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Mini protein reactor applied to preparation of proteome sample and application of mini protein reactor

A sample preparation and proteome technology, applied in the field of analytical chemistry, can solve the problems of undetectable, pancreatic cancer treatment failure, etc., and achieve the effect of short time-consuming, high sensitivity and good repeatability

Active Publication Date: 2018-10-12
SUN YAT SEN MEMORIAL HOSPITAL SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, for the PDAC marker protein, the currently used detection method cannot be detected in the early stage of PDAC due to its low content, and can only be found in the late stage, which is also the main reason for the failure of pancreatic cancer treatment (10,11)

Method used

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  • Mini protein reactor applied to preparation of proteome sample and application of mini protein reactor
  • Mini protein reactor applied to preparation of proteome sample and application of mini protein reactor
  • Mini protein reactor applied to preparation of proteome sample and application of mini protein reactor

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Embodiment 1

[0054] A mini protein reactor used in proteome sample preparation, called SPMC, its structure diagram is shown in figure 1 shown. The mini protein reactor includes pipette tips (1), adapters (2), IMAC magnetic beads (3), C 18 beads (C 18 small column) (4), Eppendorf tube (Eppendorf centrifuge tube) (5); pipette tip (1) is provided with several C successively from bottom to top 18 beads (4), some IMAC magnetic beads (3); C 18 The beads (4) are in contact with the IMAC magnetic beads (3); the adapter (2) is sleeved on the pipette tip (1), and the adapter (2) is placed on the upper end of the Eppendorf tube (5); the pipette tip (1) ) into the Eppendorf tube (5).

[0055] The pipette tip is preferably a 10-200 μL pipette tip;

[0056] The Eppendorf tube is preferably a 500μL-2.0mL Eppendorf tube;

[0057] The C 18 The beads are preferably 2~5μm Empore C 18 beads; more preferably 3 μm Empore C 18 beads;

[0058] The IMAC magnetic beads are preferably 10-50 μm IMAC magneti...

Embodiment 2

[0061] The present invention is to utilize the method for preparing proteomics sample of SPMC mini-protein reactor, comprises the following steps successively:

[0062] (1) Collection of serum samples: After collecting PDAC patient serum and healthy human serum, store them in a -80°C refrigerator.

[0063] (2) Design of SPMC: such as figure 1 As shown, SPMC is achieved by combining several C 18 beads (3 μm Empore, USA) were loaded into a standard 200 μL pipette tip, and then a certain amount of 20 μm POROS IMAC magnetic beads (Applied Biosystems, USA) was introduced. Then place the SPMC tip through the adapter into a 2.0 mL Eppendorf tube. Calculation of C based on the amount of protein 18 The number of beads and the amount of IMAC magnetic beads, for example: 0.4-1.5 mg IMAC can be used for the separation of 2-20 μg protein (binding capacity of about 30 μg protein / mg beads).

[0064] (3) Enrichment: Before sample loading, several C 18 200 μL pipette tips of beads, severa...

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Abstract

The invention discloses a mini protein reactor applied to preparation of a proteome sample and an application of the mini protein reactor and relates to the field of analytical chemistry. The mini protein reactor mainly comprises IMAC (immobilized metal chelating chromatography) magnetic beads and C18 beads. Smaller than 10 mu L of serum is expected to be taken as a sample for general situation analysis of proteome and can be used for translational research. According to the method, complicated sample preparation steps in proteomics are realized with a series column separation technology and include phosphoprotein pre-concentration, reduction, alkylation and digestion as well as desalination and fractionation. The method is easy to use, consumes short time (2 hours), is high in throughput,high in sensitivity (capable of detecting more than 100 phosphoprotein) and good in repeatability (R is larger than 0.99), is an effective method which can mine the most proteome data no matter fromthe number or from the variety and is speculated to be applied to technology translational research of non-labeled quantitative phosphoproteomics.

Description

technical field [0001] The invention relates to the field of analytical chemistry, in particular to a mini protein reactor used in the preparation of proteome samples and its application. Background technique [0002] The reversible protein phosphorylation and dephosphorylation process regulates many life activities of organisms, and controls many physiological and pathological processes of living organisms, such as cell proliferation, growth, differentiation and apoptosis, and cell signal transduction, cell Apoptosis, etc., are vividly called the switch of life activities; on the other hand, the abnormality of phosphorylation process can lead to a series of common diseases, including tumor cancer, heart disease and Alzheimer's disease, etc. (1), phosphorylated kinase inhibition The role of antitumor agents in reducing antitumor therapy has been widely recognized (2). For example, major receptors for VEGF signaling include the fms-like tyrosine kinase VEGFR-1 and the fetal ...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06
CPCG01N30/02G01N30/06
Inventor 黄炳培孟琼
Owner SUN YAT SEN MEMORIAL HOSPITAL SUN YAT SEN UNIV
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