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Double-channel SPE column and application of double-channel SPE column in quantitative proteomics

A dual-channel, protein technology, applied in measurement devices, scientific instruments, instruments, etc., can solve the problems of sample loss and pollution, inability to respond to chemically labeled samples, low sample recovery rate, etc., to achieve simple operation, avoid sample loss and recovery. high rate effect

Active Publication Date: 2014-10-01
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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Problems solved by technology

The sample pretreatment process of this method includes protein extraction, urea denaturation, dithiothreitol (DTT) reduction, iodoacetamide (IAA) alkylation, trypsin digestion and dimethylation labeling, stop labeling and C18 reaction. Compared with desalting, so many operations are prone to sample loss and contamination
The existing solution is filter-assisted sample preparation (FASP) (Nat Methods, 2009.6:359), but the sample recovery rate of this method is low and cannot be applied to the analysis of chemically labeled samples; Heck et al. realized dimethylation labeling in SPE (Nature Protocols, 2009.4:484), but this method ignores the loss of samples before labeling

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  • Double-channel SPE column and application of double-channel SPE column in quantitative proteomics
  • Double-channel SPE column and application of double-channel SPE column in quantitative proteomics
  • Double-channel SPE column and application of double-channel SPE column in quantitative proteomics

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Embodiment Construction

[0032] In order to investigate the effect of dual-channel SPE on protein quantification, the device was used to process protein samples. The specific steps are as follows:

[0033] 1. The production process of dual-channel SPE: divide the 1mL SPE tube into two equal parts with a paper knife, add a sieve plate at the bottom of the channel, use a polytetrafluoroethylene film as a separator in the middle of the channel, and use a plastic glue stick to seal the The septum is sealed with the SPE column tube, and filled with C18 reversed-phase chromatography filler (40 μm particle size).

[0034] 2. Protein sample processing process: Add 100 μL of 0.1 μg / μL BSA dissolved in 8M urea to each of the two channels, and add 1 μL of 0.1mol / L dithiothreitol to react at room temperature for 1 hour, then add 2 μL of 0.1M iodoethyl The amide was reacted in the dark for 30 minutes, and then diluted 10 times with 50mM phosphate buffer (pH8.0), then added trypsin according to 1:20 (enzyme / protein...

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Abstract

The invention relates to a novel solid phase extraction (SPE) device capable of achieving the whole process of quantitative analysis pretreatment of protein. According to the device, an SPE tube is partitioned into two chambers with the same capacity through a partition plate, and after the chambers are filled with chromatographic packing, the whole process of sample treatment in quantitative proteomics can be achieved in situ, wherein the treatment comprises denaturation, reduction, alkylation, enzymolysis, isotope labeling and mixing of protein samples. The device is easy and convenient to manufacture and low in manufacturing cost, and has the advantages that the recovery rate is high, operation is easy, the accuracy of quantitative results is good and the precision is high when the samples are treated.

Description

technical field [0001] The present invention designs a dual-channel SPE device, which can realize the whole process of quantitative protein sample processing such as protein sample in-situ denaturation, reduction, alkylation, enzymatic hydrolysis, labeling, desalting and mixing. Background technique [0002] Quantitative proteomics is a hot topic in systems biology research. It provides extremely important data for discovering disease biomarkers, discovering protein-protein interactions, drug-protein interactions, explaining the mechanism of disease, explaining biological metabolic processes, etc. support. Protein is the embodiment and bearer of life activities. It is extremely rich in organisms and participates in all metabolic processes of living organisms. Therefore, changes in protein content will lead to physiological changes, and for humans, it is reflected in the occurrence of diseases. Therefore Accurate quantification of proteins is extremely important for understa...

Claims

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Application Information

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IPC IPC(8): G01N30/60
Inventor 张丽华周愿赵群张珅杨开广张玉奎
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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