Methods for proteomic profiling using non-natural amino acids
AssigneeCalifornia Institute of Technology
InventorsDaniela Dieterich, David Tirrell, Erin Schuman, Aaron Link
Life of this patent
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Abstract
The invention provides methods, reagents and systems for incorporating non-natural amino acids into proteins, preferably in vivo, using the endogenous protein synthesis machinery of an organism. The incorporated non-natural amino acids contain reactive groups for further chemical reagents, which may serve as a “handle” to enrich the proteins or fragments thereof in a number of uses, such as proteomic analysis, imaging of diseased tissues/cells, etc.
Claims25
A method for determining protein expression pattern in a cell or a tissue, comprising:
- (1) contacting the cell or the tissue with a non-natural amino acid comprising a first reactive group, under a condition where the non-natural amino acid is incorporated into the newly synthesized proteins of the cell or the tissue;
- (2) contacting proteins or fragments thereof from the cell or the tissue with an affinity reagent comprising a second reactive group and an affinity moiety, wherein the first and second reactive groups react to label the non-natural amino acid with the affinity reagent;
- (3) isolating the newly synthesized proteins or fragments thereof comprising the non-natural amino acid labeled with the affinity reagent via the affinity moiety;
- and,
- (4) determining protein expression pattern by identifying the isolated proteins or fragments thereof.
A method for monitoring protein expression in a cell or a tissue, comprising:
- (1) contacting the cell or the tissue with a non-natural amino acid comprising a first reactive group, under a condition where the non-natural amino acid is incorporated into the newly synthesized proteins of the cell or the tissue;
- (2) contacting the cell or the tissue with a fluorescent reagent comprising a second reactive group and a fluorescent moiety or a reagent that can be subsequently coupled to a fluorescent reagent, wherein the first and second reactive groups react to label the non-natural amino acid with the fluorescent reagent, said fluorescent reagent is substantially more fluorescent after the first and second reactive groups react;
- (3) monitoring the intensity of fluorescence in the cell or the tissue, thereby monitoring protein expression in said cell or said tissue.
A functional reagent comprising:
- (1) a reactive group for reacting with a non-natural amino acid incorporated into a protein;
- (2) an affinity moiety or a fluorescent moiety;
- and,
- (3) one or more cleavable groups situated between the reactive group and the affinity moiety or the fluorescent moiety.
Dependencies are inferred from the claim text.