The broad long-term goals of this project are to develop more powerful proteomics detection technology with greatly improved sensitivity, that will reveal the global patterns of biologically stimulated changes (differential detection) in protein levels, posttranslational modifications and enzyme activities simultaneously (using 6-12 channel multicolor fluorescent dye (Zdye) detection), develop ways to use the patterns of protein isoform levels to recognize changing physiological states, and to more efficiently target and obtain the variable proteins for detailed mass spectral analysis.
The specific aims and research design of the present proposal are to: (1) scale up the synthesis of amino-reactive Zdye and Z2dyes that were developed and shown feasible during the current Phase I proposal; (2) prepare and test highly water soluble Zdye saturation labels that react with protein thiols to avoid precipitation of labeled proteins (which is a problem with available thiol saturation labels for 2D gel detection), to facilitate spot picking or targeted electroelution of the proteins of each type in the saturation-labeled spots of interest, and to provide additional differential detection sensitivity, compared to Zdye amino labels (>30x total sensitivity increase compared to currently available amino labels for multicolor 2D gel detection); (3) develop and test cleavable thiol-reactive Zdyes that leave small isotopic tags behind after cleavage, which will allow quantitative mass spectral analysis of the relative amounts of each protein of interest, even when two or more proteins are present in the gel spots targeted, and demonstrate fluorescence enhanced targeted-SILAC (stable isotope labeling with amino acids in cell culture) to pin-point proteins for mass spectral analysis-- resulting in more efficient FET-SILAC experiments; (4) develop and test several new Zdyes colors for expanded multi-channel multiplexing, to enable single gel studies of global changes accompanying, for example, multi-point dose-response curves, multipoint kinetic curves, and simultaneous, global comparisons of several parallel samples; and (5) develop and test new dye chromophore frameworks for the Zdye and the Z2Dye designs that will be substantially easier and cheaper to produce, provide more affordable products to the end-user, may be feasible to use in 1D gel applications, and will embody the same design concepts proved feasible in the first or second generation Zdyes. This project has strong health-relatedness, since a large fraction of proteins and protein posttranslationally modified isoforms are not reliably detectable with current proteomics technology. Protein posttranslational modifications are at the heart of most biological mechanisms, since PTMs control protein activity, subcellular localization and protein-protein interactions. ? ?
Cellular mechanisms controlling health and disease depend primarily on proteins and a wide variety of modifications of proteins to regulate cellular activities, but current proteomics technology remains inadequate to visualize all the protein forms present and to determine all the forms that change during biological responses. This Phase II STTR proposal builds on the successful foundation of Phase I to synthesize and demonstrate a new family of different-colored, highly water soluble fluorescent dyes (Zdyes) that take greatly expanded advantage of the unique ability of multiplexed 2D gels to reveal global changes in the relative amounts of proteins and modifications of proteins. The greatly improved sensitivity and simultaneous comparison of a larger number of sample conditions, provided by the Zdye technology, will reveal the changes in the patterns of protein species that occur down to the limit of single protein copies per cell, to more clearly expose the inner workings of biological mechanisms. ? ? ?
| Maaty, Walid S; Lord, Connie I; Gripentrog, Jeannie M et al. (2013) Identification of C-terminal phosphorylation sites of N-formyl peptide receptor-1 (FPR1) in human blood neutrophils. J Biol Chem 288:27042-58 |
