26 results about "Protein sequencing" patented technology

The present invention relates to the field of biochemistry, more particularly to proteomics, more particularly to protein sequencing, even more particularly to single molecule peptide sequencing. The invention discloses means and methods for single molecule protein sequencing and / or amino acid identification using cleavage inducing agent. Said cleavage inducing agents which are not specific for one particular amino acid, cleave polypeptides step by step from the N-terminus onwards and provide information on the identity of the cleaved amino acids based on the kinetics of said reaction.

Disclosed herein are methods for preparing a biological sample for single-cell analysis which will simultaneously provide single-cell RNA, single-cell extracellular protein, single-cell cytoplasmic protein, and single-cell transcription factor readouts while preserving cellular heterogeneity. Some methods disclosed herein allow for the detection of proteins including the detection of the level of proteins and / or the detection of post translational modifications of proteins.

This application provides a method, related methods, and apparatus for processing protein sequencing data, relating to the field of protein sequencing technology. The method for processing protein sequencing data involves acquiring protein sequencing data of a target protein from a target slice, determining the frequency of each protein signal intensity value in the sequencing data, and calculating the probability of each protein signal intensity value based on the frequency; calculating a protein signal intensity threshold to classify protein signal intensity based on the protein signal intensity value and probability; and identifying sequencing sites with protein signal intensity values ​​greater than the protein signal intensity threshold as foreground sites corresponding to the target protein. Since the protein signal intensity threshold is calculated based on the protein signal intensity value and probability, using the protein signal intensity value and probability as defining factors for the protein signal intensity threshold helps to reasonably and accurately determine the threshold used to distinguish positive and negative signals, thereby facilitating more precise identification of foreground sites.

This invention belongs to the field of protein sequencing, specifically disclosing a novel solid-state sub-nanopore fabrication method and its application in protein sequencing. The technical solution involves: reliably and easily fabricating pores with a diameter less than 1 nm on a 5 nm thick pure silicon film; reconstructing the spatial structure of the pores in three dimensions at the atomic scale through transmission electron microscopy imaging and multilayer image simulation comparison; and then incorporating key geometric parameters into finite element multiphysics modeling to simulate the electric field distribution within the pores and calculate the conductivity of the sub-nanopores, thereby evaluating the application potential and performance of this fabrication method in protein single-molecule sequencing. The sensing region depth of this novel nanopore can be less than 1 nm, giving it an advantage over other similar nanopores in developing protein de novo sequencing technology with single-point residue specificity (kmer=1), providing a new approach for the single-point residue specificity detection of protein variants.

The application provides a key preparation method of bird's nest peptide and bird's nest peptide for pregnant women, and the preparation method of the bird's nest peptide comprises the following steps: soaking dry bird's nest in pure water at 0-4 DEG C for 2 h at a material-to-liquid ratio of 1:10, beating and then drying to obtain soaked bird's nest; mixing the soaked bird's nest with pure water at a mass ratio of 1:15-20, sealing and then stewing at 121-123 DEG C for 12-15 min, repeating the stewing once after cooling to room temperature to obtain fresh stewed bird's nest; carrying out in-vitro digestion experiment on the fresh stewed bird's nest to simulate the digestion process of the fresh stewed bird's nest in the gastrointestinal tract, and obtaining digested bird's nest; centrifuging the digested bird's nest to obtain supernatant, treating the supernatant, and then carrying out protein sequencing; comparing the protein sequencing with an online database, screening out a peptide segment with high matching degree and a response value greater than 10 9 The bird's nest peptide can significantly improve the proliferation rate of nerve cells, and provides a cell basis for the development of the nervous system.

The present application discloses a protein marker for early pregnancy detection of mammals, and particularly relates to a hidden marker protein serum amyloid A of early pregnancy test paper. Protein sequencing used in the present application adopts liquid chromatography-mass spectrometry (LC-MS) technology, sample correlation is compared through Venn, sample correlation, PCA and PLS-DA analysis; protein function annotation is carried out through GO, KEGG, COG and Pfam, and subcellular localization prediction is carried out; difference protein, protein set and difference metabolite are calculated and counted; correlation analysis of proteome and transcriptome is carried out, difference protein of sheep serum in early pregnancy is obtained, and a key marker protein of early pregnancy of sheep is screened, which provides a basis for unknown early pregnancy detection of sheep.

Compositions and methods for analysis of protein expression at the single cell level are described. The single cell proteomic technology described herein implements peptide barcode molecules to uniquely label all of the proteins and / or polypeptides present in a single cell, whereby all of the proteins / polypeptides (proteome) present in a single intact cell are uniquely labeled with a peptide barcode molecule in a partition. Polypeptides from a single cell can be uniquely barcoded and via protein sequencing, such as nanopore sequencing. Proteomic analysis of the protein sequencing data elucidates the entire proteome that was expressed in the single cell.

