title: Bioorthogonal Noncanonical Amino Acid Tagging (BONCAT) Enables Time-Resolved Analysis of Protein Synthesis in Native Plant Tissue.
abstract: Proteomic plasticity undergirds stress responses in plants, and understanding such responses requires accurate measurement of the extent to which proteins levels are adjusted to counter external stimuli. Here, we adapt bioorthogonal noncanonical amino acid tagging (BONCAT) to interrogate protein synthesis in vegetative Arabidopsis (Arabidopsis thaliana) seedlings. BONCAT relies on the translational incorporation of a noncanonical amino acid probe into cellular proteins. In this study, the probe is the Met surrogate azidohomoalanine (Aha), which carries a reactive azide moiety in its amino acid side chain. The azide handle in Aha can be selectively conjugated to dyes and functionalized beads to enable visualization and enrichment of newly synthesized proteins. We show that BONCAT is sensitive enough to detect Arabidopsis proteins synthesized within a 30-min interval defined by an Aha pulse and that the method can be used to detect proteins made under conditions of light stress, osmotic shock, salt stress, heat stress, and recovery from heat stress. We further establish that BONCAT can be coupled to tandem liquid chromatography-mass spectrometry to identify and quantify proteins synthesized during heat stress and recovery from heat stress. Our results are consistent with a model in which, upon the onset of heat stress, translation is rapidly reprogrammed to enhance the synthesis of stress mitigators and is again altered during recovery. All experiments were carried out with commercially available reagents, highlighting the accessibility of the BONCAT method to researchers interested in stress responses as well as translational and posttranslational regulation in plants.
authors.affiliation: Division of Chemistry and Chemical Engineering (W.S.G., S.E.S., S.H.H., D.A.T.), and Proteome Exploration Laboratory (M.J.S., A.M., S.H.), California Institute of Technology, Pasadena, California 91125; and.|Center for Plant Cell Biology, University of California, Riverside, California 92521 (J.B.-S.).
authors.forename: Weslee S
authors.identifier: 0000-0001-7733-7291
authors.initials: WS
authors.lastname: Glenn
authors.affiliation: Division of Chemistry and Chemical Engineering (W.S.G., S.E.S., S.H.H., D.A.T.), and Proteome Exploration Laboratory (M.J.S., A.M., S.H.), California Institute of Technology, Pasadena, California 91125; and.|Center for Plant Cell Biology, University of California, Riverside, California 92521 (J.B.-S.).
authors.forename: Shannon E
authors.identifier: 0000-0002-6617-3874
authors.initials: SE
authors.lastname: Stone
authors.affiliation: Division of Chemistry and Chemical Engineering (W.S.G., S.E.S., S.H.H., D.A.T.), and Proteome Exploration Laboratory (M.J.S., A.M., S.H.), California Institute of Technology, Pasadena, California 91125; and.|Center for Plant Cell Biology, University of California, Riverside, California 92521 (J.B.-S.).
authors.forename: Samuel H
authors.identifier: 0000-0001-7647-0752
authors.initials: SH
authors.lastname: Ho
authors.affiliation: Division of Chemistry and Chemical Engineering (W.S.G., S.E.S., S.H.H., D.A.T.), and Proteome Exploration Laboratory (M.J.S., A.M., S.H.), California Institute of Technology, Pasadena, California 91125; and.|Center for Plant Cell Biology, University of California, Riverside, California 92521 (J.B.-S.).
authors.forename: Michael J
authors.identifier: 0000-0003-0878-3831
authors.initials: MJ
authors.lastname: Sweredoski
authors.affiliation: Division of Chemistry and Chemical Engineering (W.S.G., S.E.S., S.H.H., D.A.T.), and Proteome Exploration Laboratory (M.J.S., A.M., S.H.), California Institute of Technology, Pasadena, California 91125; and.|Center for Plant Cell Biology, University of California, Riverside, California 92521 (J.B.-S.).
authors.forename: Annie
authors.initials: A
authors.lastname: Moradian
authors.affiliation: Division of Chemistry and Chemical Engineering (W.S.G., S.E.S., S.H.H., D.A.T.), and Proteome Exploration Laboratory (M.J.S., A.M., S.H.), California Institute of Technology, Pasadena, California 91125; and.|Center for Plant Cell Biology, University of California, Riverside, California 92521 (J.B.-S.).
authors.forename: Sonja
authors.identifier: 0000-0002-5904-9816
authors.initials: S
authors.lastname: Hess
authors.affiliation: Division of Chemistry and Chemical Engineering (W.S.G., S.E.S., S.H.H., D.A.T.), and Proteome Exploration Laboratory (M.J.S., A.M., S.H.), California Institute of Technology, Pasadena, California 91125; and.|Center for Plant Cell Biology, University of California, Riverside, California 92521 (J.B.-S.).
authors.forename: Julia
authors.identifier: 0000-0002-8568-7125
authors.initials: J
authors.lastname: Bailey-Serres
authors.affiliation: Division of Chemistry and Chemical Engineering (W.S.G., S.E.S., S.H.H., D.A.T.), and Proteome Exploration Laboratory (M.J.S., A.M., S.H.), California Institute of Technology, Pasadena, California 91125; and tirrell@caltech.edu.|Center for Plant Cell Biology, University of California, Riverside, California 92521 (J.B.-S.) tirrell@caltech.edu.
authors.forename: David A
authors.identifier: 0000-0003-3175-4596
authors.initials: DA
authors.lastname: Tirrell
mesh_terms: D000409:Alanine / Q000031:analogs & derivatives* / Q000737:chemistry; D017360:Arabidopsis / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D029681:Arabidopsis Proteins / Q000096:biosynthesis*; D001386:Azides / Q000737:chemistry*; D002853:Chromatography, Liquid; D004396:Coloring Agents / Q000737:chemistry*; D055864:Droughts; D006358:Hot Temperature; D015151:Immunoblotting; D008027:Light; D014176:Protein Biosynthesis; D015203:Reproducibility of Results; D036226:Seedlings / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects; D012965:Sodium Chloride / Q000494:pharmacology; D013194:Staining and Labeling / Q000379:methods; D013312:Stress, Physiological; D053719:Tandem Mass Spectrometry; D013997:Time Factors
chemical_list: D029681:Arabidopsis Proteins; D001386:Azides; D004396:Coloring Agents; C506098:azidohomoalanine; D012965:Sodium Chloride; D000409:Alanine