title: Size-matched alkyne-conjugated cyanine fluorophores to identify differences in protein glycosylation.
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.
authors.affiliation: Biology Department, University of Nevada, Reno, NV, USA.
authors.forename: Amanda R
authors.initials: AR
authors.lastname: Burnham-Marusich
authors.forename: Anna M
authors.initials: AM
authors.lastname: Plechaty
authors.forename: Patricia M
authors.initials: PM
authors.lastname: Berninsone
mesh_terms: D000480:Alkynes / Q000737:chemistry*; D002232:Carbocyanines / Q000737:chemistry*; D057930:Click Chemistry / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes / Q000737:chemistry*; D006023:Glycoproteins / Q000032:analysis* / Q000737:chemistry; D006031:Glycosylation
chemical_list: D000480:Alkynes; D002232:Carbocyanines; D005456:Fluorescent Dyes; D006023:Glycoproteins
keywords: Azido-GalNAc; Click chemistry; DIGE; Glycosylation; Posttranslational modification