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Open-tubular capillary column enriching phosphoeptide or phosphorylated protein and method

A phosphorylated protein, open-tube capillary column technology, applied in the field of analytical chemistry, can solve problems such as the inability to meet the large-scale, high-throughput analysis of proteomics, the difficulty of processing multiple samples, and the limitation of further applications, and achieve easy reuse. , Wide application range and low cost effect

Inactive Publication Date: 2010-03-31
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these enrichment methods are either very cumbersome in operation steps, and it is difficult to process multiple samples at the same time
Either due to manual operation in the centrifuge tube, the transfer process leads to sample loss, and only a few samples can be processed at most at a time, which cannot meet the requirements of large-scale and high-throughput analysis of proteomics
These shortcomings limit its further application in large-scale phosphoproteome analysis

Method used

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  • Open-tubular capillary column enriching phosphoeptide or phosphorylated protein and method

Examples

Experimental program
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Effect test

Embodiment 1

[0029]Push the 0.1M HCl solution into the capillary column with a micro-sampling pump, wash it for 10 minutes at a flow rate of about 1 μL / min, then fill the capillary with HCl solution, seal the end with a rubber stopper, and let it stand at room temperature for 30 minutes. Push out the HCl solution, rinse with water until the pH of the effluent is about 7, inject 0.1M NaOH solution into the capillary, rinse at a flow rate of about 1 μL / min for 10 minutes, then fill the capillary with NaOH solution, seal the end with a rubber stopper, and let it stand at room temperature 2h. Push out the NaOH solution and rinse with water until the pH of the effluent is about 7.

[0030] Push 10% 3-aminopropyltriethoxysilane (APES) methanol solution (v / v) into the capillary, seal both ends of the capillary, and immerse the capillary in a 70° C. water bath for 8 h. Then push out the APES solution, rinse the capillary with 20 μL of methanol solution, and dry it with nitrogen gas.

[0031] Qui...

Embodiment 2

[0034] After diluting the digestion solution of the standard phosphorylated protein α-casein with 50% ACN, 0.1% TFA solution to 2 pmol / μL, use a micro-sampling pump to push into the zirconium phosphate group-modified opening at a flow rate of 0.2 μL / min. A total of 20 μL was injected into a capillary column (inner diameter 50 μm, length 150 cm). Wash the inner wall of the capillary column successively with 50 μL of 50% ACN, 0.1TFA% solution and 50 μL of pure water at a flow rate of 1.0 μL / min to remove impurities such as non-phosphopeptides and salts adhering to the inner wall. Inject 10 μL of 0.1M ammonia solution into the capillary column at a flow rate of 0.2 μL / min to elute the specifically adsorbed phosphopeptides, spot the eluted solution on the MALDI-TOF target, and after natural air drying, spot the DHB solution (50% ACN , 0.1%H 3 PO 4 ), followed by mass spectrometry analysis. When not enriched, many non-phosphopeptides appeared, and only 7 phosphopeptides were det...

Embodiment 3

[0036] The zirconium phosphate group-modified open-tube capillary column was used to load the β-casein hydrolyzate, and the operation in Example 2 was repeated for enrichment and MALDI-TOF-MS analysis. When not enriched, the signal-to-noise ratio is very low, but after enrichment, most of the non-phosphopeptides disappear, and two phosphopeptides are detected, which become the two strongest peaks in the spectrum, such as image 3 shown in a-b.

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Abstract

The invention relates to a method for enriching phosphoeptide or phosphorylated protein in an off-line way and an on-line way for a functionally modified open-tubular capillary column, which comprisesthe following steps: preparing an open-tubular capillary column, an inner wall of which can selectively adsorb the zirconium phosphate functional group of phosphoeptide or phosphorylated protein, enriching the phosphoeptide or phosphorylated protein after loading samples, and then, using a cleaning buffer solution to remove non-phosphoeptide or non-phosphorylated protein and salt; and after the cleaning process is finished, using eluent to elute the phosphoeptide or phosphorylated protein, wherein the eluted phosphoeptide or phosphorylated protein can be marked on the MALDI target to carry out normal MALDI-TOF-MS analysis, or the eluted phosphoeptide or phosphorylated protein can be inoculated on the liquid phase part of microliter or nanoliter LC-ESI-MS to be combined with ESI-MS / MS to automatically enrich and analyze the phosphoeptide or phosphorylated protein in the on-line way.

Description

technical field [0001] The invention belongs to the field of analytical chemistry, and is a method for selectively enriching phosphopeptides or phosphorylated proteins before using mass spectrometry technology to analyze and identify them, that is, a functionally modified open-tube capillary column to realize off-line and An online method for the selective enrichment of phosphopeptides or phosphorylated proteins. Background technique [0002] Phosphorylation of protein is one of the important covalent modifications in organisms, and it plays an important role in the regulation of cell proliferation, differentiation, signal transduction, apoptosis and other life processes. In recent years, mass spectrometry (MS) has been widely used for the identification of phosphorylated proteins. Due to the low stoichiometry of phosphorylated proteins in cells / tissues and the signal suppression of phosphopeptides by non-phosphorylated peptides during mass spectrometry analysis, direct mas...

Claims

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Application Information

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IPC IPC(8): G01N1/40G01N30/08
Inventor 钱小红张养军薛彦峰卫军营
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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