CN106000102B - Time of Fluff Slurry collects and preserves the method and device of protein in urine as absorber - Google Patents
Time of Fluff Slurry collects and preserves the method and device of protein in urine as absorber Download PDFInfo
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- CN106000102B CN106000102B CN201610388290.0A CN201610388290A CN106000102B CN 106000102 B CN106000102 B CN 106000102B CN 201610388290 A CN201610388290 A CN 201610388290A CN 106000102 B CN106000102 B CN 106000102B
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
Abstract
The invention discloses a kind of Time of Fluff Slurry to collect and preserve the method and device of protein in urine as absorber.Described method includes following steps：(1) urine is collected using Time of Fluff Slurry as absorber；(2) protein in urine that the Time of Fluff Slurry absorbs is eluted, and is preserved, that is, realize the collection and preservation of protein in urine；The mode of the preservation is that the eluent afforded is adsorbed on adsorbed film.The method of the elution is as follows：1) Time of Fluff Slurry and eluent are mixed and stirred for obtaining mixed liquor；2) mixed liquor is filtered, the mixed liquor is made to flow through the adsorbed film, then protein is adsorbed on the adsorbed film in urine.The method of the present invention is simple and easy to do, and expense is low；It does not need any organic solvent, is conducive to environmental protection, it is most important that protein is preserved in the state of being completely dried, and the degradation of protein is avoided, can long-term preservation urine protein at room temperature.
Description
Technical field
The present invention relates to a kind of method and apparatus of protein in collection and preservation urine, and in particular to a kind of Time of Fluff Slurry work
The method and device of protein in urine is collected and preserved for absorber.
Background technology
Biomarker refer to the relevant variation monitored of physiology or pathophysiological process, essence be variation.
Important component of the blood plasma as organismic internal environment is adjusted various physical and chemical compositions by homeostatic mechanism and keeps relative constant, that is, become
Change range is small, it is few to accommodate biomarker；And metabolic waste of the urine as body, including the more letter of reflection body variation
Breath, i.e. very more biomarkers.In addition, urine specimen acquisition is noninvasive, can continuously, largely collect, by enzymolysis shadow
Sound is small.Therefore, the same subject can be repeated as many times and sample, to the variation of convenient dynamic monitoring disease.Urine protein
The opposite blood of composition is simpler, background is low.Urine protein histone matter content dynamic range is substantially 106, to blood plasma and
In the case that urine uses duplicate protein identification analysis strategy, more protein can be identified in urine.Urine
70% protein of Proteomics comes from urinary system, and another 30% is generated through glomerular filtration by blood plasma.Therefore urine protein
The physiology and pathophysiological change of urinary system can not only directly be reacted；The disease of the other systems of body can also be reacted.It is comprehensive
Upper described, urine may be the source for excavating biomarker more better than blood.
According to the information that Urinary Protein Biomarker Database are included, it can much reflect different pathological
The urine protein candidate markers of physiological status are reported, but are being previously required to face this on a large scale applied to clinic
Bed verification.Therefore, urine is as important biological sample, if can protect it together with each stage of disease with clinical case history
It leaves and, then in the following retrospective and perspective study for finding biomarker, especially verified in larger scale clinical
In experiment, it will play the role of immeasurable.
Group without automatic micturition function, such as infant and the patient of loss of consciousness stupor, during medical diagnosis on disease
Accurate main suit cannot be provided, to make the diagnosis of disease more depend on laboratory examination.And blood test belongs to invasive
It checks, therefore finds disease marker from urine and provide valuable clue, especially infant for the diagnosis of disease.In view of
This, there are some difficulties when collecting without automatic micturition function human urine, it is desirable to provide a kind of simply to collect and preserve in urine
The method and device of protein.
Invention content
The object of the present invention is to provide a kind of method and apparatus that simply can collect and preserve protein in urine, especially
Protein suitable for the urine of the group (infant and the patient etc. of loss of consciousness stupor) of no automatic micturition function, this hair
Bright method being capable of protein in urine collected by long-term preservation.
The method of protein, includes the following steps in collection and preservation urine provided by the present invention：
(1) fresh urine is collected as absorber using Time of Fluff Slurry；
(2) protein in urine that the Time of Fluff Slurry absorbs is eluted, and is preserved, that is, realize egg in urine
The collection and preservation of white matter.
