Method and means for sample preparation

A sample, a technique in a sample, applied in the field of depleting undesired molecules and/or enriching desired molecules, separation materials comprising magnetic particles, and depletion of high-abundance large proteins

Active Publication Date: 2014-01-08
CYTIVA SWEDEN AB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the removal of albumin may have a profound impact on the final quantitative performance of the proteomic profile
Therefore, other methods have to be used

Method used

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  • Method and means for sample preparation
  • Method and means for sample preparation

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Example 1: -SO in bead-based cores 3 - Ligand preparation of agarose magnetic shell medium

[0034] This example illustrates figure 1 Synthesis of the indicated beads.

[0035] Preparation of magnetic agarose beads

[0036] Approximately 50 g of iron oxide particles (particle size 1.5 μm) were added to an agarose solution containing agarose (117 g) and water (500 g). Adjust the solution to 95 °C. This solution was then added to a solution of toluene (1420ml) and ethylcellulose (106g) in a vessel equipped with a stirrer while maintaining the temperature at 75°C. Increase mixer speed until desired particle size is achieved. The emulsion was then cooled to room temperature. Wash the beads with ethanol and water.

[0037]Add water (100 mL) to the washed beads (500 mL), Na 2 SO 4 (74g), 50% NaOH (6mL) and NaBH 4 (0.5g). The temperature was raised to 50°C, and epichlorohydrin (61 mL) and sodium hydroxide (42 mL) were added. After the addition was complete, the ...

Embodiment 2

[0047] Example 2: Based on -SO in the bead core 3 - Ligand preparation of dextran magnetic shell media

[0048] This example illustrates figure 2 Synthetic procedure for the indicated beads. The synthesis was designed to obtain beads with a porosity comparable to Sephadex G-50 (separation range from 1500 to 30000 g / mol) but with iron oxide particles and ligands in the bead core.

[0049] Production of dextran magnetic beads with porosity comparable to Sephadex G-50

[0050] Add approximately 20 g of iron oxide particles (particle size 1.5 μm) to a solution containing water (200 mL), 50% NaOH (13 ml), NaBH 4 (0.5g) and dextran TF (94g) in a dextran solution. The solution was heated to 50°C and then added to a solution of dichloroethane (200ml) and cellulose acetate butyrate (12g) in a vessel equipped with a paddle stirrer. Increase mixer speed until desired particle size is achieved. Epichlorohydrin (13 mL) was then added and the reaction was continued at 50°C for 16 ...

Embodiment 3

[0053] Example 3: Chromatographic evaluation of the samples described in Example 1

[0054] To confirm that proteins with a molecular weight greater than about 10000 g / mol are excluded from the magnetic beads produced according to Example 1, the breakthrough capacity of a number of proteins was tested. A sample based on magnetic agarose beads (see Example 1) is packed into a suitable column through which the protein solution is pumped. Adjust mobile phase conditions to acidic pH to allow all proteins to adsorb to the nuclear ligand (-SO 3 - ).

[0055] The magnetic agarose shell medium (sample produced in Example 1) to be studied in terms of breakthrough capacity was filled into a HR5 / 2 column, and after equilibrating with buffer, the sample solution was pumped through the column at a flow rate of 0.2 mL / min. The breakthrough capacity was evaluated at 10% of the highest UV detector signal (280 nm). The maximum UV signal was estimated by pumping the test solution directly i...

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Abstract

The present invention relates to a method for depletion of undesired molecules and / or enrichment of desired molecules from a sample comprising high abundant as well as low abundant molecules, comprising the following steps: a) providing a separation material comprising a solid phase (beads) comprising an inner porous core material comprising magnetic particles and an outer porous shell with a porosity equal or denser than that of the shell; b) adding the sample to the separation material; c) adsorbing a first fraction of molecules with a molecular weight of 500-50000 Da in the core and simultaneously excluding a second fraction of molecules from binding to the core and the shell, wherein the molecular weight of the second fraction molecules is at least 5 preferably 10 times higher than the molecular weight of the first fraction and d) eluting the desired molecules from the separation material, wherein step d) and optionally step c)is performed using an oscillating power / field applied over the separation material. The first fraction of molecules are for example drugs with a mw of about 700 Da, small proteins / peptides with an mw of about 7000 Da or proteins with a mw of about 40000 Da.

Description

field of invention [0001] The present invention relates to methods and devices for sample preparation. More precisely, the present invention relates to methods for depleting undesired molecules and / or enriching desired molecules from a sample, e.g. depleting high abundant large peptides from low abundant small proteins / peptides such as biomarkers protein. The invention also relates to a separation material comprising magnetic particles for use in said method. Background of the invention [0002] High-throughput quantitative serum profile methods are an important method for biomarker discovery. In addition to using proteomics to identify protein sequences, modern proteomics aims to find new methods that have direct impact in clinical diagnosis, new drug design, clinical trials, and treatment control. Regardless of research goals, proteomics methods must include a series of work steps, including sample preparation, quantitative analysis, data acquisition, database retrieval...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/50B01D15/20B01D15/34
CPCB01J20/3204B01D15/34B01J20/285B01D15/3866G01N2030/143B01J20/3227G01N30/14G01N1/405B01J20/3274B01J20/3293B01J20/28009B01D15/3885
Inventor G·格拉德B-L·约翰逊J-L·马洛伊塞
Owner CYTIVA SWEDEN AB
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