Establishment of Dimethyl Labeling-based Quantitative Acetylproteomics in Arabidopsis

Shichang Liu,Fengchao Yu,Zhu Yang,Tingliang Wang,Hairong Xiong,Caren Chang,Weichuan Yu,Ning Li

doi:10.1074/mcp.ra117.000530

Abstract

Protein acetylation, one of many types of post-translational modifications (PTMs), is involved in a variety of biological and cellular processes. In the present study, we applied both CsCl density gradient (CDG) centrifugation-based protein fractionation and a dimethyl-labeling-based 4C quantitative PTM proteomics workflow in the study of dynamic acetylproteomic changes in Arabidopsis. This workflow integrates the dimethyl chemical labeling with chromatography-based acetylpeptide separation and enrichment followed by mass spectrometry (MS) analysis, the extracted ion chromatogram (XIC) quantitation-based computational analysis of mass spectrometry data to measure dynamic changes of acetylpeptide level using an in-house software program, named Stable isotope-based Quantitation-Dimethyl labeling (SQUA-D), and finally the confirmation of ethylene hormone-regulated acetylation using immunoblot analysis. Eventually, using this proteomic approach, 7456 unambiguous acetylation sites were found from 2638 different acetylproteins, and 5250 acetylation sites, including 5233 sites on lysine side chain and 17 sites on protein N termini, were identified repetitively. Out of these repetitively discovered acetylation sites, 4228 sites on lysine side chain (i.e. 80.5%) are novel. These acetylproteins are exemplified by the histone superfamily, ribosomal and heat shock proteins, and proteins related to stress/stimulus responses and energy metabolism. The novel acetylproteins enriched by the CDG centrifugation fractionation contain many cellular trafficking proteins, membrane-bound receptors, and receptor-like kinases, which are mostly involved in brassinosteroid, light, gravity, and development signaling. In addition, we identified 12 highly conserved acetylation site motifs within histones, P-glycoproteins, actin depolymerizing factors, ATPases, transcription factors, and receptor-like kinases. Using SQUA-D software, we have quantified 33 ethylene hormone-enhanced and 31 hormone-suppressed acetylpeptide groups or called unique PTM peptide arrays (UPAs) that share the identical unique PTM site pattern (UPSP). This CDG centrifugation protein fractionation in combination with dimethyl labeling-based quantitative PTM proteomics, and SQUA-D may be applied in the quantitation of any PTM proteins in any model eukaryotes and agricultural crops as well as tissue samples of animals and human beings.

Highlights

LC-mass spectrometry (MS)/MS and Computational Analysis of Arabidopsis Acetylproteomes—A comprehensive dimethyl-labeling-based quantitative acetylproteomics has been performed on a model plant organism Arabidopsis (Fig. 1), which consists of ethylene treatment of plants (Fig. 1B-I), protein fractionation by a detergent-free cesium chloride (CsCl) density gradient centrifugation followed by trypsin digestion (Fig. 1B-II), dimethyl labeling and mixing of peptides (Fig. 1A; Fig. 1B-III), chromatographic separation and affinity chromatographic enrichment of acetylpeptides (Fig. 1B-IV) followed by LC-MS/MS analysis (Fig. 1B-V)
The results of our comprehensive and quantitative post-translational modifications (PTMs) proteomics workflow have substantially expanded the database of Arabidopsis protein acetylation sites, revealing 4245 acetylation sites that were not described in previous publications [48, 65,66,67]
Acetylproteins of the endoplasmic reticulum (ER), Golgi apparatus, plasma membrane, and mitochondria were found to be concentrated in the fraction t and b, whereas acetylproteins of the cytosol, nucleus, and ribosome were concentrated in the fraction m instead

Summary

Introduction

Using SQUA-D software, we have quantified 33 ethylene hormone-enhanced and 31 hormone-suppressed acetylpeptide groups or called unique PTM peptide arrays (UPAs) that share the identical unique PTM site pattern (UPSP) This CDG centrifugation protein fractionation in combination with dimethyl labeling-based quantitative PTM proteomics, and SQUA-D may be applied in the quantitation of any PTM proteins in any model eukaryotes and agricultural crops as well as tissue samples of animals and human beings. In vitro dimethyl labeling of peptides has emerged as one of the fastest and most dependable chemical-labeling strategies for quantitative proteomics since its introduction in 2003 [1] This approach has the advantages of high cost-effectiveness, high labeling efficiency, and minimal side reactions [2,3,4]. Given the importance of acetylation, thousands of acetylation sites have been previously found and the acetylation levels have been quantified through either isotopic labeling or label-free quantification strategies in bacteria [37, 38], mammalian cell lines (39 – 41), malaria parasites [42], mouse hearts [43], mouse livers (44 – 46), and human cells [47]

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Conclusion