In above-mentioned method, the mode of the preservation is that the eluent afforded is adsorbed on adsorbed film, i.e.,
Protein in urine is preserved from being transferred in Time of Fluff Slurry on adsorbed film.
In above-mentioned method, the method for the elution is as follows：
1) Time of Fluff Slurry and eluent are mixed and stirred for obtaining mixed liquor；
2) mixed liquor is filtered, the mixed liquor is made to flow through the adsorbed film, then protein adsorbs in urine
In on the adsorbed film.
In above-mentioned method, the Time of Fluff Slurry can quickly and effectively absorb urine as absorber, and not with urine
In most protein generate permanent and firmly combine, so as to which urine protein is eluted from absorber.
The material and fibre length of the Time of Fluff Slurry and distribution do not influence its assimilation effect.
In above-mentioned method, the eluent can be disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution, the phosphoric acid hydrogen two
The concentration of sodium-phosphate sodium dihydrogen buffer solution can be 1M, and pH value can be 6 (being adjusted by adding sodium hydroxide), have certain acid
Basicity buffer capacity can adjust the pH value of urine；
The dosage of the eluent is can submerge the Time of Fluff Slurry and the Time of Fluff Slurry can be made to stir evenly.
In above-mentioned method, when the suction filtration, make the mixed liquor followed by isolation film, the adsorbed film and filter paper；
The isolation film, the adsorbed film and the filter paper are sequentially overlapped together.
In above-mentioned method, the suction filtration step carries out in bottle,suction, can specifically be carried out under the action of vacuum filtration,
To accelerate the filtration of mixed liquor.
In above-mentioned method, the isolation film is pvdf membrane；
The number of plies of the isolation film superposition is 1 layer；
Some granule foreigns are isolated for that can be 0.22 μm, with removal in the aperture of the isolation film；
The effect of the isolation film is：When the mixed liquor passes through, isolation film does not combine urine protein, and
The cell and cell fragment obstructed in the fluff pulp fibers and urine is contacted with adsorbed film combination；
The adsorbed film is that nitrocellulose filter or the pvdf membrane of activation, the activation are referred to pvdf membrane in methyl alcohol
It impregnates several seconds；
The number of plies of the adsorbed film superposition is 1 layer；
The aperture of the adsorbed film is 0.22~0.45 μm, concretely 0.22 μm；
The number of plies of the filter paper overlaid can be 3~8 layers, such as 4~6 layers；
Medium speed filter paper can be selected in the filter paper.
When the isolation film, the adsorbed film and the filter paper are overlapped successively, water infiltration is used first, to prevent bubble
Generation.
In above-mentioned method, the method further includes that the adsorbed film for having adsorbed protein in urine is dried
Step,
The drying carries out in the baking oven not higher than 56 DEG C or to spontaneously dry at room temperature, and the room temperature refers to 20~
25℃。
When the adsorbed film that the present invention collected adsorbed protein in urine preserves, itself and record there can be urine
The label paper of liquid sample information is put into independent vacuum seal bag, in -80 DEG C or room temperature preservation.
The present invention also provides a kind of devices of protein in preservation urine, it includes a bottle,suction and matches with the bottle,suction
The funnel of conjunction；The bottom of the funnel is equipped with filter paper, adsorbed film and isolation film successively from the bottom to top.
In above-mentioned device, the number of plies of the filter paper can be 3~8 layers, concretely 4~6 layers；
The number of plies of the adsorbed film is 1 layer；
The number of plies of the isolation film is 1 layer；
The isolation film can be pvdf membrane.
In above-mentioned device, the funnel is Buchner funnel.
In above-mentioned device, the bottle,suction is connected with Vacuum filtration device, can be filtered by vacuum, to accelerate to mix
Liquid passes through.
The method that the present invention collects and preserves protein in urine, uses Time of Fluff Slurry to collect fresh urine as absorber first
Liquid, then Protein elution in urine that absorber absorbs is got off by Suction filtration device, and adsorb and be incorporated on adsorbed film, by film
After drying, urine protein is preserved in the form of vacuum, drying.
The present invention collects and preserves the method and device of protein in urine, and urine egg is preserved in the form of vacuum, drying
White matter.The method of the present invention is simple and easy to do, and expense is low；It does not need any organic solvent, is conducive to environmental protection, it is most important that
Protein is preserved in the state of being completely dried, and the degradation of protein is avoided, can long-term preservation urine egg at room temperature
White matter.
