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Towards Visualizing Citrullinated Proteins in SDS‐PAGE Gels Using Phenylglyoxal‐Based Chemistries
Author(s) -
Thompson Kourtland A.,
Grant Jennifer E.
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.791.9
Subject(s) - citrullination , citrulline , chemistry , biochemistry , arginine , amino acid
Citrullination is the deimination of arginine, a post‐translational modification that is the root cause of rheumatoid arthritis and multiple sclerosis. The result of this modification in these diseases is an aberrant immune response to the citrullinated proteins which results in the disease phenotype. Citrullination is notoriously difficult to visualize due to the minimal mass upon modification, and the limited options for chemical derivatization of citrulline over arginine. Furthermore, amino acid specificity patterns defining protein citrullination remain obscure, although protein secondary structure maintain an influence (Olson et al., 2017, Comp. Biol. Chem., 70:107–115). The goal of this project is to design an assay system that allows visualization of citrulline‐modified proteins on an SDS‐PAGE gel, to aid in understanding of citrullination patterns within proteins and the discovery of citrullinated proteins. A model system has been designed to detect citrullinated peptides using SDS‐PAGE and related technologies. Peptides likely to be highly citrullinated by PAD 4 , like histone H3, were synthesized; citrullinated versions of peptides were also prepared to serve as positive controls. We also examined the feasibility of actively citrullinating full‐length proteins as part of the methods development process. The basis of the assay is to use PAD 4 to citrullinate peptides and proteins in an in vitro system, and then employ phenylglyoxal‐based chemistry to detect citrullination events. The design of peptides and proteins, optimization of conditions for citrullination proteins in vitro by PAD 4 , and use of SDS‐PAGE gels in visualizing citrullinated proteins, are presented. In sum, developing more direct methods for visualization of citrulline, like the SDS‐PAGE method, will facilitate identification of proteins susceptible to citrullination events. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .