Affinity capture of specific DNA-binding proteins for mass spectrometric identification.
We describe a general approach for affinity microcapture of site-specific, nucleic acid-binding proteins. The major difficulties to developing this method into a widely applicable protocol derived from the need for a massive enrichment and the inadvertent, extensive binding of nonspecific proteins to the bait. On the basis of a detailed analysis, we propose (i) a one-step fractionation of crude extracts on P11 phosphocellulose, followed by (ii) a discrete series of positive/negative selections on wild-type and site-mutated ligand DNA in a magnetic microparticulate format, with cobalt magnets, concatamerized and biotinylated ligands, selective salt conditions, and improved competitor DNAs. We also present rules for determining the precise number and order of selections. The approach and protocol allowed isolation of four, low-abundance transcription factors and repressors from 2 x 10(9) cultured leukemia cells. Captured proteins were 10-20,000-fold enriched from the nuclear extract, in a form and amounts that permitted facile MALDI-TOF and TOF/TOF MS-based protein identification. This is 1-2 orders of magnitude better than many previous efforts and in a fraction of the time (approximately 1 factor/week). The method can be applied to any protein that binds DNA, including those with modest to low affinity, and bridges functional-biochemical studies on replication, transcriptional regulation, and DNA repair with the analytical power of mass spectrometry-based proteomics.
Yaneva
Mariana
M
Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, and Weill Graduate School of Medical Sciences, Cornell University, New York, New York 10021, USA.
Tempst
Paul
P
D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D004268:DNA-Binding Proteins