Assessing Protein Synthesis and Degradation Rates in Arabidopsis thaliana Using Amino Acid Analysis

Ishihara H, Moraes TA, Arrivault S, Stitt M - Current Protocols, 2022

Abstract

Plants continually synthesize and degrade proteins, for example, to adjust protein content during development or during adaptation to new environments. In order to estimate global protein synthesis and degradation rates in plants, we developed a relatively simple and inexpensive method using a combination of ¹³CO₂ labeling and mass spectrometry–based analyses. Arabidopsis thaliana plants are subjected to a 24-hr ¹³CO₂ pulse followed by a 4-day ¹²CO₂ chase. Soluble alanine and serine from total protein and glucose from cell wall material are analyzed by gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) and their ¹³C enrichment (%) is estimated. The rate of protein synthesis during the ¹³CO₂ pulse experiment is defined as the rate of incorporation of labeled amino acids into proteins normalized by a correction factor for incomplete enrichment in free amino acid pools. The rate of protein degradation is estimated as the difference between the rate of protein synthesis and the relative growth rate calculated using the ¹³C enrichment of glucose from cell wall material. Degradation rates are also estimated from the ¹²CO₂ pulse experiment. The following method description includes setting up and performing labeling experiments, preparation and measurement of samples, and calculation steps. In addition, an R script is provided for the calculations. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC.

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