Description of the drawings
Fig. 1 is the structural schematic diagram that the present invention preserves the device of protein in urine.
It is respectively marked in Fig. 1 as follows：
1 vacuum filtration bottle, 2 Buchner funnels, 3 rubber stoppers, 4 thin necks.
Fig. 2 is the schematic diagram that the present invention collected and preserved urine collecting process in the method for protein in urine.
Fig. 3 is that urine protein is saved in nitrocellulose filter in the method for protein in present invention collection and preservation urine
The schematic diagram of upper process.
Fig. 4 is the SDS-PAGE analysis charts of protein in urine in the embodiment of the present invention 3.
Fig. 5 is the schematic diagram of the coefficient of variation of protein abundance in urine in the embodiment of the present invention 3.
Specific implementation mode
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Embodiment 1, the device for collecting protein in urine
The structural schematic diagram of the device of protein is as shown in Figure 1 in collection urine provided by the invention.
Apparatus of the present invention include a vacuum filtration bottle 1 and the Buchner funnel 2 with the vacuum filtration bottle 1 sealing cooperation, cloth
Cooperation is sealed by rubber stopper 3 between family name's funnel 2 and vacuum filtration bottle 1.In the bottom (not shown) of Buchner funnel 2
It is equipped with filter paper, adsorbed film and isolation film successively from the bottom to top, before being laid with, is first soaked with water, to prevent from producing
Anger bubble.Filter paper is laid with 4~6 layers, is Medium speed filter paper.Adsorbed film concretely nitrocellulose filter is laid with one layer, and aperture is
0.22 μm, for adsorbing protein in urine.Isolation film concretely pvdf membrane, aperture is 0.22 μm, for obstructing Time of Fluff Slurry
Cell and cell fragment in fiber and urine are combined with nitrocellulose filter to be contacted, and it does not combine urine protein.For
Acceleration filtrate passes through, and the thin neck 4 that bottle 1 is filtered by vacuum is connected with Vacuum filtration device (not shown) (such as vacuum pump)
It connects.
(Time of Fluff Slurry collects urine, nitrocellulose filter to protein as absorber in embodiment 2, collection and preservation urine
Preserve urine protein)
The process schematic of urine collecting is as shown in Figure 2 in the present embodiment.
It is as shown in Figure 3 to be saved in the process schematic on nitrocellulose filter for urine protein in the present embodiment.
The collection that protein in urine is carried out using the device that embodiment 1 provides, is as follows：
1,30ml is mixed urine to be poured on Time of Fluff Slurry absorber, 30min is placed at 20 DEG C, the part for having urine will be inhaled
It cuts, takes out Time of Fluff Slurry and be placed in clean beaker.
2,80ml eluents (1M disodium hydrogen phosphates/phosphate sodium dihydrogen buffer solution (pH6)) are added in beaker to be totally submerged
Time of Fluff Slurry, about 80ml, are stirred with glass bar.
3, the circular filter paper (middling speed) that 4 pure water infiltrations of use are placed on Buchner funnel 2, will be with the nitre of pure water infiltration
Acid cellulose film is placed on circular filter paper, will use the pvdf membrane that pure water soaks as isolation film be placed in nitrocellulose filter it
On, the generation of bubble is avoided in whole process.
4, vacuum filtration bottle 1 is installed, the mixture that step 2 obtains is poured into.
5, vacuum filtration bottle 1 is connected with vacuum pump, so that liquid is sequentially passed through pvdf membrane, nitric acid by adjusting vacuum pressure
It drips dropwise after cellulose membrane and filter paper, with the accumulation of time, gradually increases vacuum pump pressure, until after liquid all filters
, total time is about 30 minutes or so.
6, it waits for that liquid filtration finishes, removes nitrocellulose filter, spontaneously dried at 20 DEG C.
By the nitrocellulose filter for combining urine protein and urinate night sample information recording sheet (such as：Sample number into spectrum, when taking urine
Between, routine urinalysis number etc.) be put into independent vacuum seal pack interior room temperature and preserve.
The extraction process of protein is as follows in the urine preserved on the present embodiment nitrocellulose filter：
1, the nitrocellulose membrane of adsorbing urine protein matter is shredded and is placed in 2mL centrifuge tubes, sequentially add 1.7mL acetone,
0.5% ammonium bicarbonate aqueous solutions of 0.2mL.
