title: Proteomic Profiling of De Novo Protein Synthesis in Starvation-Induced Autophagy Using Bioorthogonal Noncanonical Amino Acid Tagging.
abstract: Autophagy is an intracellular degradation process activated by stress factors such as nutrient starvation to maintain cellular homeostasis. There is emerging evidence demonstrating that de novo protein synthesis is involved in the autophagic process. However, up-to-date characterizing of these de novo proteins is technically difficult. In this chapter, we describe a novel method to identify newly synthesized proteins during starvation-mediated autophagy by bioorthogonal noncanonical amino acid tagging (BONCAT), in conjunction with isobaric tagging for relative and absolute quantification (iTRAQ)-based quantitative proteomics. l-azidohomoalanine (AHA) is an analog of methionine, and it can be readily incorporated into the newly synthesized proteins. The AHA-containing proteins can be enriched with avidin beads after a "click" reaction between alkyne-bearing biotin and the azide moiety of AHA. The enriched proteins are then subjected to iTRAQ™ labeling for protein identification and quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). By using this technique, we have successfully profiled more than 700 proteins that are synthesized during starvation-induced autophagy. We believe that this approach is effective in identification of newly synthesized proteins in the process of autophagy and provides useful insights to the molecular mechanisms and biological functions of autophagy.
authors.affiliation: Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
authors.forename: J
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore; National University of Singapore, Singapore, Singapore; Interdisciplinary Research Group in Infectious Diseases, Singapore-MIT Alliance for Research & Technology (SMART), Singapore.
authors.forename: J
authors.initials: J
authors.lastname: Wang
authors.affiliation: National University of Singapore, Singapore, Singapore.
authors.forename: Y-M
authors.initials: YM
authors.lastname: Lee
authors.affiliation: National University of Singapore, Singapore, Singapore.
authors.forename: T-K
authors.initials: TK
authors.lastname: Lim
authors.affiliation: National University of Singapore, Singapore, Singapore. Electronic address: dbslinqs@nus.edu.sg.
authors.forename: Q
authors.initials: Q
authors.lastname: Lin
authors.affiliation: Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore; NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore. Electronic address: phsshm@nus.edu.sg.
authors.forename: H-M
authors.initials: HM
authors.lastname: Shen
mesh_terms: D000409:Alanine / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis / Q000378:metabolism*; D000818:Animals; D001343:Autophagy; D018929:Cell Culture Techniques / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D057930:Click Chemistry / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D014176:Protein Biosynthesis; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods
chemical_list: D000596:Amino Acids; D011506:Proteins; C506098:azidohomoalanine; D000409:Alanine
keywords: AHA; Autophagy; BONCAT; De novo protein synthesis; Mass spectrometry; Metabolic labeling; Proteomics; Starvation; iTRAQ