title: Comprehensive quantification of monolignol-pathway enzymes in Populus trichocarpa by protein cleavage isotope dilution mass spectrometry.
abstract: The economic value of wood/pulp from many tree species is largely dictated by the quantity and chemical properties of lignin, which is directly related to the composition and linkages of monolignols comprising the polymer. Although much is known regarding the monolignol biosynthetic pathway, our understanding is still deficient due to the lack of quantitative information at the proteomic level. We developed an assay based on protein cleavage isotope dilution mass spectrometry (PC-IDMS) for the determination of all potential, primary enzymes involved in the biosynthesis of monolignols and the peroxidases responsible for their polymerization to form lignin in the model tree species, Populus trichocarpa. Described is the identification of quantitative surrogate peptides through shotgun analysis of native and recombinant proteins, optimization of trypsin proteolysis using fractional factorial design of experiments, and development of a liquid chromatography-selected reaction monitoring method for specific detection of all targeted peptides. Of the 25 targeted enzymes, three were undetected in the normal xylem tissues, and all but two of the detectable species showed good day-to-day precision (CV < 10%). This represents the most comprehensive assay for quantification of proteins regulating monolignol biosynthesis and will lead to a better understanding of lignin formation at a systems level.
authors.affiliation: W.M. Keck FT-ICR Mass Spectrometry Laboratory, Department of Chemistry, and ‡Forest Biotechnology Group, Department of Forestry and Environmental Resources, North Carolina State University , Raleigh, North Carolina 27695, United States.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Shuford
authors.forename: Quanzi
authors.initials: Q
authors.lastname: Li
authors.forename: Ying-Hsuan
authors.initials: YH
authors.lastname: Sun
authors.forename: Hsi-Chuan
authors.initials: HC
authors.lastname: Chen
authors.forename: Jack
authors.initials: J
authors.lastname: Wang
authors.forename: Rui
authors.initials: R
authors.lastname: Shi
authors.forename: Ronald R
authors.initials: RR
authors.lastname: Sederoff
authors.forename: Vincent L
authors.initials: VL
authors.lastname: Chiang
authors.forename: David C
authors.initials: DC
authors.lastname: Muddiman
mesh_terms: D000595:Amino Acid Sequence; D053898:Biosynthetic Pathways; D007553:Isotope Labeling; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D032107:Populus / Q000201:enzymology*; D020543:Proteome / Q000737:chemistry / Q000378:metabolism; D012015:Reference Standards; D021241:Spectrometry, Mass, Electrospray Ionization / Q000592:standards; D052584:Xylem / Q000201:enzymology*
chemical_list: D010940:Plant Proteins; D020543:Proteome