Identifying true protein complex constituents in interaction proteomics: the example of the DMXL2 protein complex.
A typical high-sensitivity antibody affinity purification-mass spectrometry experiment easily identifies hundreds of protein interactors. However, most of these are non-valid resulting from multiple causes other than interaction with the bait protein. To discriminate true interactors from off-target recognition, we propose to differentially include an (peptide) antigen during the antibody incubation in the immuno-precipitation experiment. This contrasts the specific antibody-bait protein interactions, versus all other off-target protein interactions. To exemplify the power of the approach, we studied the DMXL2 interactome. From the initial six immuno-precipitations, we identified about 600 proteins. When filtering for interactors present in all anti-DMXL2 antibody immuno-precipitation experiments, absent in the bead controls, and competed off by the peptide antigen, this hit list is reduced to ten proteins, including known and novel interactors of DMXL2. Together, our approach enables the use of a wide range of available antibodies in large-scale protein interaction proteomics, while gaining specificity of the interactions.
Department of Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research, Neuroscience Campus Amsterdam, VU University, Amsterdam, The Netherlands. ka.wan.li@cncr.vu.nl
Ka Wan
KW
Li
Ning
N
Chen
Patricia
P
Klemmer
Frank
F
Koopmans
Ramesh
R
Karupothula
August B
AB
Smit
D000818:Animals; D000941:Antigens / Q000378:metabolism; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D046912:Multiprotein Complexes / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping; D040901:Proteomics; D025262:Vacuolar Proton-Translocating ATPases / Q000378:metabolism
D000941:Antigens; C576980:DMXL2 protein, mouse; D046912:Multiprotein Complexes; D009419:Nerve Tissue Proteins; D010455:Peptides; D025262:Vacuolar Proton-Translocating ATPases