title: Rapid assessment of RNAi-mediated protein depletion by selected reaction monitoring mass spectrometry.
abstract: We describe the use of a targeted proteomics approach, selected reaction monitoring (SRM) mass spectrometry, to detect and assess RNAi-mediated depletion or "knockdown" of specific proteins from human cells and from Drosophila flies. This label-free approach does not require any specific reagents to confirm the depletion of RNAi target protein(s) in unfractionated cell or whole organism extracts. The protocol described here is general, can be developed rapidly, and can be multiplexed to detect and measure multiple proteins at once. Furthermore, the methodology can be extended to any tandem mass spectrometer, making it widely accessible. This methodology will be applicable to a wide range of basic science and clinical questions where RNAi-mediated protein depletion needs to be verified, or where differences in relative abundance of target proteins need to be rapidly assessed between samples.
authors.affiliation: Departments of Genome Sciences and Pathology, University of Washington, Seattle, Washington 98195, USA.
authors.forename: Veronika A
authors.initials: VA
authors.lastname: Glukhova
authors.forename: Daniela M
authors.initials: DM
authors.lastname: Tomazela
authors.forename: Geoffrey D
authors.initials: GD
authors.lastname: Findlay
authors.forename: Raymond J
authors.initials: RJ
authors.lastname: Monnat
authors.forename: Michael J
authors.initials: MJ
authors.lastname: MacCoss
mesh_terms: D000818:Animals; D002460:Cell Line; D004330:Drosophila; D055785:Gene Knockdown Techniques; D006801:Humans; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods*; D034622:RNA Interference; D053719:Tandem Mass Spectrometry
chemical_list: D010446:Peptide Fragments; D011506:Proteins