The invention describes methods and reagents useful for sequencing polypeptide molecules. The method comprises affixing a polypeptide to a substrate and contacting the polypeptide with a plurality of probes. Each probe selectively binds to an N-terminal amino acid or an N-terminal amino acid derivative. Probes bound to the polypeptide molecule are then identified before cleaving the N-terminal amino acid or N-terminal amino acid derivative of the polypeptide. Also provided are methods for the sequencing a plurality of polypeptide molecules in a sample and probes specific for N-terminal amino acids or N-terminal amino acid derivatives.

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

The present application relates to the field of protein sequencing, more particularly to protein profiling using massively parallel sequencing with single-molecule sensitivity. Methods, assays and reagents are provided for sequencing individual protein or polypeptide molecules. Also provided are methods and assays for the parallel sequencing of proteins or polypeptides. To this end, particular labeled probes are used that are reactive with the N-terminal amino acid of the polypeptide molecules and can be detected while still associated with the polypeptide(s).

Methods and systems for improved sample detection in mass spectroscopy are generally described. These are particularly useful, for example, for identifying a protein, a part of a protein, or a peptide when present in a low amount. In some embodiments, these can be useful to allow high-throughput proteomics studies for many samples, e.g., in series or in tandem. For example, certain embodiments are directed to novel approaches for identification of samples at the MS 1 level. In some cases, these improvements can be realized due to improvements in mass spectrometry instrumentation to better than the 1 ppm level for m/z measurements. Examples of improvements include, but are not limited to, improving internal mass standards, super-resolution peak fitting, isotopic labelling, Edman degradation and/or chromatography for proteins or peptides, and/or machine learning to predict peptide behavior, e.g., when exposed to such improvements.

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 with 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.

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.

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.

A sample is dissolved in a reagent 4 containing an organic solvent in a conversion vessel 11. A gas is supplied from a first gas supply part into the conversion vessel 11 via a reagent introduction tube 17, and thus the interior of the conversion vessel 11 is pressurized. A gas is supplied from a second gas supply part into the reagent 4 in the conversion vessel 11 via a reagent discharge tube 18, and thus gas bubbles 41 are formed in the reagent 4.

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 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.