Novel methodology for transcription factor enrichment and identification using mass spectrometry

Date
2014
Authors
Nagore, Linda I.
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Abstract

This thesis presents a method for the analysis of low abundant DNA-binding proteins using mass spectrometry (MS), a technology commonly used in proteomics for identification and quantitation of complex samples. This area of research has gained a great deal of attention since proteins, specifically transcription factors (TF), regulate gene expression. Unfortunately, the low abundance of TFs make it difficult to characterize each promoter and current strategies tend to be time intensive. Our intent was to exploit the high affinity of TFs for DNA response elements prior to analysis to improve the dynamic range of the proteome. We present a method that provides a route for efficient enrichment and detection termed T3 for its ability to achieve T argeted purification on a T arget plate and analyzed with T argeted proteomics. A polyvinylpyrrolidone-coated plate was constructed with the capability to enrich, desalt, and analyze a sample on a single platform by having the unique characteristic of binding DNA but not protein. T3 combined with matrix assisted laser desorption ionization (MALDI- MS) we have the capabilities of preparing and analyzing 384 samples in three days. Our contribution comes in three stages. First we examine Promoter Trap-LC-MS as a benchmark to later evaluate the quality of the T3 technique. Second, we present a proof-of-concept, which incorporates fusion protein GFP-C/EBP and its corresponding DNA sequence to demonstrate that the surface specifically binds DNA and the DNA-protein complex is stable through sample preparation steps. In the final stage, we confirm the real world applicability by using the hTERT promoter and nuclear extract to enrich for TFs.

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Keywords
MALDI-TOF-MS, mass spectrometry, proteomics, transcription factors
Citation
Department
Chemistry