title: T(3): targeted proteomics of DNA-binding proteins.
abstract: A technique that allows the inclusion of a specific DNA to enrich and direct proteomic identification of transcription factors (TFs) while providing a route for high-throughput screening on a single platform would be valuable in investigations of gene expression and regulation. Polyvinylpyrrolidone binds DNA avidly while binding negligible amounts of protein. This observation is used in a proof-of-concept method to enrich for TFs by combining nuclear extract with a specific DNA sequence and immobilizing the DNA-protein complex on a polyvinylpyrrolidone (PVP)-coated MALDI (matrix-assisted laser desorption/ionization) plate. Any unbound proteins are washed away and further processed for analysis in a MALDI-TOF/TOF (tandem time-of-flight) mass spectrometer. Enrichment on a PVP-coated plate gives the unique advantage of purification, enzymatic digestion, and analysis on a single platform. The method is termed T(3) because it combines Targeted purification on a Target plate with Targeted proteomics. Validation was achieved in model experiments with a chimeric fusion protein, green fluorescent protein-CAAT enhancer binding protein (GFP-C/EBP), with an oligonucleotide containing the CAAT sequence. Both domains were identified with an expectation value of less than 10(-15) and more than 15% sequence coverage. The same oligonucleotide mixed with HEK293 cell nuclear extract allowed the unambiguous identification of native human C/EBP alpha with 24.3% sequence coverage.
authors: Department of Chemistry, University of Texas at San Antonio, San Antonio, TX 78249, USA.
authors: Linda I
authors: LI
authors: Nagore
authors: Department of Chemistry, University of Texas at San Antonio, San Antonio, TX 78249, USA. Electronic address: harry.jarrett@utsa.edu.
authors: Harry W
authors: HW
authors: Jarrett
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism; D002417:Cattle; D002457:Cell Extracts; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011205:Povidone / Q000737:chemistry; D011485:Protein Binding; D040901:Proteomics / Q000379:methods*; D051381:Rats; D012710:Serum Albumin, Bovine / Q000378:metabolism; D034421:Sus scrofa
chemical_list: D022762:CCAAT-Enhancer-Binding Proteins; D002457:Cell Extracts; D004268:DNA-Binding Proteins; D049452:Green Fluorescent Proteins; D012710:Serum Albumin, Bovine; D004247:DNA; D011205:Povidone
keywords: DNA-binding proteins; MALDI–MS; Mass spectrometry; Proteomics; Transcription factors
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