title: Absolute quantitation of isoforms of post-translationally modified proteins in transgenic organism.
abstract: Post-translational modification isoforms of a protein are known to play versatile biological functions in diverse cellular processes. To measure the molar amount of each post-translational modification isoform (P(isf)) of a target protein present in the total protein extract using mass spectrometry, a quantitative proteomic protocol, absolute quantitation of isoforms of post-translationally modified proteins (AQUIP), was developed. A recombinant ERF110 gene overexpression transgenic Arabidopsis plant was used as the model organism for demonstration of the proof of concept. Both Ser-62-independent (14)N-coded synthetic peptide standards and (15)N-coded ERF110 protein standard isolated from the heavy nitrogen-labeled transgenic plants were employed simultaneously to determine the concentration of all isoforms (T(isf)) of ERF110 in the whole plant cell lysate, whereas a pair of Ser-62-dependent synthetic peptide standards were used to quantitate the Ser-62 phosphosite occupancy (R(aqu)). The P(isf) was finally determined by integrating the two empirically measured variables using the following equation: P(isf) = T(isf) · R(aqu). The absolute amount of Ser-62-phosphorylated isoform of ERF110 determined using AQUIP was substantiated with a stable isotope labeling in Arabidopsis-based relative and accurate quantitative proteomic approach. The biological role of the Ser-62-phosphorylated isoform was demonstrated in transgenic plants.
authors.affiliation: Division of Life Science, The Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China.
authors.forename: Yaojun
authors.initials: Y
authors.lastname: Li
authors.forename: Yiwei
authors.initials: Y
authors.lastname: Shu
authors.forename: Changchao
authors.initials: C
authors.lastname: Peng
authors.forename: Lin
authors.initials: L
authors.lastname: Zhu
authors.forename: Guangyu
authors.initials: G
authors.lastname: Guo
authors.forename: Ning
authors.initials: N
authors.lastname: Li
mesh_terms: D000465:Algorithms; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D005030:Ethylenes / Q000378:metabolism / Q000494:pharmacology; D015151:Immunoblotting; D007553:Isotope Labeling / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D010766:Phosphorylation; D010937:Plant Growth Regulators / Q000378:metabolism / Q000494:pharmacology; D030821:Plants, Genetically Modified / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods; D012694:Serine / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; C576881:ERF110 protein, Arabidopsis; D005030:Ethylenes; D009587:Nitrogen Isotopes; D010937:Plant Growth Regulators; D020033:Protein Isoforms; D014157:Transcription Factors; D012694:Serine; C036216:ethylene