Size‐matched alkyne‐conjugated cyanine fluorophores to identify differences in protein glycosylation

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.