Abstract: Provided herein are modified dipeptide cleavases for removing amino acid(s) from peptides, polypeptides, and proteins. Also provided are methods of using the modified dipeptide cleavases for treating polypeptides, and kits comprising the modified dipeptide cleavase. In some embodiments, the methods and the kits also include other components for macromolecule sequencing and/or analysis.

Abstract: The present disclosure relates to a selective and efficient method to connect a polypeptide containing at least one lysine residue to a cargo moiety, where one step of the method involves reaction of a semicarbazide group with an ortho-acylphenyl boronic acid, forming a cyclic diazaborine ring fused to phenyl. Conditions for formation of the cyclic diazaborine are sufficiently mild for the method to be used in the presence of sensitive biomolecules such as polynucleotides. A substituent group on the phenyl can be used to link the cyclic diazaborine to a cargo moiety such as a polynucleotide, bead, or reactive group, providing a polypeptide—cargo moiety conjugate that is useful for various purposes, such as to analyze, identify, track, locate, detect, or immobilize the polypeptide. Also provided are polypeptide—cargo moiety conjugates, wherein the polypeptide and cargo moiety are connected via a linker that comprises a cyclic diazaborine.

Abstract: Provided herein are methods for modifying an arginine residue to provide a reactive handle suitable for attaching the arginine residue to a label, linker, or other target molecule. The methods utilize an Arnold Salt to convert the guanidine group of the arginine residue into a formylpyrimidine, and are efficient and very selective. Provided herein are mild reaction conditions for this arginine modification, making it suitable for use with complex biomolecules like proteins. The methods can include one or more additional steps to attach the modified arginine to a linker for further manipulation or for connecting the arginine residue to a target compound. Provided methods are especially useful for covalently linking an arginine-containing peptide to a target molecule such as a polynucleotide.

Abstract: Provided herein are modified cleavases for removing amino acids from peptides, polypeptides, and proteins. Also provided are methods of using the modified cleavases for treating polypeptides, and kits comprising the modified cleavase. In some embodiments, the methods and the kits also include other components for macromolecule sequencing and/or analysis.

Abstract: The present invention relates to methods to use cyclohexan-1,2-dione (CHD) groups to attach labels, linkers, and other molecules to a target compound comprising a CHD-reactive group such as a guanidine, amidine, urea, thiourea and the like. Methods of the invention include milder conditions than those previously known for promoting reaction of CHD with CHD-reactive groups, which makes the methods suitable for use with base-sensitive compounds and complex biomolecules. Methods of the invention are especially useful for attaching linking and labeling groups to a peptide that comprises at least one arginine residue, and can also be used to link such peptides to other target molecules such as nucleic acids. The invention also provides CHD-containing conjugation reagents and compositions comprising CHD-containing intermediates, and precursors useful for making CHD-containing compounds that can be used in the methods of the invention.

Abstract: Provided herein are modified cleavases for removing amino acids from peptides, polypeptides, and proteins. Also provided are methods of using the modified cleavases for treating polypeptides, and kits comprising the modified cleavase. In some embodiments, the methods and the kits also include other components for macromolecule sequencing and/or analysis.

Abstract: The present disclosure relates to a binder that specifically binds to an N-terminally modified polypeptide through interaction with a modified N-terminal amino acid. Also provided herein is a method and related kits for treating a polypeptide using or comprising the binder and/or modified cleavase. In some embodiments, also provided herein is a method and related kits for transferring information using a plurality of enzymes, including for performing a ligation, extension, and cleavage reaction with nucleic acid molecules associated with the polypeptide for analysis.

Abstract: The present disclosure relates to methods and kits for high-throughput, highly parallel polypeptide identification employing labeling of specific amino acid residues, barcoding and nucleic acid encoding of the labeled residues. The workflow and architecture described herein allow identification of polypeptides in a sample in a cyclic manner based on encoding of their specific amino acid residues and without use of protein-based or aptamer-based binding agents. Successful “binder-free” encoding takes advantage of the high affinity and specificity of nucleic acid tags, as well as the specific chemistry of certain amino acid side chains.

