LC-MS Analysis of Proline Metabolite

LC–MS analysis of proline was conducted according to the method of Klupczynska et al. [34 (link)]. Methanol-extracted proline was subjected to Agilent 1260 Infinity HPLC system coupled to Agilent 6530 Q-TOF mass spectrometry detector (Agilent Technologies, Santa Clara, CA, USA) with electrospray ionization as an ion source in positive ionization mode. L-proline-d3 (Sigma Aldrich, Saint Louis, MO, USA) was used as an internal standard. A HILIC column (Luna HILIC, 2 × 100 mm, 3 µm, Phenomenex, Torrance, CA, USA) was applied to separate the tested mixture. The samples were collected in three biological repeats, injected in duplicates, and randomized before analysis. Total protein concentration was used for normalization.