Enhanced Sensitivity Employing Zwitterionic and pI Balancing Dyes (Z-CyDyes) Optimized for 2D-Gel Electrophoresis Based on Side Chain Modifications of CyDye Fluorophores. New Tools For Use in Proteomics and Diagnostics

Abstract

The CyDye family of fluorescent dyes is currently the overwhelming choice for applications in proteomic analysis, using two-dimensional difference gel electrophoresis (2D-DIGE). Protein labeling with CyDyes is hampered by protein precipitation and gel smearing when used above minimal labeling. The solubility of labeled protein may be improved by introducing water solubilizing groups on the dye such as cysteic acids. However, addition of a negatively charged functionality will have the undesired effect of shifting the pI in relation to the unlabeled protein. These limitations have been addressed through the synthesis of highly water-soluble and pI balancing zwitterionic CyDye fluorophores (Z-CyDyes). The new dyes feature a cysteic acid motif, a titratable amine functionality and a NHS activated ester group. In side by side 2D-DIGE comparisons of Z-CyDyes and CyDyes, the new dyes significantly enhanced protein spot volume and the number of spots that were detected. Z-CyDyes have the potential to enhance the depth of proteome coverage and provide a general strategy for improving the performance of protein tagging reagents.

Intensive herbicide use has selected for constitutively elevated levels of stress‐responsive mRNAs and proteins in multiple herbicide‐resistant Avena fatua L

“…Two‐dimensional fluorescence difference gel electrophoresis (2‐D DIGE) was initiated by minimal fluorescent labeling of lysine side‐chains (1 per 100) with N ‐hydroxysuccinimide ester cyanine dyes (Z‐CyDyes Z‐Cy3, Z‐Cy5 and Z‐Cy2, according to the manufacturer's protocol (GE Healthcare Bio‐Sciences Corp., Piscataway, NY)). Briefly, extracts containing 50 µg of protein were labeled separately on ice with 400 pmol of either Z‐Cy3 or Z‐Cy5 dissolved in DMF.…”

Proteomic and biochemical assays of glutathione-related proteins in susceptible and multiple herbicide resistant Avena fatua L.

“…Two-dimensional difference gel electrophoresis (2D-DIGE) was initiated by minimal fluorescent labeling of lysine side-chains (1 per 100) with N-hydroxysuccinimide ester cyanine dyes (Z-CyDyes; Z-Cy3, Z-Cy5, Z-Cy2; [21]) according to the manufacturer's protocol (GE Healthcare Bio-Sciences Corp. Piscataway, NY). Briefly, GSH affinity chromatography extracts containing 50 μg protein, were labeled separately on ice with 400 pmol of either Z-Cy3 or Z-Cy5 Cydye DIGE fluors dissolved in DMF.…”

“…Of these, five proteins were detected at higher levels in MHR4 plants, including three tau class GSTs (OsGSTU1, OsGSTU5, OsGSTU27), one phi class GST (OsGSTF3), and an unclassified GST (Table 1). In addition to GSTs, eight other protein spots (13,17,(19)(20)(21)(22)(23)30) corresponding to five unique proteins were identified as GSH-binding proteins from non-plant systems (Table 1). DIGE gels also revealed 17 differential spots corresponding to 14 unique proteins with no known GSH-binding activity in plants (Table A.1).…”

Discovery of plasma protein biomarkers related to Alzheimer’s disease, sex and APOE genotype

“…We also employed a pooling strategy to incorporate a larger population in the study to reduce effects of individual variability and to emphasize changes that are related to disease (39,40). Using recently developed Zdyes (41,42) for covalent labeling and differential in-gel comparison of AD and control plasma proteins, we conducted a proteomic screen of protein isoforms in samples obtain from the Australian imaging and biomarker lifestyle study of ageing (AIBL). We have confirmed several previously observed findings (23,24) and report additional plasma protein signatures of AD that vary with APOE ε4 gene dose and sex.…”