15N Ammonium Labeling Experiment and Amino Acid Analysis Using Hydrophilic Liquid Chromatography MS

Synchronized cultures of Chlamydomonas were grown as described in detail previously (Jüppner et al., 2018). Before the end of 3rd cycle, the parent cultures were sampled as unlabeled reference for 0 h; thereafter, they were fed with fresh medium containing ¹⁵N-labeled ammonium chloride (Sigma-Aldrich), together with the treatment with DMSO or rapamycin to a final concentration of 5 µM. Samples were harvested after 0.25 h in five replicates for each condition.

Amino acids from ¹⁵N labeling experiments were analyzed from the polar fraction of the previously published methyl tert-butyl ether extraction method (Jüppner et al., 2017). Samples extracted from 10 million cells and reference amino acid standards (1 µg/mL) were resuspended in 100 μL 80% acetonitrile in water (Biosolve). For the analysis, 2 μL of each sample or standard was injected onto a BEH amide column (100 × 2.1 mm using 1.8-µm particles; Waters) using an Acquity UPLC system (Waters). The UPLC separation was performed using buffer A (0.1% formic acid in acetonitrile) and buffer B (0.1% formic acid in water). The gradient started with 95% buffer A, which was linearly decreased to 50% A within the first 6 min. Within the next minute, the concentration of A was further decreased linearly to 30%, which was held for 0.1 min. The column was then reequilibrated for 5 min with 95% A, before the next sample was injected.

The samples from the UPLC were measured using an Exactive high-resolution mass spectrometer, which was operated as described previously (Giavalisco et al., 2011), monitoring a mass range between m/z 50 and 500. To annotate the measured amino acids, mass spectra from samples were compared with spectra obtained from reference amino acids. For this purpose, accurate m/z (mass precision below 5 ppm) and retention time values (within 0.15 min) of all amino acids were matched.

For the analysis of isotope enrichment, the ratios of monoisotopic (only ¹⁴N-containing peaks) and the ¹⁵N-labeled mass peaks were determined using Xcalibur (version 2.2; Thermo Fisher Scientific). The extracted m/z and RT pair for monoisotopic peaks and the isotopologs of each amino acid were transferred into a ToxID template file, according to the vendor’s description, which was then used for the automated peak extraction using ToxID (version 1.2; Thermo Fisher Scientific). The ToxID extraction was performed using the accurate mass scans mode, setting the retention time tolerance to 0.15 min and the m/z tolerance to 5 ppm. The resulting output was normalized by the internal standard ampicillin (Giavalisco et al., 2011), which was added to the initial extraction mixture (Jüppner et al., 2017). Relative isotopolog ratios and ¹⁵N enrichment were calculated according to the absolute intensities of the detected peaks.