Compositions and methods for enrichment and detection of ubiquitin and ubiquitin conjugates
Inventors
ZHANG, Mengwen • Berk, Jason • Hochstrasser, Mark
Assignees
Publication Number
US-12320815-B2
Publication Date
2025-06-03
Expiration Date
2040-10-01
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Abstract
Described herein are compositions and methods for detecting the presence of ubiquitination in a sample. The compositions include synthetic peptides containing a high affinity ubiquitin binding domain and an additional peptide sequence that can be coupled to materials such as resins and dyes.
Core Innovation
The present invention provides compositions and methods for detecting the presence of ubiquitination in a sample. The core of the invention is a synthetic peptide containing a high-affinity ubiquitin-binding domain (OtUBD), derived from an effector protein of the bacterium Orientia tsutsugamushi, and an additional peptide sequence or synthetic linker. These ubiquitin-binding peptides can be coupled to materials such as resins or dyes, allowing their application in enrichment and detection protocols.
The problem addressed by this invention arises from challenges in ubiquitin research: specifically, enriching monoubiquitinated substrates as well as polymers with any type of ubiquitin chain linkage, and detecting ubiquitinated proteins with high specificity and cost-effectiveness. Existing enrichment tools are engineered mainly for poly-ubiquitin chain recognition, and antibodies used for detection have notable drawbacks such as poor immune response due to ubiquitin's high sequence and structural conservation, high production costs, and limited renewability.
This invention provides ubiquitin-binding peptides with unprecedented binding affinity for mono-ubiquitin (KD in the low nanomolar range), enabling the enrichment of both mono- and polyubiquitylated proteins irrespective of chain linkage. The invention further allows for economical recombinant production, stable storage, modification (e.g., dye labeling or resin coupling), and flexibility in applications such as affinity purification and detection via various analytical methods. The compositions can be tailored by varying the peptide sequence or adding specific linkers to facilitate the desired coupling chemistry.
Claims Coverage
There are five independent inventive features disclosed in the independent claims, each focusing on compositions, reaction products, or methods involving high-affinity ubiquitin-binding peptides.
High-affinity ubiquitin-binding peptide with defined sequence and linker
An ubiquitin-binding peptide having the sequence of SEQ ID NO: 1 or SEQ ID NO: 5, with a variable N- or C-terminal segment Z which is either: - (Xaa)⁶, a sequence of six amino acids or analogs selected from a defined set (including cysteine and related thiol-containing residues as required), or - L, a chemical moiety chosen from a specifically listed group of linkers (e.g., beta-alanine, aminohexanoic acid, PEG derivatives, azido- and propargyl-containing linkers, and others as enumerated), with the requirement that at least one Xaa is a thiol- or selenium-containing residue, and the n value for the peptide extension is defined as 6.
Ubiquitin-binding peptide wherein Z is (Xaa)⁶
The peptide where the variable segment Z is explicitly (Xaa)⁶, a sequence of six amino acids or analogs from the defined group, and with at least one Xaa being cysteine or a related residue.
Ubiquitin-binding peptide with at least one cysteine in (Xaa)⁶
The peptide wherein at least one of the Xaa residues in (Xaa)⁶ is cysteine.
Reaction product of the ubiquitin-binding peptide and a resin
A composition consisting of the defined ubiquitin-binding peptide covalently attached to a resin which includes at least one reactive group capable of reacting with a sulfhydryl (SH) or selenohydryl (SeH) functionality. The resin can include agarose and/or an iodoacetyl group.
Reaction product of the ubiquitin-binding peptide and a dye
A composition of the ubiquitin-binding peptide covalently attached to a dye containing a reactive group that can react with a sulfhydryl (SH) or selenohydryl (SeH) functionality. The dye can be a cyanine type and/or utilize a maleimide reactive group.
Method for detecting ubiquitinated substances using the peptide
A method comprising the steps of: 1. Contacting a sample containing at least one ubiquitinated substance with the defined ubiquitin-binding peptide; 2. Detecting in the sample the presence of the ubiquitin-binding peptide bound to the ubiquitinated substance.
The claims broadly and specifically protect high-affinity ubiquitin-binding peptides with unique sequence and linker configurations, their covalent conjugation to resins or dyes with specified chemistries, and the use of these compositions and methods for the detection of ubiquitinated substances.
Stated Advantages
The ubiquitin-binding peptides can be economically produced as recombinant proteins with high yields.
These peptides allow detection and enrichment of both monoubiquitinated and polyubiquitinated proteins in an unbiased manner, regardless of ubiquitin linkage type.
The compositions and methods provide high specificity and sensitivity for detecting ubiquitinated proteins.
The use of synthetic peptides circumvents drawbacks of antibodies, including cost, limited supply, variable titers, and ethical concerns.
The presence of N-terminal cysteines enables facile conjugation to various detection moieties (e.g., dyes) and resins without compromising binding activity.
The peptides display high affinity for mono-ubiquitin (nanomolar KD), surpassing previously known ubiquitin-binding domains.
The peptides are stable upon storage at 4°C and can be expressed and purified in E. coli.
Documented Applications
Enrichment and detection of ubiquitinated proteins from biological samples using affinity purification and analytical techniques such as electrophoresis, far-western blotting, and mass spectrometry.
Use as a tool in ubiquitin proteomics to identify ubiquitin conjugation sites and linkage types in complex samples.
Detection of both monoubiquitinated and polyubiquitinated proteins, including those not enriched by polyubiquitin-specific tools.
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