Publication Information
Liu et al., 2018
Abstract
Mol Cell Proteomics. 2018 May;17(5):1010-1027. doi: 10.1074/mcp.RA117.000530.
Epub 2018 Feb 13.
Establishment of Dimethyl Labeling-based Quantitative Acetylproteomics in
Arabidopsis.
Liu S(1), Yu F(2)(3), Yang Z(1)(4), Wang T(5), Xiong H(6), Chang C(7), Yu
W(8)(3), Li N(9)(4).
Author information:
(1)From the ‡Division of Life Science, Energy Institute, Institute for the
Environment, The Hong Kong University of Science and Technology, Hong Kong SAR,
China.
(2)§Division of Biomedical Engineering, The Hong Kong University of Science and
Technology, Hong Kong SAR, China.
(3)¶Department of Electronic and Computer Engineering, The Hong Kong University
of Science and Technology, Hong Kong SAR, China.
(4)‖The Hong Kong University of Science and Technology, Shenzhen Research
Institute, Shenzhen, Guangdong, 518057, China.
(5)**Tsinghua-Peking Joint Center for Life Sciences, Center for Structural
Biology, School of Life Sciences and School of Medicine, Tsinghua University,
Beijing 100084, China.
(6)‡‡College of Life Science, South-central University for Nationalities, Wuhan,
430074, China.
(7)§§Department of Cell Biology and Molecular Genetics, University of Maryland,
Maryland 20742-5815.
(8)§Division of Biomedical Engineering, The Hong Kong University of Science and
Technology, Hong Kong SAR, China; boningli@ust.hk.
(9)From the ‡Division of Life Science, Energy Institute, Institute for the
Environment, The Hong Kong University of Science and Technology, Hong Kong SAR,
China; boningli@ust.hk.
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.
© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.
DOI: 10.1074/mcp.RA117.000530
PMCID: PMC5930405
PMID: 29440448 [Indexed for MEDLINE]
