title: Identification and quantification of newly synthesized proteins translationally regulated by YB-1 using a novel Click-SILAC approach.
abstract: Messenger RNA-binding translational regulatory proteins determine in large part the spectrum of transcripts that are translated under specific cellular contexts. Y-box binding protein-1 (YB-1) is a conserved eukaryotic translational regulator that is implicated in cancer progression. To identify specific proteins that are translationally regulated by YB-1, we established a pulse-labelling approach combining Click chemistry and stable isotope labelling by amino acids in cell culture (SILAC). The proteome of TC32 human Ewing sarcoma cells, which robustly express YB-1, was compared with or without YB-1 siRNA knockdown. Cells labelled with light or heavy isotopologs of Arg and Lys were then cotranslationally pulsed with the methionine derivative, azidohomoalanine (AHA). Cells were lysed and newly synthesized proteins were selectively derivatized via a Click (3+2 cycloaddition) reaction to add an alkyne biotin tag. They were then affinity purified and subjected to liquid chromatography-tandem mass spectrometry. This combined Click-SILAC approach enabled us to catalog and quantify newly synthesized proteins regulated by YB-1 after only 45 min of labelling. Bioinformatic analysis revealed that YB-1 regulated proteins are involved in diverse biological pathways. We anticipate that this Click-SILAC strategy will be useful for studying short-term protein synthesis in different cell culture systems and under diverse biological contexts.
authors.affiliation: Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, Canada BC V6T 1Z4.
authors.forename: Syam Prakash
authors.initials: SP
authors.lastname: Somasekharan
authors.forename: Nikolay
authors.initials: N
authors.lastname: Stoynov
authors.forename: Barak
authors.initials: B
authors.lastname: Rotblat
authors.forename: Gabriel
authors.initials: G
authors.lastname: Leprivier
authors.forename: Jason D
authors.initials: JD
authors.lastname: Galpin
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Ahern
authors.forename: Leonard J
authors.initials: LJ
authors.lastname: Foster
authors.forename: Poul H B
authors.initials: PH
authors.lastname: Sorensen
mesh_terms: D001859:Bone Neoplasms / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007553:Isotope Labeling; D009363:Neoplasm Proteins / Q000096:biosynthesis*; D014176:Protein Biosynthesis; D020543:Proteome / Q000096:biosynthesis*; D040901:Proteomics / Q000379:methods; D012512:Sarcoma, Ewing / Q000378:metabolism* / Q000473:pathology; D051840:Y-Box-Binding Protein 1 / Q000096:biosynthesis*
chemical_list: D009363:Neoplasm Proteins; D020543:Proteome; D051840:Y-Box-Binding Protein 1; C070856:YBX1 protein, human