The application provides a polypeptide and protein sequencing method, a kit and application. The polypeptide sequencing method comprises the following steps: S1, attaching a target polypeptide to a nanopore; S2, introducing a recognition molecule specifically recognizing a terminal amino acid of the target polypeptide into a nanopore sequencing system; under the action of an electric field, the detection of the terminal amino acid of the target polypeptide is realized by utilizing the electric signal change generated in the binding and dissociation process of the recognition molecule and the target polypeptide. By utilizing the unique electric signal change generated in the binding and dissociation process of the recognition molecule and the terminal amino acid of the target polypeptide, the detection of a single amino acid is realized.

The invention relates to an anthrax protein nanopore system and application thereof in protein sequencing, and belongs to the technical field of nanopore detection. The invention aims to solve the problems of low flux, insufficient accuracy and limited nanopore resolution in the existing protein sequencing technology. According to the invention, an anthrax protein PA63 heptamer nanopore electrochemical detection device is constructed, an asymmetric pH buffer system is adopted, Trypsin, Calpain-1, Pancreative Elatase, CarboxyceptidaseA enzyme and the like are added step by step for polypeptide enzyme digestion, voltage is applied to drive amino acid to pass through nanopores, current signals are collected, amplitude, retardation time and frequency characteristics are analyzed, and polypeptide amino acid sequence detection is realized. The nanopore is small in opening current (fA level) and high in resolution ratio, detection accuracy and sensitivity are enhanced by combining multi-enzyme step-by-step cutting and carving, and a direct and efficient sequencing means is provided for protein function research.

The application provides a single-molecule protein sequencing device based on a quantum tunneling electrode, a method and application thereof. First, a tunneling electrode pair with a gap of less than 5 nanometers is prepared, and then an exopeptidase is bridged between the electrodes in a directional manner through asymmetric modification. When the exopeptidase cuts amino acids from the end of a substrate peptide chain, the conformational change of the exopeptidase modulates the tunneling current. The current signal is collected in real time, and the amino acid sequence can be analyzed by using a machine learning algorithm to analyze characteristic fingerprints. Based on this, in the future, the method can be developed into a label-free and real-time single-molecule protein sequencing technology, which can be used for low-abundance proteome analysis, post-translational modification identification and single-cell protein research.

The application discloses a bacillus amyloliquefaciens genome-scale metabolic network model, a construction method and application, and belongs to the field of system biology. The method comprises the following steps: firstly, a rough model of bacillus amyloliquefaciens is automatically constructed according to protein sequencing results of bacillus amyloliquefaciens in a Uniport database; secondly, the rough model is manually refined and the synthesis reaction of acetoin is added according to a literature database, a metabolic pathway diagram and a biochemical database, so as to construct a genome-scale metabolic network model; thirdly, the genome-scale metabolic network model is converted into a computer-readable mathematical model; and finally, the mathematical model is analyzed by using a flux balance analysis method, and compared with experimental values in the literature. The method has the advantages of short construction time, simple operation and high accuracy, can systematically predict the nutritional conditions for the growth of bacillus amyloliquefaciens, and provides a feasible basis for improving the yield of acetoin and industrial production.

The present invention provides methods for identifying and sequencing proteins, polypeptides, and amino acids, as well as compositions useful for them. [Solution] The present invention provides amino acid recognition molecules, such as amino acid-binding proteins and their fusion polypeptides, which differentially associate with different types of amino acids to generate detectable characteristic signatures that serve as indicators of the amino acid sequence of polypeptides. The present invention also provides amino acid recognition molecules that include a shielding element to enhance photostability during polypeptide sequencing reactions.

Described herein are techniques for detecting amino acid variants in peptides using data from a sequencing device. Sequencing data is generated from traces of light pulses output by the sequencing device from detecting light emissions by fluorescently tagged N-terminal amino acid (NAA) recognizers during sequencing of at least one peptide. The sequencing data comprising light pulse durations and inter-pulse durations between successive light pulses. The techniques generate read(s) each comprising a sequence of recognition segments indicating time periods in which fluorescently tagged NAA recognizers were binding to particular NAAs of the peptide, assign fluorescently tagged NAA recognizers to recognition segments, align read(s) to reference peptide sequences to obtain peptide alignments, and detect amino acid variants using the peptide alignments.

