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. 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Setting up the 13 CO 2 labeling system and stable isotope labeling of Arabidopsis thaliana rosette leaves Basic Protocol 2: Extraction of soluble amino acids for GC-TOF-MS analysis Basic Protocol 3: Preparation of amino acids from total protein for GC-TOF-MS analysis Basic Protocol 4: Preparation of sugars from cell wall material for GC-TOF-MS analysis Basis Protocol 5: GC-TOF-MS analysis of 13 C-labeled samples and estimation of 13 C enrichment (%) Basis Protocol 6: Estimation of protein synthesis and degradation rates.

Plant phenotyping relevance

植物のタンパク質合成・分解速度という生理状態を定量する標識・質量分析法を開発し、試料調製と計算手順まで体系的に記載した方法論研究である。

Code and data availability

公開論文であることは確認できましたが、現在の公式API・許可済み取得経路では本文を自動取得できませんでした。

No evidence-backed public reproduction asset is currently recorded.