Harry Jarrett
University of Texas San Antonio, USA
Title: New method for analyzing transcription factors and other proteins involved in transcription using MALDI-TOF mass spectrometry
Biography
Biography: Harry Jarrett
Abstract
MALDI plates coated with polyvinylpyrrolidone do not bind protein but avidly bind DNA and DNA-protein complexes. The DNA sequence determines which proteins bind. When DNA, either oligonucleotides or longer DNAs such as core promoter sequences, is mixed with nuclear extract and spotted on the plate, DNA-protein complexes are bound while other proteins can be simply washed away. The samples are then digested with trypsin and matrix is added on the plate and the plate is then ready for mass spectrometry analysis. Highest signal-to-noise is obtained with core promoters (in this case a 300 bp human telomerase (hTERT) promoter) than with 20-mer duplex oligonucleotides but usable data is obtained with either. Using overlapping oligonucleotides representing the entire hTERT promoter, binding sites for TCF23 (an E-box binding protein), SP1, and AP2 were all localized to specific sequence regions. When the intact promoter is used, SP1 and AP2 are also found but instead of TCF23, USF2 is found. This suggests that USF2 binds to the E-boxes in the promoter context. Other components of the RNA polymerase 2 transcription complex such as TF2H were also characterized. The method allows as many as 384 samples to be analyzed on a single plate in a matter of three days and thus the method is capable of high-throughput analysis of DNA-binding proteins.
