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Method for continuously enriching and separating phosphoeptide at high throughput

A phosphopeptide, high-throughput technology, applied in peptide preparation methods, chemical instruments and methods, peptides, etc., can solve difficult phosphopeptide continuous, high-throughput separation and enrichment, time-consuming and laborious, difficult to achieve selectivity and other problems, to achieve high-throughput separation and enrichment, to avoid manual operation, and to achieve the effect of simple operation

Inactive Publication Date: 2012-08-29
DONGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, all these methods currently used for phosphopeptide enrichment require repeated manual operations, which is not only time-consuming and laborious, but also difficult to achieve continuous, high-throughput separation and enrichment of phosphopeptides. Ineffective in separating and enriching phosphopeptides in samples
In addition, even with the above-mentioned new methods, the isolation and enrichment of phosphopeptides from complex samples is sometimes difficult to achieve the desired selectivity, and often depends on the solvents and additives used in the loading and washing solutions.

Method used

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  • Method for continuously enriching and separating phosphoeptide at high throughput
  • Method for continuously enriching and separating phosphoeptide at high throughput
  • Method for continuously enriching and separating phosphoeptide at high throughput

Examples

Experimental program
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Effect test

Embodiment 1

[0042] Use a ruler to measure a quartz capillary with a length of 8 cm, an inner diameter of 530 μm, and an outer diameter of 660 μm. Put 0.05M zinc acetate and 0.05M hexamethylenetetramine solutions into two syringes placed on the micro-injection pump respectively, set the pushing speed at 25 μL / min, and transport the two solutions to the oven at 90 °C at the same time. In the capillary microchannel in , the fluid delivery time was controlled to be 2h, and vertically grown ZnO nanorods were prepared on the inner surface of the microchannel. Then, 65 μg / mL of TiO was delivered into the microchannel at a syringe pump speed of 5 μL / min and an oven temperature of 80 °C. 2 Sol, control the fluid delivery time for 12h, and obtain vertically grown TiO after drying 2 / ZnO nanorod arrays. figure 1 The TiO grown on the inner surface of the microchannel obtained for this embodiment 2 / ZnO nanorod array field emission scanning electron microscope photograph, it can be seen that the n...

Embodiment 2

[0046] Adjust enrichment residence time to be 30s and continue 3min, other conditions are the same as embodiment 1. Figure 5 is the MALDI-TOF MS spectrum after enrichment of 500fmolα-casein hydrolyzate in this example, wherein The number is the characteristic peak of phosphopeptide. It is not difficult to find that when the enrichment residence time is 30s, the signals of the three characteristic peaks of α-casein phosphopeptides in the spectrum are enhanced, and the signals of non-phosphorylated peptides are reduced, indicating that the microfluidic device can detect phosphopeptides Perform fast and efficient enrichment.

Embodiment 3

[0048] Use a ruler to measure a quartz capillary with a length of 12 cm, an inner diameter of 530 μm, and an outer diameter of 660 μm. Put the 0.05M zinc acetate and 0.05M hexamethylenetetramine solutions into two syringes placed on the micro-injection pump respectively, set the pushing speed at 10 μL / min, and transfer the two solutions to the oven at 90 °C at the same time. In the capillary microchannel in , the fluid delivery time was controlled to be 5h, and vertically grown ZnO nanorods were prepared on the inner surface of the microchannel. Then, 65 μg / mL of TiO was delivered into the microchannel at a syringe pump speed of 5 μL / min and an oven temperature of 70 °C. 2 Sol, control the fluid delivery time for 12h, and obtain vertically grown TiO after drying 2 / ZnO nanorod arrays.

[0049] Take two polytetrafluoroethylene microtubes with an inner diameter of 300 μm and a length of 40 cm as fluid delivery channels, and glue them to one end of the quartz capillary with epo...

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Abstract

The invention relates to a method for continuously enriching and separating phosphoeptide at high throughput. The method comprises the following steps: assembling a microfluidic device; cleaning the microfluidic device for 1 to 3 minutes with two kinds of cleaning solution in the staying time of between 30 and 60 seconds in sequence by using a syringe pump as a fluid transportation power source, and feeding proteolysis products to the microchannel of the device; and enriching, cleaning and eluting to prepare the enriched product. The separation method has simple operation, and can regulate and control the stay time of the fluid in the microchannel by regulating the pushing speed of the syringe pump, namely automatically control the enriching condition by regulating the contact time of thefluid with adsorbing materials, thereby avoiding repeated manual operation required by the conventional methods, and realizing continuous, quick and high-throughput separation and enrichment of the phosphoeptide by means of inherent advantages of the microfluidic device.

Description

technical field [0001] The invention belongs to the field of enrichment and separation of phosphopeptides, in particular to a method for continuous high-throughput enrichment and separation of phosphopeptides. Background technique [0002] Protein post-translational modifications (PTMs) are an important topic in current proteomics research. Protein phosphorylation is the most common and important post-translational modification of proteins (according to statistics, about one-third of proteins in mammals are considered to be phosphorylated), and it almost regulates the entire life activity. Processes, including cell signal transduction, cell differentiation, proliferation, neural activity, muscle contraction, metabolism, transcription and translation, protein degradation, etc. The important role of phosphorylated protein in cell life activities makes it a hot spot in proteomics research. [0003] At present, biological mass spectrometry has become the main tool for identify...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/22
Inventor 王宏志何中媛李耀刚张青红
Owner DONGHUA UNIV
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