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Rapid treatment method for phosphoproteome sample

A technology for phosphorylating proteins and processing methods, applied in the preparation of test samples, etc., can solve the problems of high non-specific adsorption performance of immobilized enzymes, no generalization, inconvenient operation, etc., and achieve high-quality spectral identification coverage. Effect

Inactive Publication Date: 2015-09-30
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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AI Technical Summary

Benefits of technology

In this new technology described by our researchers, it suggests quickly analyzing large amounts (>10 grams) of proteins with specific properties called Phosphoprotein or Protease K). These techniques are useful because they allow us to study how these molecules work together more efficiently during various biological processes like metabolism, signal transduction pathways, gene regulation, etc., which could lead to important applications ranging from drug development to diagnositic purposes.

Problems solved by technology

This patents describes various technical solutions aim at improving the speed and accuracy of analyzing large amounts of data collected from experiments involving studying specific aspects of cells or tissues during their physiological processes like growth, gene expression dynamics, histochemistry, molecular imaging, and drug discovery. Current techniques involve multiple stages of liquid phase separation followed by enzymic activity measurement after each stage. These techniques require expensive equipment and consume considerable amount of time. Additionally, they may result in reduced sensitivity due to poor substrate selectivity towards lysochlorophyll breakdown.

Method used

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  • Rapid treatment method for phosphoproteome sample
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  • Rapid treatment method for phosphoproteome sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Investigation of the reaction system of cell lysis-proteolysis: 100,000 HeLa cell samples were placed in the following five reaction systems, namely: 1) 2M urea buffer solution; 2) 0.8% NP-40 buffer solution; 3) 12mM sodium deoxycholate (SDC) buffer solution; 4) 2M urea and 0.8% NP-40 buffer solution; 5) 2M urea and 12mM SDC buffer solution, these five buffer solutions contain 10mM DTT and phosphatase inhibitor respectively (ie 1mM NaF and 1mM Na3VO4 ), then add 300ng / μL trypsin to the solution, vortex and shake on the shaker for tens of seconds until all the cell membranes are broken, then place the mixture in a 37°C water bath sonicator for ultrasonic incubation for 1h, and finally, control it by centrifugal force A GELoader Tip centrifuge filled with 1 mg Ti-IMAC material was used for enrichment of phosphopeptides. The obtained phosphopeptides were lyophilized at room temperature and redissolved in 100 μL 0.1% (v / v) formic acid for RP LC-MS / MS For analysis, qualitati...

Embodiment 2

[0033] The effect of high-concentration trypsin on the enrichment of phosphopeptides and identification by mass spectrometry: Add a series of trypsin with different enzyme / protein ratios to 200 μg of HeLa hydrolyzate, mix well, and enrich with 2 mg of Ti-IMAC material. The enrichment steps of phosphopeptides are as follows: After resuspending the material with 80% ACN6%TFA, mix it with the enzymatic hydrolysis solution at a volume ratio of 1:1, shake at room temperature for 30 minutes, and after fully enriched, at 13500rpm Centrifuge for 3 minutes, discard the supernatant, then wash with 50% ACN6%TFA, 200mM NaCl and 30% ACN6%TFA in sequence, shake for 30 minutes, remove the supernatant by high-speed centrifugation as above, and finally wash with 10% NH 3 ·H 2 The phosphopeptide was eluted twice with O solution and subsequently identified by mass spectrometry as described in Example 1.

[0034] Such as image 3 As shown, the numbers on the axis of abscissa represent the exces...

Embodiment 3

[0036] Optimization of the reaction conditions for the rapid processing method of phosphorylated proteome samples: the optimal trypsin concentration and reaction time required for the reaction were investigated respectively. Add four groups of 100000 HeLa cell samples into 50 μL 0.8% NP-40 reaction system, then add 20ng / μL, 100ng / μL, 300ng / μL, 1.0μg / μL trypsin respectively, and vortex the solution on the shaker Rotate and oscillate for tens of seconds until all the cell membranes are broken, then place the mixture solution in a 37°C water bath ultrasonicator and incubate for 1 hour, and finally, use a GELoader Tip centrifuge with controllable centrifugal force and filled with 1mg Ti-IMAC material for phosphoric acid Peptide enrichment, as described in Example 1, was followed by mass spectrometric identification.

[0037] As mentioned above, after the optimal concentration of trypsin was investigated, four groups of 100,000 HeLa cell samples were added to the reaction system of...

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Abstract

The invention relates to a rapid treatment method for a phosphoproteome sample and develops a novel sample treatment method integrating cell splitting decomposition and protein enzymolysis by using the advantage that protein rapid enzymolysis can be promoted through high-concentration trypsin, and application of the novel sample treatment method in analysis of phosphoproteomics. Benefiting from the high-concentration trypsin, a deep mechanism of the cell splitting decomposition and the protein enzymolysis is promoted; a cell sample can finish rapid conversion of a peptide fragment mixture in one step; and in a phosphoeptide enriching process, a non-phosphorylated peptide fragment and other substances with incompatible mass spectrums can be removed. The rapid treatment method can realize the rapid conversion from cells to the peptide fragment by 25 minutes; in a contrast normal group, the simplest method also needs at least 16 hours; and a relatively good mass spectrum identification coverage rate of the contrast group is obtained through analyzing a HeLa actual cell sample, and the accuracy and the high efficiency of the sample pre-treatment method are proved.

Description

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Claims

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Application Information

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Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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