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Urine proteomics sample pretreatment method and applications thereof

A technology for proteomics and sample pretreatment, applied to scientific instruments, measuring devices, instruments, etc., can solve the problems of cumbersome experimental steps, and achieve the effects of increasing the number of identifications, low cost, and easy operation

Inactive Publication Date: 2018-01-09
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

(Multidimensional) liquid chromatography can solve the shortcomings of two-dimensional gel electrophoresis, and the combination with mass spectrometry has greatly improved the number of protein identifications, but the experimental steps are cumbersome and require corresponding instruments and equipment (Proteomics, 2005, 5, 4994 -5001; Mol. Cell. Proteomics, 2006, 5, 560-562)

Method used

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  • Urine proteomics sample pretreatment method and applications thereof
  • Urine proteomics sample pretreatment method and applications thereof

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Experimental program
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Effect test

Embodiment 1

[0044] Preparation and Zeta Potential Measurement of Amphoteric Carrier-Modified Polymer Microspheres

[0045] Weigh 30mg polyacrylate particles (aperture particle size 5 μm), and were washed three times with ethanol and phosphate buffer (pH 8.0), and reacted with 1 mL of 15% glutaraldehyde phosphate buffer (pH 8.0) at room temperature for 8 hours, then water and phosphate buffer (pH 8.0) wash the particles separately.

[0046] Measure 400 μL of amphoteric carrier (a mixture of aliphatic polyamine polycarboxylate, composed of isomers and homologues, pI=3-10, trade name Pharmalyte TM ) liquid, dissolved in a mixture of 8 mg sodium cyanoborohydride and 600 μL phosphate buffer (pH 8.0) to obtain a 40% amphoteric carrier solution, and reacted the liquid with the above particles at room temperature for 24 hours. Abandon the supernatant, use the mass-volume percentage concentration of 1.5% glycine and the mass-volume percentage concentration of 1.2% sodium cyanoborohydride mixed ...

Embodiment 2

[0051] 1. Sample handling process

[0052] Urine samples were first centrifuged at 1,500 g for 10 minutes (4°C), and then concentrated with a filter membrane with a molecular weight cut-off of 3 kDa to obtain a sample (Ur) with a protein concentration of 15 mg / mL. The above sample (10 mg) was added to the amphoteric carrier-modified polymer particles and incubated at room temperature for 2 h. Centrifuge and remove supernatant. After washing the particles with PBS, the supernatant was removed by centrifugation again, and the obtained substances were washed with 2M NaCl, 200mMGly-HCl (pH 2.5) and 30% ACN (containing 0.1% TFA) respectively, specifically:

[0053] (1) Wash the obtained substance with 2M aqueous sodium chloride solution, wash 3 times, and shake for 20 min each time, and the eluents after three washings are combined to obtain eluent 1,

[0054] (2) Wash the material obtained in step (1) with 200mM Gly-HCl (pH 2.5), wash 3 times, shake for 20min each time, and comb...

Embodiment 3

[0064] Preparation and Zeta Potential Measurement of Amphoteric Carrier-Modified Polymer Microspheres

[0065] Weigh 10mg of polyacrylate particles (aperture particle size of 20 μm), and washed three times with ethanol and phosphate buffer (pH 7.4), reacted with 1 mL of 8% glutaraldehyde phosphate buffer (pH 7.4) at room temperature for 5 h, then washed with water and phosphate buffer (pH 7.4) wash the particles separately.

[0066] Measure 200 μL of amphoteric carrier (a mixture of aliphatic polyamine polycarboxylate, composed of isomers and homologues, pI=3-10, trade name Pharmalyte TM ) liquid, dissolved with a mixture of 5 mg sodium cyanoborohydride and 800 μL phosphate buffer (pH 7.4) to obtain a 20% amphoteric carrier solution, and reacted the liquid with the above particles for 8 hours at room temperature. The supernatant was discarded, and the mass-volume percentage concentration was 0.5% glycine and the mass-volume percentage concentration was 0.5% sodium cyanoboro...

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Abstract

The present invention relates to a urine proteomics sample pretreatment method and applications thereof, and belongs to the technical field of analysis. According to the present invention, the methodcan be used for enriching proteins in urine samples so as to achieve the deep analysis of proteomics in urine samples; the method is the novel urine sample pre-treatment method; the urine sample is subjected to primary enrichment through the material, and then a stepwise elution method is performed, such that the complexity of the urine sample can be effectively reduced; compared to the traditional urine sample pretreatment method, the method of the present invention has characteristics of convenience, easy performing, low cost and the like; and the method has good application prospect and practical value in proteomics.

Description

technical field [0001] The present invention relates to a urine proteomics sample pretreatment method and its application, specifically a method for reducing the complexity of urine samples by step-by-step elution, and realizing sample pretreatment for proteomics analysis in urine samples method. Background technique [0002] Proteomic analysis of urine samples is of great significance for clinical diagnosis and pathological classification. Compared with blood tests and biopsies, urine samples can be obtained in large quantities and are harmless to the human body. The proteomic analysis of urine samples can not only reflect the health of the urinary system, but also monitor the physiological status of the human system (Expert Rev. Proteomics, 2007, 4, 39-50). At present, a variety of urine proteomic sample pretreatment methods have been developed, through protein separation and mass spectrometry detection technology, the urine proteome is deeply analyzed (J.Am.Soc.Nephrol....

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06G01N30/96
Inventor 张丽华陈远波邓楠吴琪梁振随志刚李云峰张晓丹杨开广张玉奎
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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