Method for detecting degradation of protein in cells

Abstract

The invention belongs to the field of biochemistry, and relates to a method for detecting the degradation of protein in cells. The method comprises the following steps: (1) marking methionine of newly synthesized protein in the cells with azide small molecule groups by utilizing an methionine analogue; (2) lysing the cells at different time points, and coupling specific molecules which can be identified to the azide small molecule groups through CuAAC reaction; and (3) quantifying target protein which is coupled with the molecules which can be identified in the step (2) by directly utilizing an HTRF (Homogeneous Time-Resolved Fluorescence) resonance principle in a reaction system so as to determine the degradation rate of the target protein. The method is a new protein degradation measurement method which is free from the need of an isotope or cytotoxin, is compatible with high-throughput screening, and can be used for screening related disease protein degradation medicines in pharmaceutical industry, screening specific related protein degradation signals in academia, and the like.

Description

technical field [0001] The invention belongs to the field of biochemistry and relates to a method for detecting intracellular protein degradation, in particular to a detection method for intracellular protein degradation based on CuAAC reaction and energy transfer homogeneous time-resolved fluorescence resonance. Background technique [0002] It is well known in the art that protein degradation and its related parameters (degradation rate and half-life, etc.) are of great significance for understanding protein function, the expected effect of small molecule regulators on protein, and developing drugs for the degradation of specific disease proteins. At present, the protein degradation pathways reported mainly include proteasomal degradation and lysosomal degradation. Studies have shown that when proteins are degraded abnormally through the above two pathways, diseases may occur. For example, a variety of neurodegenerative diseases (such as Huntington's disease, etc.) are ca...

AI Technical Summary

Problems solved by technology

Practice has shown that the main defects of this classic protein degradation detection method are as follows: 1. It needs to use isotopes, so it has a certain impact on the health and safety of experimenters and has high requirements for the laboratory; 2. The operation process is complicated and requires High-throughput incompatible experimental steps such as immunoprecipitation and electrophoretic separation make it impossible to use this method for high-throughput screening for protein degradation; Quantification of rates is noisy and imprecise in many cases

[0004] In recent years, some studies have used the protein translation blocker cycloheximide to study protein degradation, which has the advantages of convenient operation and the possibility of high-throughput screening; however, there are still the following defects: 1. Cycloheximide blocks all proteins Translation, strong non-specificity; 2. Cycloheximide has high cytotoxicity, and it will cause large-scale cell death after 9 hours of treatment. Therefore, it is not suitable for proteins with slower degradation rates; on the other hand, in fact many Slow protein degradation in neurodegenerative diseases

However, the operating procedure used is similar to the traditional pulse-trace experiment, except that the azide small molecule group is used instead of the isotope 35 S markers, therefore, still cannot achieve high-throughput and accurate quantification

Images