. Amino acid standards were dissolved in water and diluted to a range of concentrations to generate standard curves used to quantify Pro and Hyp in samples. Source data can be found in Figure 5—source data 1.