Identifying proteins and peptides, and more specifically large-scale sequencing of single peptides in a mixture of diverse peptides at the single molecule level is an unmet challenge in the field of protein sequencing. Herein are methods for identifying amino acids in peptides, including peptides comprising unnatural amino acids. In one embodiment, the N-terminal amino acid is labeled with a first label and an internal amino acid is labeled with a second label. In some embodiments, the labels are fluorescent labels. In other embodiments, the internal amino acid is Lysine. In other embodiments, amino acids in peptides are identified based on the fluorescent signature for each peptide at the single molecule level.

In some aspects, the invention comprises a method for determining an amino acid sequence or fingerprint of a polypeptide. In some embodiments, the method comprises providing a solid state substrate comprising a cis side and a trans side, the substrate comprising a reaction well that defines a reaction volume and comprises (i) a proximal throughhole extending between the cis side and the trans side of the substrate, (ii) one or more side walls, and (iii) a distal opening. The solid state substrate further comprises an opaque metal layer that substantially blocks excitation light from penetrating into the reaction volume and from penetrating to the cis side of the substrate. Also provided is a carrier particle comprising a fluorescently labeled polypeptide strand that is attached to the carrier particle. The fluorescently labeled polypeptide strand comprises (i) a proximal end that is attached to the carrier particle, (ii) a distal end that is cleavable by an exopeptidase, and (iii) at least one fluorescently labeled amino acid comprising a fluorescent label. The carrier particle is located on the cis side of the substrate, but does not pass through the throughhole, such that the attached fluorescently labeled polypeptide strand protrudes through the throughhole so that the distal end of the fluorescently labeled strand is in the reaction volume. The trans side of the substrate is illuminated with excitation light to create a fluorescence excitation zone adjacent to the distal opening of the reaction well. While the substrate is illuminated, the fluorescently labeled polypeptide strand is reacted with an exopeptidase so that amino acids are released serially from the distal end of the stand and diffuse through the fluorescence excitation zone, so that fluorescently labeled amino acids in the excitation zone emit fluorescent signals. The fluorescent signals are detected as a function of time, whereby an amino acid sequence is determined from the time order of fluorescent signals detected from the released fluorescently labeled amino acids.

Provided herein are methods and systems for sequencing proteins. One or more methods disclosed herein may use linkers comprising an amino acid-reactive group and an additional reactive moiety that may be used to couple a polymerizable molecule. The linker may couple to a polymerizable molecule and an amino acid of a peptide and a capture moiety via the polymerizable molecule, followed by cleavage of the amino acid from the peptide. Further processing and analysis may be conducted using, for example, nanopores or nanogaps.

Provided herein are methods and systems for sequencing proteins. One or more methods disclosed herein may use linkers comprising an amino acid-reactive group and an additional reactive moiety that may be used to couple a polymerizable molecule. The linker may couple to a polymerizable molecule and an amino acid of a peptide and a capture moiety via the polymerizable molecule, followed by cleavage of the amino acid from the peptide. Further processing and analysis may be conducted using, for example, nanopores or nanogaps.

Methods for nanopore-based protein analysis are provided. The methods address the characterization of a target protein analyte, which has a dimension greater than an internal diameter of the nanopore tunnel, and which is also physically associated with a polymer. The methods further comprise applying an electrical potential to the nanopore system to cause the polymer to interact with the nanopore tunnel. The ion current through the nanopore is measured to provide a current pattern reflective of the structure of the portion of the polymer interacting with the nanopore tunnel. This is used as a metric for characterizing the associated protein that does not pass through the nanopore.

An object of the present invention is to provide a method for efficiently identifying a polyubiquitinated substrate which is generally not easily identified. The method for identifying a polyubiquitinated substrate includes (1) a step of expressing a trypsin-resistant polyubiquitin chain-binding protein and a ubiquitin ligase in a cell, (2) a step of isolating a complex that contains the trypsin-resistant polyubiquitin chain-binding protein from the cell having undergone the step (1), (3) a step of subjecting the complex isolated by the step (2) to trypsin digestion, and (4) a step of identifying a peptide that has a ubiquitination site from a digested material obtained by the step (3).

The present invention is a method for measuring the amount of at least one molecule in a biological sample, the method comprising a) combining the sample, or a derivative thereof, with one or more aptamers and allowing one or more molecules in the sample to bind to the aptamer(s); b) separating bound from unbound molecules; and c) quantifying the molecule(s) bound to the or each aptamer, wherein quantification of the bound molecule(s) is carried out by sequencing at least part of the or each aptamer. Uses of and products derived from the method are also contemplated.

Provided herein are methods of single-cell polypeptide and/or polynucleic acid sequencing, which facilitate the direct sequencing of a single cell without amplification. Also provided herein are compositions, kits and devices useful for the same.

The invention further relates to a process for the enzymatic synthesis of an (oligo)peptide. The invention relates to a method for designing an enzymatic synthesis process of an (oligo)peptide, comprising identifying two or more (oligo)peptide fragments of an (oligo)peptide, which fragments are (oligo)peptides suitable for preparing the (oligo)peptide by enzymatic condensation of the two or more peptide fragments using a ligase. The invention relates to a method for designing an enzymatic synthesis process of a cyclic (oligo)peptide, comprising identifying a non-cyclic (oligo)peptide from which the cyclic (oligo)peptide can be prepared by cyclisation, catalysed by a cyclase. The invention further relates to a process for the enzymatic synthesis of an (oligo)peptide.

This invention relates to the in vitro production of cyclic peptides using cyanobacterial enzymes, such as patellamide biosynthesis enzymes. Linear peptide substrates are cyclized using an isolated cyanbacterial macrocyclase, such as PatG from Prochloron spp. Before cyclisation, residues in the linear peptide substrates may be heterocyclised using isolated cyanbacterial heterocyclasses, such as PatD or TruD heterocyclase. Methods of the invention may be useful, for example, for the production of cyclic peptidyl molecules, including cyclotides, such as katalas, and cyanobactins, such as patellamides and telomestatins, for example for use in the development of therapeutics.