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Qualitative analysis of proteins

A qualitative, protein-based technique used in the characterization and analysis of proteins

Pending Publication Date: 2020-12-01
菲奈克瑟斯公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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  • Qualitative analysis of proteins
  • Qualitative analysis of proteins
  • Qualitative analysis of proteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0114] Example 1. Protein Analysis - Example Workflow for Biologics Characterization

[0115] 1. Purification of Proteins by Affinity Chromatography

[0116] 2. Buffer Exchange by Gel Filtration

[0117] 3. Optionally denature, reduce, alkylate, then dilute

[0118] 4. Protease digestion by solid-phase enzymes followed by dilution

[0119] 5. Desalting with Ion-Pair Reverse Phase

[0120] 6. Chromatography-mass spectrometry analysis

[0121] Protein drugs and protein drug candidates differ from conventional small molecule drugs in that they are not chemically synthesized. In contrast, protein drugs called biologics are recombinant proteins synthesized using the cellular machinery of living mammalian cells.

[0122] Therefore, a major challenge in manufacturing biologics is maintaining post-translational modifications, protein molecular weight, charge homogeneity, and other physical attributes. In summary, these analytical measurements require high-performance, low-volu...

Embodiment 2

[0124] Example 2. Example Workflow for Protein Complex Disease Marker Discovery and Diagnostics

[0125] 1. Add His-tag recombinant protein to the sample

[0126] 2. Swap native protein with tagged recombinant protein

[0127] 3. Purification of Proteins by Affinity Chromatography

[0128] 4. Buffer Exchange by Gel Filtration

[0129] 5. Optionally denature, reduce, alkylate, then dilute

[0130] 6. Protease digestion by solid phase enzyme followed by dilution

[0131] 7. Desalting with Ion-Pair Reverse Phase

[0132] 8. Chromatography-mass spectrometry analysis

[0133] In the case of functional cells, proteins rarely act as individual proteins. Instead, proteins associate with many different proteins and form protein complexes. Sometimes these complexes are transient interactions and can be difficult to detect. Protein complexes are a viable source of disease information, and components of protein complexes are potential disease markers. Recent advances in the fie...

Embodiment 3

[0136] Example 3. Example Workflow for Peptide Mapping Analysis

[0137] 1. Provide samples

[0138] 2. Buffer Exchange by Gel Filtration

[0139] 3. Denaturation, reduction, alkylation

[0140] 4. Dilute with buffer

[0141] 5. Protease digestion by solid-phase trypsin, Glu-C, Lys-N, Lys-C, Asp-N, Arg-C and / or chymotrypsin digestion enzymes

[0142] 6. Dilution

[0143] 7. Add ion-pair reagent and desalt with ion-pair reverse phase

[0144] 8. Chromatography-mass spectrometry analysis

[0145] Discover protein drug candidates by screening libraries against druggable targets. Once positive interactions are found, drug candidates are optimized in terms of their binding effects. The process first requires the identification of specific peptides on the target to which the drug candidate binds, a process known as epitope mapping. A major challenge in epitope mapping is to generate robust, reproducible, and complete methods.

[0146] Drug targets are recombinant proteins...

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Abstract

The present invention relates to a method for qualitative analysis of a sample protein, which method comprises the steps of providing a water swollen gel column bed comprising bound protease and a sample protein in liquid buffer, digesting the sample protein into polypeptides by contact with the gel bed and subjecting the polypeptides to mass spectrometry (MS). The method is advantageously performed using back and forth flow of the sample protein including two or more repeats, and may be performed at a temperature of less than about 37 DEG C, such as a temperature of less than about 30 DEG C.The invention also includes an automated method as well as a device and a kit for performing mass-based analysis of proteins with higher speed than the prior art while maintaining conditions that are tolerable to the protease.

Description

technical field [0001] The present invention relates to the characterization and analysis of proteins, and in particular to the enzymatic cleavage of sample proteins into their complex peptides using proteases. The methods of the invention efficiently provide for the identification of proteins under conditions tolerable to the reagents employed. Furthermore, the present invention also includes automated methods, devices and kits based on and utilizing the enzymatic cleavage described herein. Background technique [0002] Characterization, identification and analysis of proteins by mass spectrometry can be achieved by enzymatic cleavage of proteins into their complex peptides or polypeptides. Peptides can be separated by ion pairing chromatography and introduced into a mass spectrometer instrument. Using computer-based software, the mass information of the polypeptide fragments is used to "recombine" the original protein to identify the protein. In some cases, proteins can...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68C07K1/16
CPCG01N33/6848C07K1/16G01N30/72
Inventor 李·黄徐東瑩道格拉斯·T·杰尔德
Owner 菲奈克瑟斯公司