The identification of nucleic acid-interacting proteins using a simple proteomics-based approach that directly incorporates the electrophoretic mobility shift assay.
Proteins that interact with nucleic acids are central to numerous cellular processes, and their continuing characterization represents one of the foremost challenges in the postgenomic era. Here we describe a simple proteomics-based approach for the identification by mass spectrometry of proteins in crude extracts that interact with nucleic acids. It incorporates the electrophoretic mobility shift assay and is based on the finding that when a protein forms a complex with nucleic acid its electrophoretic mobility is affected as well as that of the nucleic acid. Our method should greatly reduce and in some cases may even eliminate the need for extensive protein purification and as such should contribute significantly to the functional annotation of the proteome. Furthermore it requires no prior knowledge of the molecular mass, quaternary structure, or pI of the interacting protein. Proof of principle is demonstrated using a recently discovered transcription factor; however, the approach should also have application in the identification of proteins that interact with RNA.
Stead
Jonathan A
JA
Astbury Centre for Structural Molecular Biology and Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom.
Keen
Jeff N
JN
McDowall
Kenneth J
KJ
D000595:Amino Acid Sequence; D001667:Binding, Competitive; D002846:Chromatography, Affinity; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D007525:Isoelectric Focusing / Q000379:methods; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D040901:Proteomics; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014157:Transcription Factors / Q000032:analysis* / Q000378:metabolism
D004268:DNA-Binding Proteins; D014157:Transcription Factors; D004247:DNA