2, by mixture room temperature intense oscillations 20s, 55 DEG C of dry bath device 60min are placed in, and stop heating strongly every 20min
Vibrate 30s.
3,4 DEG C of jogs 2h, 12 000r/min, 18 DEG C of centrifugation 15min, abandon supernatant, are placed at room temperature for 10min, dry.
4,150 μ L lysis buffers (7M urea, 2M thiocarbamides, 40mM Tris, 25mM dithiothreitol (DTT)s) are added, after piping and druming
Ultrasonic 3min.
5,12 000r/min, 18 DEG C of centrifugation 15min, take supernatant.
6, Bradford methods survey protein concentration after, -80 DEG C freeze it is spare.
Protein in comparative example 1, acetone precipitation extraction urine
Acetone precipitation urine protein method reference literature (Thongboonkerd V, Mcleish K, Arthur J, et
al.Proteomic analysis of normal human urinary proteins isolated by acetone
precipitation or ultracentrifugation.Kidney Int,2002,62(4):1461-1470.) it carries out, mixes
Urine (identical as urine in embodiment 1) 3500g/min, 4 DEG C of centrifugation 30min are closed, supernatant 12000g/min, 4 DEG C of centrifugations are taken
30min takes supernatant 20ml that the acetone of 60mL precoolings, -20 DEG C of precipitation 4h are added.Bradford methods survey -80 DEG C of guarantors of protein concentration
It deposits spare.
Protein in the urine that protein and comparative example 1 are extracted in the urine that embodiment 3, the embodiment of the present invention 2 preserve
Analysis
1, SDS- polyacrylamide gels are analyzed
Separation gel 4%~12%, coomassie brilliant blue staining.
Analysis result：Each sample takes 25 μ g urine proteins progress SDS-PAGE analyses, and the results are shown in Figure 4, by Fig. 4
As can be seen that the present invention uses Time of Fluff Slurry to collect urine, the method for nitrocellulose filter preservation urine protein as absorber, with
Control group is done with the protein example prepared by acetone precipitation to compare, and covers most same straps, without apparent egg
White matter is degraded, and illustrates that the method for the present invention has good technology repeatability.
2, urine protein digestion and LC-MS/MS are analyzed
100 μ g urine proteins are taken, by (Wisniewski JR, Zougman A, the Nagaraj N, et such as Wisniewski
al.Universal sample preparation method for proteome analysis.Nat Methods,
2009,6(5):359-363.) method digestion, peptide fragment Oasis pillar desalinations after digestion.Peptide fragment is dissolved in 0.1% first after desalination
Acid, chromatographic isolation (Thermo EASY-nLC 1200)：Elution time 60min, 0.3 μ L/min of column flow rate, gradient
For 4%~28% Mobile phase B (+20% water of+79.9% acetonitrile of 0.1% formic acid).Polypeptide under reversed-phase column elution is used
(ThermoOrbitrap Fusion Lumos) carries out scanning of the mass spectrum.Each 3 technologies of sample repeat.
Data analysis：All second order ms results Mascot (Perkins DN, Pappin DJC, Creasy DM, et
al.Probaility-based protein identification by searching sequence databases
using mass spectrometry data.Electrophoresis,1999,20(18):3551–3567.)(Matrix
Science companies, version 2 .4.0) software progress database retrieval.Database used is Swissprot_human database
(data were ended on May 3rd, 2013).Search condition：Pancreatin digestion；Allow most 2 leakages enzyme sites；Fixation is modified to half Guang
The ureidomethy of propylhomoserin；Parent ion and product ion mass accuracy are 0.05Da.Using Progenesis (Nonlinear
Company, edition 4 .1) software quantifies albumen, method reference literature (Hauck S, Dietter J, Kramer R, et
al.Deciphering membrane-associated molecular processes in target tissue of
autoimmune uveitis by label-free quantitative mass spectrometry.Mol Cell
Proteomics,2010,9(10):2292–2306.).Select to carry out quantitative spectral peak charge number be+2 ,+3 ,+4；Mascot peptides
Section scoring (Ions score)>30, false positive rate (FDR)<1%；If albumen only has a peptide fragment to be identified, that is, think the egg
Do not have quantitative information in vain.
