Proteomics mass spectrometric detection method aiming at urine sample

A proteomics, urine sample technology, applied in the field of biological sample detection, can solve the problems of low abundance marker information loss, poor repeatability, poor quantitative accuracy, etc., to achieve high accuracy, high reproducibility, The effect of wide application prospects

Inactive Publication Date: 2018-08-10
SOUTH UNIVERSITY OF SCIENCE AND TECHNOLOGY OF CHINA
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  • Application Information

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Problems solved by technology

Data Dependent Acquisition (DDA), which is the main method for the analysis of non-target compounds by tandem mass spectrometry, is prone to loss of information on low-abundance markers, and has the disadvantages of poor repeatability and poor quantitative accuracy.

Method used

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  • Proteomics mass spectrometric detection method aiming at urine sample
  • Proteomics mass spectrometric detection method aiming at urine sample
  • Proteomics mass spectrometric detection method aiming at urine sample

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Embodiment 1

[0037] This embodiment provides a proteomics mass spectrometry detection method for urine samples, the method is

[0038] (1) Instruments and reagents

[0039] Orbitrap Fusion three-in-one mass spectrometer, EASY-nLC 1000 nano-flow ultra-high performance liquid chromatography (ThermoFisher Scientific). Acetonitrile and formic acid used were purchased from Shanghai Anpu Co., Ltd., and other reagents were purchased from Sigma-Aldrich Company.

[0040] (2) Sample preparation

[0041] Take 4 mL of urine sample and use ultrafiltration centrifuge tube with molecular weight cut-off of 10K ( Ultra-4 10K, Millipore) was concentrated by ultrafiltration, and the concentration of urine samples was determined by Bradford method for protein concentration. Add DTT (final concentration 5mmol / L) to the protein extract, shake at 55°C for 25min. Add iodoacetamide (final concentration 15mmol / L), and place in the dark for 30min at room temperature. After the reaction, 2 μL of 1mol / L DTT was ...

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Abstract

The invention provides a proteomics mass spectrometric detection method aiming at a urine sample. The method uses a variable window data independence mass spectrometric collection method; each circulation scanning mode comprises one time of primary full scanning and 30 times of variable window data independence secondary scanning; all parent ions in each window are subjected to secondary crushingand detection, so that all fragment information of all ingredients in the sample is obtained without leakage and differences. The identical number of parent ions in each window can be ensured by usedvariable windows; the selectivity and the sensitivity of the method are considered to the maximum degree; thousands of urine protein can be quantitated in a reproduce manner in the 80min chromatography gradient; the quantitated protein quantity is 2.5 times of that of a conventional data dependence collection method in the same analysis time. The method has the characteristics of high flux, high reproduce performance and high accuracy, is suitable for the analysis of large-scale clinic urine samples and the disease mark detection and has wide application prospects.

Description

technical field [0001] The invention belongs to the technical field of biological sample detection, and relates to a proteomics mass spectrometry detection method for urine samples. Background technique [0002] Urine is an ideal source for studying disease markers. Compared with blood samples, which are most commonly used for detection and analysis, it is non-invasive, can be obtained in large quantities, is not subject to homeostasis regulation, and can accommodate and accumulate more changes. Since part of the composition of urine comes from blood filtrate, it can not only be used as an ideal source for the study of urinary system biomarkers, but also reflect the state of blood and the whole body to a certain extent. In recent years, research on urine proteomics has received more and more attention. However, due to the low protein content, wide span of protein abundance, and large individual differences in urine samples, the proteomics of urine has always been a difficul...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/06G01N30/72G01N30/88
CPCG01N30/06G01N30/72G01N30/88
Inventor 林琳
Owner SOUTH UNIVERSITY OF SCIENCE AND TECHNOLOGY OF CHINA
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