title: Simple protein complex purification and identification method for high-throughput mapping of protein interaction networks.
abstract: Most current methods for purification and identification of protein complexes use endogenous expression of affinity-tagged bait, tandem affinity tag purification of protein complexes followed by specific elution of complexes from beads, and gel separation and in-gel digestion prior to mass spectrometric analysis of protein interactors. We propose a single affinity tag in vitro pull-down assay with denaturing elution, trypsin digestion in organic solvent, and LC-ESI MS/MS protein identification using SEQUEST analysis. Our method is simple and easy to scale-up and automate, making it suitable for high-throughput mapping of protein interaction networks and functional proteomics.
authors.affiliation: Pacific Northwest National Laboratory, 902 Battelle Blvd., P.O. Box 999, Richland, Washington 99354, USA.
authors.forename: Lye Meng
authors.initials: LM
authors.lastname: Markillie
authors.forename: Chiann-Tso
authors.initials: CT
authors.lastname: Lin
authors.forename: Joshua N
authors.initials: JN
authors.lastname: Adkins
authors.forename: Deanna L
authors.initials: DL
authors.lastname: Auberry
authors.forename: Eric A
authors.initials: EA
authors.lastname: Hill
authors.forename: Brian S
authors.initials: BS
authors.lastname: Hooker
authors.forename: Priscilla A
authors.initials: PA
authors.lastname: Moore
authors.forename: Ronald J
authors.initials: RJ
authors.lastname: Moore
authors.forename: Liang
authors.initials: L
authors.lastname: Shi
authors.forename: H Steven
authors.initials: HS
authors.lastname: Wiley
authors.forename: Vladimir
authors.initials: V
authors.lastname: Kery
mesh_terms: D002853:Chromatography, Liquid; D010449:Peptide Mapping; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D011506:Proteins