Abstract: The present invention relates to methods to use cyclohexan-1,2-dione (CHD) groups to attach labels, linkers, and other molecules to a target compound comprising a CHD-reactive group such as a guanidine, amidine, urea, thiourea and the like. Methods of the invention include milder conditions than those previously known for promoting reaction of CHD with CHD-reactive groups, which makes the methods suitable for use with base-sensitive compounds and complex biomolecules. Methods of the invention are especially useful for attaching linking and labeling groups to a peptide that comprises at least one arginine residue, and can also be used to link such peptides to other target molecules such as nucleic acids. The invention also provides CHD-containing conjugation reagents and compositions comprising CHD-containing intermediates, and precursors useful for making CHD-containing compounds that can be used in the methods of the invention.

Abstract: Provided herein are modified cleavases for removing amino acids from peptides, polypeptides, and proteins. Also provided are methods of using the modified cleavases for treating polypeptides, and kits comprising the modified cleavase. In some embodiments, the methods and the kits also include other components for macromolecule sequencing and/or analysis.

Abstract: The present disclosure relates to methods and kits for performing an identification of a terminal amino acid residue of the polypeptide, or performing a polypeptide sequencing. The methods include a step of contacting the terminal amino acid residue of the polypeptide with a coupler, followed by attaching the coupler-polypeptide complex to the solid support and cleaving the coupler-polypeptide complex from the polypeptide, thereby isolating the terminal amino acid residue of the polypeptide from the remaining amino acid residues of the polypeptide in complex with the coupler, thereby enabling efficient identification of the terminal amino acid residue via recognition by binding agents capable of binding to the coupler-amino acid complex.

Abstract: The present disclosure relates to a metalloprotein binder that specifically binds to a N-terminally modified peptide. Also provided herein is a method and related kits for treating or analyzing a peptide using the metalloprotein binder and/or modified cleavase. In some embodiments, the method provided herein comprises binding metalloprotein binder-coding tag conjugates to a modified N-terminal amino acid residue of an immobilized peptide associated with a recording tag, transferring identifying information from the coding tag to the recording tag using a ligation or primer extension, and cleaving the modified N-terminal amino acid residue. The method and metalloprotein binders provided herein are useful for de novo peptide identification or sequencing.

Abstract: Provided herein are modified cleavases for removing amino acids from peptides, polypeptides, and proteins. Also provided are methods of using the modified cleavases for treating polypeptides, and kits comprising the modified cleavase. In some embodiments, the methods and the kits also include other components for macromolecule sequencing and/or analysis.

Abstract: The present invention relates to methods of cleaving the N-terminal amino acid from a polypeptide, which may be in free form or conjugated to a carrier or surface, such as a bead. It provides methods to activate the N-terminal amine of a polypeptide to promote formation of a cyclic adduct of the N-terminal amino acid, resulting in cleavage of the N-terminal amino acid from the polypeptide. The method can be used to sequence and/or analyze a polypeptide. For example, the methods can be combined with methods described herein for sequencing and/or analysis that employ barcoding and nucleic acid encoding of molecular recognition events, and/or detectable labels. The invention also provides compounds and kits useful for practicing these methods.

Abstract: The present invention relates to methods to use cyclohexan-1,2-dione (CHD) groups to attach labels, linkers, and other molecules to a target compound comprising a CHD-reactive group such as a guanidine, amidine, urea, thiourea and the like. Methods of the invention include milder conditions than those previously known for promoting reaction of CHD with CHD-reactive groups, which makes the methods suitable for use with base-sensitive compounds and complex biomolecules. Methods of the invention are especially useful for attaching linking and labeling groups to a peptide that comprises at least one arginine residue, and can also be used to link such peptides to other target molecules such as nucleic acids. The invention also provides CHD-containing conjugation reagents and compositions comprising CHD-containing intermediates, and precursors useful for making CHD-containing compounds that can be used in the methods of the invention.

Abstract: The present disclosure relates to methods of accelerating reactions involving macromolecules, e.g., peptides, polypeptides, and proteins for sequencing and/or analysis. In some embodiments, the methods include the application of radiation, e.g., electromagnetic radiation or microwave energy. In some embodiments, the methods and uses are for modifying a polypeptide or a plurality of polypeptides (e.g., peptides and proteins) for sequencing and/or analysis that employ barcoding and nucleic acid encoding of molecular recognition events, and/or detectable labels.

Abstract: Provided herein are modified cleavases for removing amino acids from peptides, polypeptides, and proteins. Also provided are methods of using the modified cleavases for treating polypeptides, and kits comprising the modified cleavase. In some embodiments, the methods and the kits also include other components for macromolecule sequencing and/or analysis.