کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1173479 1491373 2015 8 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
T3: Targeted proteomics of DNA-binding proteins
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
T3: Targeted proteomics of DNA-binding proteins
چکیده انگلیسی

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 T3 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.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytical Biochemistry - Volume 474, 1 April 2015, Pages 8–15
نویسندگان
, ,