The present invention provides a nanopore single-molecule protein sequencer, which mainly includes a nanopore array chip system, an ultra-low current detection system and a data processing and construction system; the chip mainly includes a chip of peptide charge screening, a chip of amino acid sequence reading based on a series of specific nanopores and the like, the design principle of which is as follows: a series of biological nanopores with amino acid targeted identification are designed according to properties such as hydrophilicity and hydrophobicity, polarity, and chargeability of amino acids, and characteristic ion flow signals of amino acids forming a protein to be detected in nanopore are obtained one by one; characteristic information of a protein sequence in each of the nanopores is acquired by an arrayed ultra-low current measurement system; a standard model peptide sequence information base is used for identifying, correcting, integrating and reading amino acid sequences.

Provided herein are binding agents that selectively bind to individual monomer types (e.g., amino acid types) of a polymeric analyte (e.g., a peptide). The present disclosure provides methods for producing and, optionally, engineering binding agents. Such binding agents can be used for a variety of applications, including single-molecule peptide or protein sequencing.

The application relates to a cysteine functionalized MoS2 nanopore and application thereof in single-molecule protein and post-translational modification detection. The device realizes the regulation of the interaction between molecules in the pore, the gating state and the transport rate by constructing a peptide chain valve structure fixed by cysteine in the MoS2 nanopore and introducing positively charged and aromatic amino acids. By adjusting the composition of the peptide chain and the voltage condition, the van der Waals force, the electrostatic force and the hydrogen bond force between the protein molecule and the pore can be enhanced, the single-molecule protein can be slowly transported through the pore and the high-resolution ion current detection can be realized. The application can be used for distinguishing different amino acids, protein sequence characteristics and post-translational modification sites such as phosphorylation, and has high sensitivity, high selectivity and good scalable application prospect in protein sequencing, post-translational modification positioning and biomolecular genomics analysis.

Disclosed herein are methods for preparing a biological sample for single cell analysis that will simultaneously provide single cell RNA, single cell extracellular protein, single cell cytoplasmic protein, and single cell transcription factor readings while retaining cell heterogeneity. Some methods disclosed herein allow for detection of proteins, including detection of protein levels and / or detection of protein post-translational modifications.

Provided herein are novel amino acid binding proteins that recognize aspartate and / or glutamate in protein sequencing reactions; novel amino acid binding proteins that recognize arginine in protein sequencing reactions; and novel amino acid binding proteins that recognize glycine, alanine, and / or serine in protein sequencing reactions.

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 methods of obtaining data during a degradation process of a polypeptide, and outputting a sequence representative of the polypeptide. In some aspects, the application provides amino acid recognition molecules comprising a shielding element that enhances photostability in polypeptide sequencing reactions.

The application discloses a method for homogenizing nanopore polypeptide signal and single-molecule protein sequencing. The method captures the polypeptide by using electrophoretic force (EPF) opposite to electroosmotic flow (EOF) as driving force, and determines the sequence of the polypeptide to be detected by detecting and analyzing the electric signal generated when the polypeptide to be detected passes through the nanopore. The method of the application successfully improves the nanopore electric signal of the polypeptide by capturing the polypeptide by using electrophoretic force (EPF) and overcoming the reverse electroosmotic flow (EOF), and improves the discrimination of the nanopore analysis of the polypeptide, thereby providing a new method for polypeptide discrimination, modification identification and protein polypeptide spectrum analysis.

Provided herein are novel amino acid binding proteins that recognize aspartate and / or glutamate in protein sequencing reactions; novel amino acid binding proteins that recognize arginine in protein sequencing reactions; and novel amino acid binding proteins that recognize glycine, alanine, and / or serine in protein sequencing reactions.

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.

The present description provides methods, assays and reagents useful for sequencing proteins. Sequencing proteins in a broad sense involves observing the plausible identity and order of amino acids, which is useful for sequencing single polypeptide molecules or multiple molecules of a single polypeptide. In one aspect, the methods are useful for sequencing multiple polypeptides. The methods and reagents described herein can be useful for high resolution interrogation of the proteome and enabling ultrasensitive diagnostics critical for early detection of diseases.

The invention relates to an anti-autotomy and high-specific-activity AspN enzyme mutant as well as a coding gene and application thereof. The invention aims to solve the problems that the wild type AspN enzyme is easy to self-dissect and inactivate and is poor in stability. The mutant is obtained by mutating one or more aspartic acid sites in the 94th site, the 108th site, the 132 site, the 141 site, the 182 site and the 184 site on the basis of an amino acid sequence shown in SEQ ID NO: 1. The preferred mutations include D182N, D182A, D182V, D132N, D132Q, D184E, D94G, D108T, D141G, and a combination of D182N and D132N. Compared with a wild type, the specific activity and / or the enzyme activity retention rate of the mutant are / is remarkably improved. The stability and catalytic efficiency of the AspN enzyme are effectively improved, and the method has good application prospects in protein sequencing, polypeptide preparation and mass spectrometry.