SGER: New Fluorescent Dyes for Multiplex Detection in Proteomics

Montana State University, Bozeman MT

Investigators

Abstract

Quantitative methods for the display and analysis of a complex mixture of proteins are needed to analyze changes in the proteome. Two-dimensional gels are capable of separating a large number of proteins and permit visualization of patterns of protein expression and post-translational modification (PTM) under different physiological conditions--but suffer from gel-to-gel reproducibility problems. The best post-separation protein staining methods are able to detect proteins down to the range of about 300-1,000 copies per cell, a level insufficient to monitor many important cellular control proteins that are present in low copy numbers. New fluorescent dyes that are designed to overcome major limitations in proteomic methods, as commonly practiced using two-dimensional gel electrophoresis, will be synthesized and tested in this project. The new dyes are designed to (1) enhance sensitivity about 300 fold to allow detection of proteins in the range of 1-3 copies per cell, (2) increase the solubility of dye-labeled proteins for increasing loading capacity and reduction of gel artifacts to foster improved protein identification by mass spectrometry, (3) allow multicolor detection of proteins produced by cells under different physiological conditions, and (4) permit removal of dyes from labeled proteins to enhance protease cleavage and improve identification of proteins by mass spectrometry. The novel, ultrasensitive, multiplex proteomics methods under development promise to be very widely useful in biological research and education.