Application of multiplexed cysteine-labeled complex protein sample for 2D electrophoretic gel alignment.

The analysis of cellular subproteomes by 2DE is hampered by the difficulty of aligning gel images from samples that have very different protein composition. Here, we present a sensitive and cost-effective fluorescent labeling method for analyzing protein samples that is not dependent on their composition. The alignment is guided by inclusion of a complex mixture of proteins that is co-run with the sample. Maleimide-conjugated fluorescent dyes Dy-560 and Dy-635 are used to label the cysteine residues of the sample of interest and the alignment standard, respectively. The two differently labeled mixtures are then combined and separated on a 2D gel and, after selective fluorescence detection, an unsupervised image registration process is used to align the protein patters. In a pilot study, this protocol significantly improved the accuracy of alignment of nuclear proteins with total cellular proteins.