Simple protein complex purification and identification method for high-throughput mapping of protein interaction networks.
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.
Markillie
Lye Meng
LM
Pacific Northwest National Laboratory, 902 Battelle Blvd., P.O. Box 999, Richland, Washington 99354, USA.
Lin
Chiann-Tso
CT
Adkins
Joshua N
JN
Auberry
Deanna L
DL
Hill
Eric A
EA
Hooker
Brian S
BS
Moore
Priscilla A
PA
Moore
Ronald J
RJ
Shi
Liang
L
Wiley
H Steven
HS
Kery
Vladimir
V
D002853:Chromatography, Liquid; D010449:Peptide Mapping; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization
D011506:Proteins