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Preparation method of urinary protein and detection method for urinary proteome

A urine protein and protein technology, which is applied in the detection field of urine proteome, can solve the problems of low throughput of quantitative deep urine proteome, etc., and achieve the effects of improving accuracy and repeatability, short mass spectrometry detection time, and simple preparation process

Pending Publication Date: 2018-07-27
北京松果天目健康管理有限公司
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Problems solved by technology

[0005] In view of the deficiencies in the prior art, in order to solve the low-throughput problem of quantitative deep urine proteome detection, the inventors systematically improved the urine protein preparation method based on centrifugation technology, and formed a method that can be used for high-throughput quantitative Sample preparation method for deep urine proteome testing

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  • Preparation method of urinary protein and detection method for urinary proteome
  • Preparation method of urinary protein and detection method for urinary proteome
  • Preparation method of urinary protein and detection method for urinary proteome

Examples

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Embodiment 1

[0055] Embodiment 1 urine protein preparation method

[0056] (1) Centrifuge 1ml of urine sample at 20°C for 75 minutes with a centrifugal force of 200,000g, discard the supernatant, and keep the precipitate;

[0057] (2) Transfer the precipitate to a 1.5ml centrifuge tube, add 400 μl of resuspension buffer (50mM Tris, 250mM sucrose, pH 8.5) to the centrifuge tube, let it stand at room temperature for 30 minutes, and blow the weight with a pipette. suspended sediment;

[0058] (3) Dithiothreitol was added to the above resuspended pellet to a final concentration of about 100mM, and heated at 65°C for 30 minutes to remove most of the uromodulin (UMOD, see figure 1 );

[0059] (4) Add cleaning buffer (10mM triethanolamine, 100mM sodium chloride, pH7.4) to 4ml, then centrifuge at 20°C for 75 minutes with a centrifugal force of 200000g, discard the supernatant, and leave the precipitate;

[0060] (5) Redissolve the precipitate with 30 μl of digestion buffer (such as 10 mM Tris o...

Embodiment 2

[0064] Embodiment 2 urine protein preparation method

[0065] (1) Centrifuge 10ml of urine sample at 4°C for 20 minutes at a centrifugal force of 100,000, discard the supernatant and keep the precipitate;

[0066] (2) Transfer the above precipitate to a centrifuge tube, add 60ul of resuspension buffer (50mM Tris, 250mM sucrose, pH8.5) into the centrifuge tube, let it stand at room temperature for 10 minutes, and blow the resuspended precipitate fully with a pipette ;

[0067] (3) Add dithiothreitol to the above resuspended pellet to a final concentration of 50 mM, and heat at 80°C for 10 minutes to remove most of the uromodulin protein in the sample;

[0068] (4) Add cleaning buffer (10mM triethanolamine, 100mM sodium chloride, pH7.4) to 400ul, then centrifuge for 20 minutes under 4 conditions with a centrifugal force of 100000, discard the supernatant, and leave the precipitate;

[0069] (5) Use one-dimensional electrophoresis (SDS-PAGE) for protein separation, dissolve the...

Embodiment 3

[0070] Embodiment 3 A method for enriching urinary protein,

[0071] Including the following steps:

[0072] (1) Add 10 mg of diatomaceous earth to 1 ml of urine and shake well;

[0073] (2) Heating at a temperature between 37-100°C for 5 minutes, then cooling to room temperature;

[0074] (3) Rotate and mix at room temperature for 30 minutes, centrifuge at 12,000 rpm for 5 minutes, and discard the supernatant;

[0075] (4) Add 30-100 microliters of digestion buffer to carry out in-solution digestion of enriched urinary protein.

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Abstract

The invention discloses a detection method for urinary proteome, which includes the steps of preparation of urinary protein, separation at protein or peptide level, and mass spectrum identification. The method of preparation of urinary protein includes steps of: 1) performing ultracentrifugation to a urine sample at normal temperature for a certain time and rejecting the supernatant and maintaining a precipitate; 2) adding a proper amount of resuspending buffer liquid to the precipitate in the step 1) to re-suspend the precipitate; 3) adding a reducing agent, which can break a disulfide bond,to the resuspension liquid and heating the resuspension liquid for 10-60 min at 37-80 DEG C; 4) adding a cleaning buffer solution to the solution in the step 3) and performing high-speed centrifugation for a certain time, and rejecting the supernatant and maintaining a precipitate; 5) re-dissolving the precipitate in the step 4) with a digestive buffer solution, and performing in-solution enzymolysis to proteins or performing protein separation through one-dimensional electrophoresis (SDS-PAGE). The method simplifies preparation process of urinary protein and improves accuracy and repeatability of detection. The method can be used for high-throughput quantitative deep detection of the urinary proteome.

Description

technical field [0001] The invention relates to a urine protein preparation method and a urine proteome detection method, in particular to a urine protein preparation method and a urine proteome detection method that can be used for high-throughput quantitative deep urine proteome detection. Background technique [0002] Urine is the most commonly used body fluid sample except blood in clinical tests. The detection of bilirubin, glucose, ketone bodies, protein, blood cells and other indicators in urine routine is used for the diagnosis or efficacy monitoring of various diseases. In view of the important value of urine testing in health medicine, scientists from all over the world have been using proteomics technology to try to find new protein markers from urine for disease diagnosis, prognosis judgment, and efficacy monitoring. [0003] The detection process of urine proteome includes preparation of urine protein, separation at protein or peptide level, and identification b...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06
CPCG01N30/02G01N30/06G01N1/28G01N33/68
Inventor 秦钧冷文川倪晓天路天元甄蓓王广舜孙长青钟博文
Owner 北京松果天目健康管理有限公司
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