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

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 13 CO 2 labeling and mass spectrometry-based analyses. Arabidopsis thaliana plants are subjected to a 24-hr 13 CO 2 pulse followed by a 4-day 12 CO 2 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 13 C enrichment (%) is estimated. The rate of protein synthesis during the 13 CO 2 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 13 C enrichment of glucose from cell wall material. Degradation rates are also estimated from the 12 CO 2 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.

Combination of long-term 13CO2 labeling and isotopolog profiling allows turnover analysis of photosynthetic pigments in Arabidopsis leaves

“…When combined with hydroponic cultivation, dual labeling with 13 CO 2 and 15 N (in the form of 15 NH 4 NO 3 , 15 NH 4 15 NO 3 or K 15 NO 3 in nutrient solution) enables simultaneous tracking of C and N allocation [ 41 ]. While 15 N feeding was successfully applied to unveil leaf proteome turnover [ 2 , 3 ], attempts are being made with 13 CO 2 to concomitantly analyze turnover of metabolites and proteins [ 42 , 43 ]. Chambers for long-term 13 CO 2 labeling typically have a large volume to treat multiple plants in parallel [ 41 – 44 ].…”

Remodelled Ribosomes Synthesise a Specific Proteome in Proliferating Plant Tissue during Cold

“…Translation is related mainly to protein biosynthesis and approaches to study translational dynamics typically involve the use of stable isotope mass spectrometry as an empirical measure of protein synthesis (K 𝑠 ) and degradation (K 𝑑 ) (Nelson et al, 2014b,a). The calculation of plant protein K 𝑠 using a stable isotope pulse depends on several parameters that must be considered (Ishihara et al, 2015(Ishihara et al, , 2021Li et al, 2017). The amount of protein fraction that has taken up the externally supplied stable isotope tracer can be determined by isotopolog analysis of proteinogenic amino acids from isolated and hydrolyzed protein fractions or individual purified proteins.…”

“…The first two variables are protein content and relative growth rate (RGR), which are required to adjust K S rates between plant systems that differ in these characteristics. A third set of variables describes the dynamics of tracer incorporation into soluble amino acid pools, and label incorporation into these pools may differ between the physiological conditions being compared and between individual proteinogenic amino acids ( Ishihara et al, 2021 ). All of these variables are necessary to correct the isotopic envelopes of proteins or digested peptides based on knowledge of their individual amino acid sequences.…”