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Novel proteome analysis method and devices therefor

A technology of proteomics and analysis methods, applied in the field of database itself, can solve the problems of backward purification and separation research, time-consuming, complex multi-step, shortening of detection time, etc., and achieve the effect of shortening operation time.

Inactive Publication Date: 2004-05-12
PROTOSERA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0017] Third, there is no effective idea, method or technique for grouping total proteins contained in biological samples
[0018] Fourth, in conventional proteome research, before MS analysis, it is necessary to cut the gel into small fragments and use a special solution to extract proteins from each fragment, which is a time-consuming, multi-step complex operation
The complex operation of the method cannot realize the miniaturization of the device, the shortening of the detection time, the processing of multiple samples, or the automation of the whole set of devices
[0019] The fifth problem is that there are many kinds of so-called "low-abundance proteins"
[0021] Therefore, the object of the present invention is to provide an idea, method and technology for solving the problems faced by current proteome research, thus making it possible for the first time to study the proteome of membrane proteins, which has been considered It is a vital part of all proteins for the elucidation of biological functions and diseases, but it is far behind in research due to the great difficulty of maintaining its function when purifying and isolating

Method used

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  • Novel proteome analysis method and devices therefor
  • Novel proteome analysis method and devices therefor
  • Novel proteome analysis method and devices therefor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0208] Preparation of Liposomes Encapsulating Urokinase Receptor

[0209] (1) Preparation of membrane fraction

[0210] Since U937 is a cell line derived from human monocytes and expresses a high concentration of urokinase receptor after being stimulated by phorbol ester (PMA), it was used as a sample of the isolated membrane fraction. After washing, cells were disrupted by treatment with Polytron 3 times at 1 min intervals for 2-5 sec each under ice-cooled conditions, while the membrane fraction was centrifuged through a 40% sucrose density gradient (95,000 g x 60 min) enriched on the interface ( Figure 6 ).

[0211] (2) Preparation of liposomes embedding membrane proteins

[0212] The purified yolk lecithin (1.25 g) and cholesterol (0.125 g) were suspended in 25 mL of physiological saline, and treated with a probe-type ultrasonic device for 15 minutes under ice-cooling. The average particle size of the obtained liposomes was 80 nm. Add the pre-prepared U937 membrane ...

Embodiment 2

[0223] Example 2: Appearance of receptor-embedded liposomes after particle diameter reduction

[0224] The particle size of liposomes embedded in membrane proteosomes was changed by extruder method, and the appearance of liposomes embedded in urokinase receptor was examined by fluorescent (FITC)-labeled urokinase. As a result, the number of liposomes embedding target receptors in the liposome solution increased significantly when the pore size of the filter passed through was not more than 0.6 μm, as shown in FIG. 16 . It is speculated to be caused by the fact that immediately after fusion the vast majority of target receptors are encapsulated in large liposomes in multilamellar liposomes, fluorescence cannot be detected, and more importantly, the smaller size of liposomes Plastids lead to a reduction in the number of receptors entrapped in a liposome, thus causing a marked difference between liposomes entrapping the target receptor and liposomes not entrapping the target rece...

Embodiment 3

[0226] Example 3: Blotting of Urokinase onto Mass Spectrometry Plates

[0227] Urokinase (10, 13, 16, 20, 33 μg) was loaded into the same well of the polyacrylamide gel at constant time intervals and electrophoresed. After migration was complete, the gel was peeled off from the glass plate, and after 5 minutes of replacing the solution in the gel with blotting buffer (5% acetonitrile / 125 mM NaCl / PBS), it was hot-blotted (diffused) onto an aluminum plate whose surface Treated with a hydrophobic substance having 16 carbon atoms. As the blotting buffer, phosphate buffer containing 5% acetonitrile and 125 mM NaCl was used for blotting at 35° C. for 4 hours. Figure 17 Shown are photographs of the mass spectrum plate after blotting and of gels stained with Coomassie Brilliant Blue before and after blotting. The urokinase band on the mass spectrometer plate cannot be seen with the naked eye, but since the urokinase band on the gel becomes weaker after blotting, it implies that a c...

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Abstract

The present invention provides a proteome analysis method including grouping a proteome into membrane proteins and compounds capable of interacting with the membrane proteins, while retaining their native structure and function, and analyzing both the membrane proteins and the compounds based on biological affinity, and devices therefor.

Description

technical field [0001] The present invention relates to methodologies, techniques and devices for functional proteomics that can be used to collectively discover and quantify membrane proteins and their ligands, and to analyze their function (interaction). The present invention also relates to novel methods for pharmacological proteome analysis of diseases using membrane proteins and their ligands as indicators, said method comprising the discovery, isolation, identification and quantification of membrane proteins and their ligands (the membrane proteins and their ligands Participate in the onset, exacerbation and cure of specific diseases), elucidate its function and establish the database of its type and quantitative value, the present invention also relates to the database itself. The present invention further relates to the establishment method of every biological membrane protein bank, and also relates to the separation method of membrane protein (the method can keep the ...

Claims

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Application Information

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IPC IPC(8): G01N27/62C40B30/04G01N21/27G01N21/78G01N27/447G01N27/64G01N33/53G01N33/543G01N33/566G01N33/567G01N33/68H01J49/16
CPCC40B30/04G01N33/567G01N33/6845G01N33/6803G01N33/5436G01N27/447G01N33/68
Inventor 田中宪次李良子向井裕通栋近公司有国尚志和美重子落合文吾
Owner PROTOSERA
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