title: Cell-specific Profiling of Nascent Proteomes Using Orthogonal Enzyme-mediated Puromycin Incorporation.
abstract: Translation regulation is a fundamental component of gene expression, allowing cells to respond rapidly to a variety of stimuli in the absence of new transcription. The lack of methods for profiling nascent proteomes in distinct cell populations in heterogeneous tissues has precluded an understanding of translational regulation in physiologically relevant contexts. Here, we describe a chemical genetic method that involves orthogonal enzyme-mediated incorporation of a clickable puromycin analogue into nascent polypeptides. Using this method, we show that we can label newly synthesized proteins in a cell-specific manner in cells grown in culture and in heterogeneous tissues. We also show that we can identify the nascent proteome in genetically targeted cell populations using affinity enrichment and tandem mass spectrometry. Our method has the potential to provide unprecedented insights into cell-specific translational regulation in heterogeneous tissues.
authors.affiliation: Vollum Institute, ‡Program in Chemical Biology, and §Department of Physiology and Pharmacology, Oregon Health & Science University , Portland, Oregon 97210, United States.
authors.forename: Ruth M
authors.initials: RM
authors.lastname: Barrett
authors.affiliation: Vollum Institute, ‡Program in Chemical Biology, and §Department of Physiology and Pharmacology, Oregon Health & Science University , Portland, Oregon 97210, United States.
authors.forename: Hui-Wen
authors.initials: HW
authors.lastname: Liu
authors.affiliation: Vollum Institute, ‡Program in Chemical Biology, and §Department of Physiology and Pharmacology, Oregon Health & Science University , Portland, Oregon 97210, United States.
authors.forename: Haihong
authors.initials: H
authors.lastname: Jin
authors.affiliation: Vollum Institute, ‡Program in Chemical Biology, and §Department of Physiology and Pharmacology, Oregon Health & Science University , Portland, Oregon 97210, United States.
authors.forename: Richard H
authors.initials: RH
authors.lastname: Goodman
authors.affiliation: Vollum Institute, ‡Program in Chemical Biology, and §Department of Physiology and Pharmacology, Oregon Health & Science University , Portland, Oregon 97210, United States.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Cohen
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D019921:Biotinylation; D057930:Click Chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D050416:Glucagon-Secreting Cells / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D050417:Insulin-Secreting Cells / Q000378:metabolism; D051379:Mice; D010399:Penicillin Amidase / Q000737:chemistry; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020543:Proteome / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011691:Puromycin / Q000031:analogs & derivatives* / Q000737:chemistry; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; C570448:O-propargyl-puromycin; D010455:Peptides; D020543:Proteome; D014443:Tyrosine; D011691:Puromycin; D010399:Penicillin Amidase; D000241:Adenosine