Novel phenyl glyoxal probes
AssigneeThe Scripps Research Institute
InventorsPaul R. Thompson, Kevin L. Bicker, Venkataraman Subramanian
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Abstract
Novel phenyl-glyoxal based anti-citrulline probes and methods of synthesis are provided. Methods of use, such as, the development of methods for monitoring substrate citrullination over time; for identifying citrullinated proteins from cells are described.
Claims40
A phenylglyoxal derivative comprising:
- an isotopically labeled, fluorophore labeled, or biotin labeled phenylglyoxal (biotin-PG).
A method of labeling citrullinated proteins protein or fragments thereof in a biological sample, comprising:
- contacting a biological sample with an effective amount of a phenylglyoxal probe, wherein the probe is detectably labeled and the detectably labeled phenylglyoxal probe is covalently bonded to a citrullinated protein or fragments thereof in the biological sample;
- thereby,
- labeling the citrullinated protein or fragments thereof in the biological sample.
A method of determining the rate of citrullination and amount of citrullination of proteins in a biological sample, comprising:
- contacting a biological sample with an effective amount of a phenylglyoxal probe wherein the probe is detectably labeled and the detectably labeled phenylglyoxal probe covalently bonds to a citrullinated protein or fragments thereof in the biological sample;
- wherein the step of contacting is conducted under varying acidic and basic pH values, thereby selectively labeling arginine and citrulline residues of the proteins in the biological sample;
- and,
- determining the rate and amount of citrullination of proteins in the sample.
A method of identifying biomarkers of a disease comprising:
- contacting a biological sample with an effective amount of a phenylglyoxal probe wherein the probe is detectably labeled and the detectably labeled phenylglyoxal probe specifically binds to a citrullinated protein or fragments thereof, in the biological sample;
- comparing protein citrullination patterns of proteins detected in the sample;
- thereby identifying biomarkers of a disease.
A method of identifying biomarkers of a disease comprising:
- contacting a biological sample with an effective amount of a phenylglyoxal probe, wherein the probe is detectably labeled and the detectably labeled phenylglyoxal probe specifically binds via a covalent bond to a citrullinated protein or fragments thereof in the biological sample;
- thereby,
- labeling the citrullinated protein or fragments thereof in the biological sample;
- and,
- identifying a citrullination profile of citrullinated proteins or fragments thereof;
- comparing the citrullination profile of citrullinated proteins or fragments thereof in control samples as compared to a patient sample;
- thereby,
- identifying biomarkers of a disease.
A method of binding and identifying citrullinated proteins from serum or cellular extracts comprising:
- contacting a biological sample with an effective amount of an isotopically labeled, fluorophore labeled, or biotin labeled phenylglyoxal (biotin-PG), wherein the isotopically labeled phenylglyoxal compound is heavy phenylglyoxal (PGH) or light phenylglyoxal (PGL) having a general structure I:
- wherein Z is 13C for heavy phenylglyoxal (PGH), Z is 12C for light phenylglyoxal (PGL) and Y is H;
- or, a phenylglyoxal probe;
- isolating the citrullinated proteins, and
- identifying the citrullinated proteins.
A method of performing immunohistochemistry comprising contacting a cell or tissue samples with an effective amount of isotopically labeled, fluorophore labeled, or biotin labeled phenylglyoxal (biotin-PG), wherein the isotopically labeled phenylglyoxal compound is heavy phenylglyoxal (PGH) or light phenylglyoxal (PGL) having a general structure I:
- wherein Z is 13C for heavy phenylglyoxal (PGH), Z is 12C for light phenylglyoxal (PGL) and Y is H;
- and,
- determining distribution and localization of citrullinated proteins within cell and tissue samples.
A method of performing sandwich ELISAs comprising contacting serum or plasma samples with an effective amount of a isotopically labeled, fluorophore labeled, or biotin labeled phenylglyoxal (biotin-PG), wherein the isotopically labeled phenylglyoxal compound is heavy phenylglyoxal (PGH) or light phenylglyoxal (PGL) having a general structure I:
- wherein Z is 13C for heavy phenylglyoxal (PGH), Z is 12C for light phenylglyoxal (PGL) and Y is H;
- or, a phenylglyoxal probe;
- and, determining levels of citrullinated proteins within serum or plasma samples.
A biomarker for diagnosis of ulcerative colitis comprising ApoA1, mutants, variants, or fragments thereof.
Dependencies are inferred from the claim text.