A method for measuring dithiothreitol content in protein solution
A technology of dithiothreitol and protein solution is applied in the field of detection, which can solve the problems of failing to meet detection requirements, destroying protein structure, and DTT being easily oxidized, and achieving the effects of short detection time, good repeatability and good accuracy.
Active Publication Date: 2022-05-10
上海明捷医药科技有限公司
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Problems solved by technology
However, DTT has a strong reducing property, which may destroy the protein structure in the body, thereby causing enzyme inactivation and other phenomena, while DTTox will change the structure of creatine kinase, and even completely inactivate creatine kinase when it reaches a certain concentration. It is necessary to detect DTT and DTTox in medicines
But when adopting traditional spectrophotometer method to measure, DTT is extremely easily oxidized, and response is unstable, and sensitivity is low, can not meet detection requirement, also useful HPLC carries out quantitative report to dithiothreitol (DTT) residue at present, but Not very specific
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[0021] A method for measuring dithiothreitol content in a protein solution, comprising the steps of:
[0022] S1. Take an appropriate amount of dithiothreitol, use water as a diluent, and prepare a dithiothreitol solution with an appropriate concentration as a dithiothreitol reference solution;
[0023] S2. Take an appropriate amount of protein solution, and use water as a solvent to prepare a test solution;
[0024] S3. Prepare a series of dithiothreitol reference substance solutions, the concentrations of which are 0.4ng / mL, 1ng / mL, 2ng / mL, 3ng / mL and 4ng / mL, and are detected by LC-MS / MS method to obtain the control respectively The peak area of dithiothreitol in product solution, and carry out standard curve fitting with solution concentration, gained data is shown in table 1, and the standard curve formula that obtains is y=13623x+749, R 2 =0.9961, the results show that dithiothreitol has a good linear relationship in the concentration range, which meets the requirement...
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Abstract
The invention provides a method for measuring the content of dithiothreitol in a protein solution, comprising the following steps: S1, taking an appropriate amount of dithiothreitol, using water as a diluent, and preparing a dithiothreitol solution with an appropriate concentration, as Dithiothreitol reference substance solution; S2, take an appropriate amount of protein solution, and use water as a solvent to prepare a test solution; S3, prepare a series of dithiothreitol reference substance solutions, which are detected by LC-MS / MS method. Obtain the peak area of dithiothreitol in the reference substance solution respectively, and carry out calibration curve fitting with solution concentration; S4, get need testing solution and detect through LC-MS / MS method, obtain the peak area of need testing solution, The content of dithiothreitol in the test solution was calculated by the external standard standard curve method. The invention adopts an alkaline system to directly oxidize DTT into DTTox, which is convenient for directly measuring the content of the oxide of DTT, and has the advantages of good specificity, high accuracy and the like.
Description
technical field [0001] The invention belongs to the technical field of detection, and in particular relates to a method for measuring the content of dithiothreitol in protein solution. Background technique [0002] Dithiothreitol (DTT) is a strong reducing agent, and its reducing properties are largely due to the conformational stability of its six-membered ring (containing disulfide bonds) in its oxidized state. One of the uses of DTT is as a reducing agent and deprotecting agent for thiolated DNA. DTT is also often used for the reduction of disulfide bonds in proteins, which can be used to prevent the formation of cysteines in proteins. or intermolecular disulfide bonds. DTTox is the oxidation state of DTT, which is a six-membered ring structure containing disulfide bonds and has high reducibility. [0003] Dithiothreitol is also used mainly in clinical and experimental fields. Adding DTT in the production process of some drugs can protect the target protein structure to...
Claims
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IPC IPC(8): G01N30/02G01N30/72
CPCG01N30/02G01N30/72
Inventor 顾凯朱子丰卢彦嵇慧卿
Owner 上海明捷医药科技有限公司