A method for analyzing macromolecules, including peptides, polypeptides, and proteins, employing nucleic acid encoding is disclosed.

Aspects of this disclosure related to methods, articles, kits, and/or systems for the preparation and/or study of one or more target molecules in a sample. In some embodiments, a target molecule is a peptide, a protein, or a fragment or derivative thereof. Through the use of methods, articles, kits, and/or systems of the instant disclosure, target molecules may, in some embodiment, be more readily sequenced or prepared for sequencing.

The present disclosure relates to methods and kits for analysis of peptides, polypeptides and proteins, employing a multi-component detection agent(s). In some embodiments, the method is useful for identifying the terminal amino acid of the peptide. In some embodiments, the multi-component detection agent includes a first detection agent and second detection agent which, when in proximity, is capable of generating a detectable signal.

The current disclosure provides a transformative concept based on nanopore technology, Sequencing-by-Hydrolysis, to identify the N-terminal amino acid and the length of each peptide fragment in a peptide ladder to reconstitute the sequence of a protein: a protein/peptide analyte will be nonspecifically hydrolyzed to generate random fragments of the analyte that are different by one amino acid with the N-terminal amino acid of each fragment modified so it generates a distinguishable fingerprint signal when tested by nanopore. The length of the fragment can be estimated by characterizing its translocation signal to back calculate the location of the amino acid in the original analyte. This approach will significantly advance the nanopore technology with single amino acid resolution for protein/peptide sequencing.

The invention, in part, includes methods of single molecule protein sequencing that include using weak binding spectra in the amino acid identification.

The present invention relates to methods for identifying amino acids in peptides. In one embodiment, the present invention contemplates labeling the N-terminal amino acid with a first label and labeling an internal amino acid 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.

Described are optical methods and reagents for sequencing polypeptides. A probe that exhibits different spectral properties when conjugated to different N-terminal amino acids is conjugated to the N-terminal amino acid of a polypeptide. Sequentially detecting one or more spectral properties of the probe conjugated to the N-terminal amino acid and cleaving the N-terminal amino acid produces sequence information of the polypeptide. The use of super-resolution microscopy allows for the massively parallel sequencing of individual polypeptide molecules in situ such as within a cell. Also described are probes comprising hydroxymethyl rhodamine green, an isothiocyanate group and a protecting group.

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.

Aspects of the application provide methods of identifying and sequencing proteins, polypeptides, and amino acids, and compositions useful for the same. In some aspects, the application provides amino acid recognition molecules, such as amino acid binding proteins and fusion polypeptides thereof. In some aspects, the application provides amino acid recognition molecules comprising a shielding element that enhances photostability in polypeptide sequencing reactions.

Provided herein are rapid and reversible methods to non-specifically immobilize peptides and proteins irrespective of their sequence, as well as small molecules, on a solid support to allow for manipulations of and reactions with these molecules in a manner that does not require purification between steps, which increases sample yield and reduces the quantity of starting material required.