Size‐matched alkyne‐conjugated cyanine fluorophores to identify differences in protein glycosylation
Year: 2014
Type: article
Abstract: Currently, there are few methods to detect differences in posttranslational modifications (PTMs) in a specific manner from complex mixtures. Thus, we developed an approach that combines the sensitivity and specificity of click chemistry with the resolution capabilities of 2D-DIGE. In "Click-DIGE", posttranslationally modified proteins are metabolically labeled with azido-substrate analogs, then size- and charge-matched alkyne-Cy3 or alkyne-Cy5 dyes are covalently attached to the azide of the PTM by click chemistry. The fluorescently-tagged protein samples are then multiplexed for 2DE analysis. Whereas standard DIGE labels all proteins, Click-DIGE focuses the analysis of protein differences to a targeted subset of posttranslationally modified proteins within a complex sample (i.e. specific labeling and analysis of azido glycoproteins within a cell lysate). Our data indicate that (i) Click-DIGE specifically labels azido proteins, (ii) the resulting Cy-protein conjugates are spectrally distinct, and (iii) the conjugates are size- and charge-matched at the level of 2DE. We demonstrate the utility of this approach by detecting multiple differentially expressed glycoproteins between a mutant cell line defective in UDP-galactose transport and the parental cell line. We anticipate that the diversity of azido substrates already available will enable Click-DIGE to be compatible with analysis of a wide range of PTMs.
Source: Electrophoresis
Institution: University of Nevada, Reno
Language: English
Subfield: Spectroscopy
Field: Chemistry
Domain: Physical Sciences
Open Access status: green
Awards: G1954737764, Cell Biology of Signaling across Membranes, Acquisition of a Hybrid LC/MS/MS to Enhance Research in Nevada, IDeA Network for Biomedical Research Excellence, G7571187106, Cell Biology of Signaling across Membranes, G9000171104, Nevada IDeA Networks of Biomedical Research Excellence (INBRE)
OpenAlex ID: W1498694213