title: In-depth quantitative proteomic analysis of de novo protein synthesis induced by brain-derived neurotrophic factor.
abstract: Measuring the synthesis of new proteins in the context of a much greater number of pre-existing proteins can be difficult. To overcome this obstacle, bioorthogonal noncanonical amino acid tagging (BONCAT) can be combined with stable isotope labeling by amino acid in cell culture (SILAC) for comparative proteomic analysis of de novo protein synthesis (BONLAC). In the present study, we show that alkyne resin-based isolation of l-azidohomoalanine (AHA)-labeled proteins using azide/alkyne cycloaddition minimizes contamination from pre-existing proteins. Using this approach, we isolated and identified 7414 BONCAT-labeled proteins. The nascent proteome isolated by BONCAT was very similar to the steady-state proteome, although transcription factors were highly enriched by BONCAT. About 30% of the methionine residues were replaced by AHA in our BONCAT samples, which allowed for identification of methionine-containing peptides. There was no bias against low-methionine proteins by BONCAT at the proteome level. When we applied the BONLAC approach to screen for brain-derived neurotrophic factor (BDNF)-induced protein synthesis, 53 proteins were found to be significantly changed 2 h after BDNF stimulation. Our study demonstrated that the newly synthesized proteome, even after a short period of stimulation, can be efficiently isolated by BONCAT and analyzed to a depth that is similar to that of the steady-state proteome.
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, ‡Departments of Cell Biology, Physiology, and Neuroscience and Psychiatry, Kimmel Center for Biology and Medicine at the Skirball Institute, New York University School of Medicine , New York, New York 10016, United States.
authors.forename: Guoan
authors.initials: G
authors.lastname: Zhang
authors.forename: Heather
authors.initials: H
authors.lastname: Bowling
authors.forename: Nancy
authors.initials: N
authors.lastname: Hom
authors.forename: Kent
authors.initials: K
authors.lastname: Kirshenbaum
authors.forename: Eric
authors.initials: E
authors.lastname: Klann
authors.forename: Moses V
authors.initials: MV
authors.lastname: Chao
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Neubert
mesh_terms: D000409:Alanine / Q000031:analogs & derivatives / Q000737:chemistry; D019208:Brain-Derived Neurotrophic Factor / Q000494:pharmacology*; D002853:Chromatography, Liquid; D057809:HEK293 Cells; D006801:Humans; D013058:Mass Spectrometry; D014176:Protein Biosynthesis / Q000187:drug effects*; D020543:Proteome / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results
chemical_list: D019208:Brain-Derived Neurotrophic Factor; D020543:Proteome; C506098:azidohomoalanine; D000409:Alanine
keywords: BDNF; BONCAT; mass spectrometry; proteomics; pulsed SILAC; translation