Methods for proteome labeling
Inventors
Yang, Andrew • Wyss-Coray, Anton • BREWER, Kyle
Assignees
US Department of Veterans Affairs • Leland Stanford Junior University
Publication Number
US-11921118-B2
Publication Date
2024-03-05
Expiration Date
2039-05-09
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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.
Core Innovation
The invention provides methods for labeling the proteomes of cells, including proteins or populations of proteins produced by cells, by introducing variant aminoacyl-tRNA synthetases that recognize noncanonical amino acids into cells. These variant synthetases activate tRNAs with noncanonical amino acids, which are then integrated into the proteome, producing a modified proteome that can be contacted with a detectable moiety to yield a labeled proteome.
Existing approaches for cell-type-specific proteome characterization rely on cell isolation or culture techniques that can perturb native proteomes, lose secretomes, and lack temporal resolution. The invention addresses the need for improved tools and methods enabling cell-type-specific and temporal labeling of proteomes and secretomes in vivo, facilitating robust and comprehensive proteomic analysis without requiring exogenous tRNAs or depletion of canonical amino acids.
Claims Coverage
The patent claims include independent claims covering methods for labeling proteomes or proteins with two or more variant aminoacyl-tRNA synthetases recognizing noncanonical amino acids, as well as corresponding compositions such as polynucleotides encoding these synthetases and cells comprising such polynucleotides.
Labeling proteomes with multiple variant aminoacyl-tRNA synthetases
A method comprising introducing into a cell two or more variant aminoacyl-tRNA synthetases (comprising ScTyrY43G, MmPheT413G, HsPheT413G) that recognize noncanonical amino acids such as 3-azido-L-tyrosine (AzY) and p-azido-L-phenylalanine (AzF), exposing the cell to these amino acids, thereby incorporating them into the proteome, and contacting the modified proteome with a detectable moiety to produce a labeled proteome.
Labeling proteins produced by cells with multiple variant synthetases
A method for labeling proteins or populations of proteins by introducing multiple variant aminoacyl-tRNA synthetases (e.g., ScTyrY43G, MmPheT413G, HsPheT413G), exposing cells to corresponding noncanonical amino acids, integrating them into proteins, and contacting with a detectable moiety to produce labeled proteins.
Encoding variant aminoacyl-tRNA synthetases in isolated polynucleotides
Isolated polynucleotides encoding variant aminoacyl-tRNA synthetases having amino acid sequences at least 85% identical to MmPheT413G or HsPheT413G, optionally codon-optimized, and capable of preferentially activating tRNAs with noncanonical amino acids.
Cells comprising polynucleotides encoding variant synthetases
Cells comprising polynucleotides encoding variant aminoacyl-tRNA synthetases with sequences at least 85% identical to MmPheT413G or HsPheT413G, optionally comprising noncanonical amino acids and detectable moieties for labeling.
Methods for identifying target cells and biomarkers
Methods comprising labeling target and reference cells with proteomes or proteins labeled via the described methods, detecting labeled proteins to generate signatures, comparing signatures, and identifying target cells or biomarkers based on differences.
The claims broadly cover methods of cell and protein labeling using multiple variant aminoacyl-tRNA synthetases recognizing specific noncanonical amino acids, compositions encoding these synthetases, cells expressing them, and applications in identifying target cells and biomarkers with improved proteome labeling specificity and coverage.
Stated Advantages
Enables robust and broader mammalian cell-type-specific proteome labeling and proteomic studies.
Uses endogenous tRNAs, removing the need for strong co-expression of exogenous tRNAs.
Allows noncanonical amino acid incorporation at non-methionine codons, expanding proteome coverage beyond methionine.
Does not require depletion of canonical amino acids, facilitating less perturbative labeling.
Permits delivery of variant aminoacyl-tRNA synthetases to tissues such as liver and brain in vivo, enabling detection of tissue-derived tagged proteins in samples like blood.
Co-expression of multiple variant synthetases enhances proteome coverage and detection confidence.
Documented Applications
Identifying target cells by labeling their proteomes or proteins and comparing to reference cells.
Identifying biomarkers of interest in target cells by detecting differences in labeled proteomes or proteins compared to reference cells.
Cell-type-specific and temporal labeling of proteomes and secretomes in vivo.
Studying proteomes and secretomes in disease contexts including cancer (e.g., melanoma), neurological diseases, immune diseases, autoimmune diseases, inflammatory diseases, and metabolic diseases.
Labeling proteomes from specific mammalian cells for cataloguing, tracking, and modulation.
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