title: Bio-orthogonal labeling as a tool to visualize and identify newly synthesized proteins in Caenorhabditis elegans.
abstract: In this protocol we describe the incorporation of bio-orthogonal amino acids as a versatile method for visualizing and identifying de novo-synthesized proteins in the roundworm Caenorhabditis elegans. This protocol contains directions on implementing three complementary types of analysis: 'click chemistry' followed by western blotting, click chemistry followed by immunofluorescence, and isobaric tags for relative and absolute quantification (iTRAQ) quantitative mass spectrometry. The detailed instructions provided herein enable researchers to investigate the de novo proteome, an analysis that is complicated by the fact that protein molecules are chemically identical to each other, regardless of the timing of their synthesis. Our protocol circumvents this limitation by identifying de novo-synthesized proteins via the incorporation of the chemically modifiable azidohomoalanine instead of the natural amino acid methionine in the nascent protein, followed by facilitating the visualization of the resulting labeled proteins in situ. It will therefore be an ideal tool for studying de novo protein synthesis in physiological and pathological processes including learning and memory. The protocol requires 10 d for worm growth, liquid culture and synchronization; 1-2 d for bio-orthogonal labeling; and, with regard to analysis, 3-4 d for western blotting, 5-6 d for immunofluorescence or ~3 weeks for mass spectrometry.
authors.affiliation: 1] Brain and Mind Research Institute, University of Sydney, Camperdown, New South Wales, Australia. [2] Institute for Integrative Neuroanatomy, Charité, Universitätsmedizin Berlin, Berlin, Germany.
authors.forename: Milena
authors.initials: M
authors.lastname: Ullrich
authors.affiliation: Brain and Mind Research Institute, University of Sydney, Camperdown, New South Wales, Australia.
authors.forename: Vanessa
authors.initials: V
authors.lastname: Liang
authors.affiliation: School of Molecular Bioscience, University of Sydney, Camperdown, New South Wales, Australia.
authors.forename: Yee Lian
authors.initials: YL
authors.lastname: Chew
authors.affiliation: School of Chemistry, University of Sydney, Camperdown, New South Wales, Australia.
authors.forename: Samuel
authors.initials: S
authors.lastname: Banister
authors.affiliation: Australian Proteome Analysis Facility, Macquarie University, Sydney, New South Wales, Australia.
authors.forename: Xiaomin
authors.initials: X
authors.lastname: Song
authors.affiliation: Australian Proteome Analysis Facility, Macquarie University, Sydney, New South Wales, Australia.
authors.forename: Thiri
authors.initials: T
authors.lastname: Zaw
authors.affiliation: School of Molecular Bioscience, University of Sydney, Camperdown, New South Wales, Australia.
authors.forename: Hong
authors.initials: H
authors.lastname: Lam
authors.affiliation: School of Molecular Bioscience, University of Sydney, Camperdown, New South Wales, Australia.
authors.forename: Slavica
authors.initials: S
authors.lastname: Berber
authors.affiliation: 1] School of Chemistry, University of Sydney, Camperdown, New South Wales, Australia. [2] Faculty of Health Sciences, Macquarie University, Sydney, New South Wales, Australia.
authors.forename: Michael
authors.initials: M
authors.lastname: Kassiou
authors.affiliation: 1] School of Molecular Bioscience, University of Sydney, Camperdown, New South Wales, Australia. [2].
authors.forename: Hannah R
authors.initials: HR
authors.lastname: Nicholas
authors.affiliation: 1] Brain and Mind Research Institute, University of Sydney, Camperdown, New South Wales, Australia. [2] Clem Jones Centre for Ageing Dementia Research (CJCADR), Queensland Brain Institute (QBI), The University of Queensland, Brisbane, Queensland, Australia. [3].
authors.forename: Jürgen
authors.initials: J
authors.lastname: Götz
mesh_terms: D000409:Alanine / Q000031:analogs & derivatives; D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D029742:Caenorhabditis elegans Proteins / Q000032:analysis* / Q000378:metabolism; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D057930:Click Chemistry / Q000379:methods*; D005455:Fluorescent Antibody Technique; D013058:Mass Spectrometry; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D000596:Amino Acids; D029742:Caenorhabditis elegans Proteins; C506098:azidohomoalanine; D000409:Alanine