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A rapid processing method for phosphorylated proteome samples

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, inconvenient operation, and no generalization, and achieve high-quality spectral identification coverage. Effect

Inactive Publication Date: 2017-12-05
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the use of immobilized enzymes has not become widespread, most likely due to the inconvenience of handling and the high non-specific adsorption properties of immobilized enzyme surfaces.

Method used

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  • A rapid processing method for phosphorylated proteome samples
  • A rapid processing method for phosphorylated proteome samples
  • A rapid processing method for phosphorylated proteome samples

Examples

Experimental program
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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, respectively: 1) 2M urea buffer solution; 2) 0.8% NP-40 buffer solution; 3) 12mM sodiumdeoxycholate ( 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 for tens of seconds on the shaker until all the cell membranes are broken, then place the mixture in a 37°C water bath ultrasonicator for 1h, and finally, through a controllable centrifugal force And the GELoader Tip centrifuge filled with 1 mg Ti-IMAC material was used for enrichment of phosphopeptides, and the obtained phosphopeptides were lyophilized at room temperature, and redissolved in 100 μL 0.1% (v / v) formic acid for RP LC-MS / MS analysis , qualita...

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 present invention relates to a rapid processing method for phosphorylated proteome samples. Utilizing the advantage that high-concentration trypsin can promote rapid enzymatic hydrolysis of proteins, a new type of sample processing method integrating cell lysis and proteolysis is developed, and its Applied to the analysis of phosphoproteomics. Thanks to the deep mechanism of simultaneous cell lysis and proteolysis promoted by the high concentration of trypsin, the cell sample can be rapidly converted into a peptide mixture in a single step. During the enrichment of phosphopeptides, non-phosphorylated peptides As well as various other mass spectrometrically incompatible impurities, can be removed. The present invention only needs 25 minutes to realize the rapid transition from cells to peptides, and in the normal control group, the simplest method also needs at least 16 hours; The excellent coverage of mass spectrum identification proves the accuracy and efficiency of this sample pretreatment method.

Description

technical field [0001] The invention belongs to the technical field of phosphoproteomics in the direction of proteomics research, and specifically relates to a sample processing method applied to phosphoproteomics. Background technique [0002] To date, protein phosphorylation has been found to be a ubiquitous protein post-translational modification that regulates approximately 30% of the eukaryotic proteome. As an important post-translational modification, reversible protein phosphorylation plays an important role in the regulation of cell metabolism, such as cell differentiation, cell proliferation, cell apoptosis and signal transduction, etc. Various intracellular and extracellular stimuli can cause abnormal changes in intracellular phosphorylation levels, and this is precisely the cause of many human diseases. Therefore, in order to systematically understand the complex and variable cellular behavior, in-depth study of the phosphoproteome has become a key task. Recentl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N1/28
Inventor 邹汉法刘芳洁叶明亮
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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