(342b) Fluorescent Zdye Platform for Differential Covalent Labeling of Proteins for 2D and 1D Gel Analysis

A new array of fluorescent dyes (Zdyes) has been developed for differential, covalent labeling of proteins in 2D and 1D gel analysis. Zdyes contain zwitterionic charge pairs, which increase the solubility of labeled proteins at the protein's isoelectric points. The absorbance and fluorescence spectra of the Zdyes allow the use of widely available laser-excited fluorescence scanners.

The amine reactive Zdyes contain a titrable tertiary amine whose pK approximates that of the epsilon amine of lysine residues, preserving the pI of the unlabeled proteins. Zdyes containing a maleimide or a pyridine disulfide moiety react with protein thiols and can be used for saturation labeling, while increasing protein solubility for more sensitive 2D gel analysis. We are also developing Zdyes and associated chemistries to covalently bind multicolor Zdyes to phosphorylated, glycosidated, and nitrosylated sites in proteins for differential detection. Multicolor Zdyes that bind to active sites of native proteins, such as proteases and protein phosphatases, are also being developed.

The combination of high quantum yields and extinction coefficients of Zdyes with their increased solubility allows a deeper view into the proteome via 2D gels than is available with alternative fluorescent dyes. In 1D gels amine-labeled protein bands containing as little as 20pg of protein can be measured with confidence. Zdyes have been implemented in several projects. Using 2D gels the serum proteome of Type II diabetic subjects relative to control groups has been probed for differences in protein levels and post-translational modifications. The effects of infection of cultured monocytes by the bacterium Coxiella burnetii are also being studied using Zdyes. Differential protein expression in epileptic and normal human brain is also being examined. Complex protein mixtures benefit from the use of long, serial IEF to increase 2D resolution and spot count especially when the amount of protein is limiting. A new hyperspectral gel scanner allows the measurement of full fluorescent spectra for each image pixel, and provides accurate deconvolution of overlapping fluorescent dye emission spectra, accurate fluorescent background for each pixel, increased sensitivity and dynamic range.

Taken together this new Zdye platform allows highly sensitive fluorescent staining of proteins based on covalent binding to amines and thiols, and to sites of posttranslational modifications, making this platform compatible with a wide range of experimental applications.