Chip-based analysis of protein-protein interactions by fluorescence detection and on-chip immunoprecipitation combined with microLC-MS/MS analysis.
A new chip-based method to identify protein-protein interactions was developed using the guanine nucleotide exchange factor GRF2 and two interacting proteins, Ras and calmodulin, as model proteins. A generic immobilization strategy for FLAG-tagged bait proteins on a protein-repellent streptavidin chip surface was implemented by presentation of an oriented anti-FLAG antibody. A flow cell device, integrating different chip surfaces, was developed, and the interaction of immobilized GRF2 with the two analytes was verified by fluorescence assays. On-chip tryptic digest assays were then performed on the capture surface and analyzed by microLC-MS/MS. The interaction of GRF2 with calmodulin and Ras was demonstrated, and the lower limit of detection was determined. We also implemented an on-chip immunoprecipitation assay to identify GRF2-binding partners from complex protein mixtures. Cells overexpressing FLAG-GRF2 were lysed and then incubated with the anti-FLAG chip. In addition to detecting GRF2, we also identified calmodulin, demonstrating that this technique can successfully identify endogenous levels of proteins, bound to recombinant bait proteins. This chip-based method has the advantage that no subsequent gel separations of protein complexes prior to LC-MS analysis are required and is therefore amenable to miniaturized high-throughput determination of protein-protein interactions.
Sydor
Jens R
JR
Zyomyx, Inc, 26101 Research Road, Hayward, California 94545, USA.
Scalf
Mark
M
Sideris
Steve
S
Mao
Guo Dong
GD
Pandey
Yash
Y
Tan
Ming
M
Mariano
Maria
M
Moran
Michael F
MF
Nock
Steffen
S
Wagner
Peter
P
D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism; D002853:Chromatography, Liquid; D005453:Fluorescence; D020735:Guanine Nucleotide-Releasing Factor 2 / Q000737:chemistry / Q000378:metabolism*; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D011233:Precipitin Tests; D040081:Protein Array Analysis / Q000379:methods*; D011485:Protein Binding; D014357:Trypsin / Q000378:metabolism; D018631:ras Proteins / Q000737:chemistry / Q000378:metabolism
D002147:Calmodulin; D020735:Guanine Nucleotide-Releasing Factor 2; D014357:Trypsin; D018631:ras Proteins