The analysis result of the coefficient of variation of Mass Spectrometric Identification protein abundance：
The present invention uses Time of Fluff Slurry as absorber collects urine, nitrocellulose filter combination urine protein method is located respectively
Manage 3 urine samples, identify 629 protein jointly, the mean of abundance CV values is 20.1%, and 62.3% protein
Abundance CV values<20%；3 urine samples are handled with acetone precipitation method, identify the abundance CV values of 730 protein jointly
Mean is 22.2%, and 56.6% protein abundance CV values<20%, as shown in Figure 5.
Above-mentioned analysis result shows that the present invention uses Time of Fluff Slurry as absorber collects urine, nitrocellulose filter preserves urine
The method of protein compares widely applied acetone precipitation and directly extracts protein act, the technology repeatability of the two from urine
There is no substantial differences.
Claims (4)
1. the method for protein, includes the following steps in a kind of collection and preservation urine：
(1) urine is collected using Time of Fluff Slurry as absorber；
(2) protein in urine that the Time of Fluff Slurry absorbs is eluted, and is preserved, that is, realize protein in urine
Collection and preservation；
The mode of the preservation is that the eluent afforded is adsorbed on adsorbed film；
The method of the elution is as follows：
1) Time of Fluff Slurry and eluent are mixed and stirred for obtaining mixed liquor；
2) mixed liquor is filtered, the mixed liquor is made to flow through the adsorbed film, then protein is adsorbed in institute in urine
It states on adsorbed film；
The eluent is disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution.
2. according to the method described in claim 1, it is characterized in that：When the suction filtration, make the mixed liquor followed by isolation
Film, the adsorbed film and filter paper；
The isolation film, the adsorbed film and the filter paper are sequentially overlapped together；
The suction filtration step carries out in bottle,suction.
3. according to the method described in claim 2, it is characterized in that：The isolation film is pvdf membrane；
The number of plies of the isolation film superposition is 1 layer；
The adsorbed film is nitrocellulose filter or the pvdf membrane of activation；
The number of plies of the adsorbed film superposition is 1 layer；
The number of plies of the filter paper overlaid is 3~8 layers.
4. method according to claim 1 or 2, it is characterised in that：The method further includes to having adsorbed albumen in urine
The step of adsorbed film of matter is dried,
The drying carries out in the baking oven not higher than 56 DEG C or to spontaneously dry at room temperature.
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| CN108344620A (en) * | 2017-01-23 | 2018-07-31 | 北京师范大学 | A kind of method and its device concentrated and preserve genomic DNA in urine specimen |
Citations (5)
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|---|---|---|---|---|
| DE2928790A1 (en) * | 1979-07-17 | 1981-02-12 | Sartorius Gmbh | Preparation of cells on microscope slides - by filtering from body fluids with a plastic membrane filter, fixing, rendering the membrane filter transparent, and staining |
| CN103163194A (en) * | 2013-03-22 | 2013-06-19 | 天津大学 | Device and method for transferring and analyzing proteins in gel on line |
| CN104075917A (en) * | 2013-03-28 | 2014-10-01 | 中国医学科学院基础医学研究所 | Method and device for collecting and storing urine proteins |
| EP2899276A1 (en) * | 2008-05-30 | 2015-07-29 | QIAGEN GmbH | Lysis, binding and/or washing reagent which can be used for isolating and/or cleaning nucleic acids |
| CN105567670A (en) * | 2014-10-11 | 2016-05-11 | 中国医学科学院基础医学研究所 | Method and apparatus for preserving nucleic acid in urine sample |
-
2016
- 2016-06-02 CN CN201610388290.0A patent/CN106000102B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE2928790A1 (en) * | 1979-07-17 | 1981-02-12 | Sartorius Gmbh | Preparation of cells on microscope slides - by filtering from body fluids with a plastic membrane filter, fixing, rendering the membrane filter transparent, and staining |
| EP2899276A1 (en) * | 2008-05-30 | 2015-07-29 | QIAGEN GmbH | Lysis, binding and/or washing reagent which can be used for isolating and/or cleaning nucleic acids |
| CN103163194A (en) * | 2013-03-22 | 2013-06-19 | 天津大学 | Device and method for transferring and analyzing proteins in gel on line |
| CN104075917A (en) * | 2013-03-28 | 2014-10-01 | 中国医学科学院基础医学研究所 | Method and device for collecting and storing urine proteins |
| CN105567670A (en) * | 2014-10-11 | 2016-05-11 | 中国医学科学院基础医学研究所 | Method and apparatus for preserving nucleic acid in urine sample |
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