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

Autor: Burnham-Marusich AR; Biology Department, University of Nevada, Reno, NV, USA., Plechaty AM, Berninsone PM
Jazyk: angličtina
Zdroj: Electrophoresis [Electrophoresis] 2014 Sep; Vol. 35 (18), pp. 2621-5. Date of Electronic Publication: 2014 Jul 24.
DOI: 10.1002/elps.201400241
Abstrakt: 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.
(© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
Databáze: MEDLINE
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