Methods for proteome labeling
AssigneesThe Board of Trustees of the Leland Stanford Junior University, The United States Government as represented by the Department of Veterans Affairs
InventorsAndrew Yang, Anton Wyss-Coray, Kyle Brewer
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Abstract
Provided herein are methods for labeling the proteomes of cells, as well as methods for labeling proteins or populations of proteins produced by cells. In some embodiments, the methods comprise introducing variant aminoacyl-tRNA synthetases and noncanonical amino acids into cells. Also provided herein are polynucleotides encoding variant aminoacyl-tRNA synthetases that recognize noncanonical amino acids. The methods and compositions provided herein are useful for, among other things, identifying target cells and identifying biomarkers of interest.
Claims18
A method for labeling a proteome of a cell or a portion of the proteome of the cell, the method comprising:
- (a) introducing into the cell two or more variant aminoacyl-tRNA synthetases that recognize one or more noncanonical amino acids, wherein the two or more variant aminoacyl-tRNA synthetases comprise two or more of ScTyrY43G of SEQ ID NO:4, the MmPheT413G of SEQ ID NO:5, HsPheT413G of SEQ ID NO:6, and wherein the one or more noncanonical amino acids comprises 3-azido-L tyrosine (AzY), p-azido-L-phenylalanine (AzF), or a combination thereof;
- (b) subsequently exposing the cell to the one or more noncanonical amino acids, thereby integrating the one or more noncanonical amino acids into the proteome or the portion of the proteome, and producing a modified proteome;
- and
- (c) contacting the modified proteome with a detectable moiety, thereby producing a labeled proteome.
A method for labeling a protein or a portion of proteins produced by a cell, the method comprising:
- (a) introducing into the cell two or more variant aminoacyl-tRNA synthetases that recognize one or more noncanonical amino acids, wherein the two or more variant aminoacyl-tRNA synthetases comprise two or more of ScTyrY43G of SEQ ID NO:4, the MmPheT413G of SEQ ID NO:5, and HsPheT413G of SEQ ID NO:6, and wherein the one or more noncanonical amino acids comprise 3-azido-L tyrosine (AzY), p-azido-L-phenylalanine (AzF), or a combination thereof;
- (b) subsequently exposing the cell to the one or more noncanonical amino acids, thereby integrating the one or more noncanonical amino acids into the protein or population of proteins, and producing a modified protein or population of modified proteins;
- and
- (c) contacting the modified protein or population of modified proteins with a detectable moiety, thereby producing a labeled protein or population of labeled proteins.
An isolated polynucleotide sequence that encodes a polypeptide with an amino acid sequence that is at least 85% identical to any one of MmPheT413G of SEQ ID NO:5 or HsPheT413G of SEQ ID NO:6.
A cell comprising a polynucleotide that encodes a polypeptide with an amino acid sequence that is at least 85% identical to any one of MmPheT413G of SEQ ID NO:5 or HsPheT413G of SEQ ID NO:6.
Dependencies are inferred from the claim text.