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-labelling-based 4C quantitative PTM proteomics workflow in the study of dynamic acetylproteomic changes in Arabidopsis. This workflow integrates the dimethyl chemical labelling 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 labelling (SQUA-D), and finally the confirmation of ethylene hormone-regulated acetylation using immunoblot analysis. Eventually, using this proteomic approach, 7,456 unambiguous acetylation sites were found from 2,638 different acetylproteins, and 5,250 acetylation sites, including 5,233 sites on lysine side chain and 17 sites on protein N-terminus, were identified repetitively. Out of these repetitively discovered acetylation sites, 4,228 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 signalling. 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 labelling-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.
Fengchao Yu 
