CN102288467B - Preparation method of animal protein sample for proteomic analysis - Google Patents
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Abstract
The invention provides a preparation method of an animal protein sample for proteomic analysis. The in-situ fixation of a protein is achieved through freezing a tissue; the microscopic capturing and cutting are carried out on a target area; and the total protein of a cut cell is extracted through splitting the tissue. The experimental flows and a sample preparation technique are improved; the loss and the degradation (or damage) of the protein and the manual modification of the protein are greatly reduced; the interaction of the protein with other biological macromolecules is reduced; the protein further can be enabled to be in a completely-denatured state; the efficient recognition of a low-abundance target protein spot is achieved; the separation effect is quite good; and the preparation method is applicable to the screening and the determination of a biological marker of the protein.
Description
(1) technical field
The present invention relates to a kind of preparation method of the animal protein quality sample for proteome analysis, especially a kind of preparation method of the animal protein quality sample for the screening of soil pollution biomarker.
(2) background technology
Proteomics is a new branch of science of rising in recent years, and its main technology is two dimensional gel electrophore-sis (2-DE) and analytical technique of mass spectrum, can disclose from aggregate level the differential expression situation of intracellular protein.Dielectrophoresis generally can only be differentiated 1000～3000 protein (spot) at present, and the albumen kind in the sample can reach more than 100,000 kinds, and therefore the processing to sample is extremely important before doing dielectrophoresis.Sample preparation is the step of most critical in the dielectrophoresis, and the quality that this step processes will directly affect 2-DE result.But just present, do not have a standard of comparison or general sample preparation methods.Sample preparation methods commonly used all adopts three-step approach now: sample fragmentation, protein precipitation and removal impurity.The broken means of the existing sample that adopts very easily cause losing of albumen in the sample or cause albumen to be modified.In addition, also very easily cause the degraded of albumen and modified in the step of albumen precipitation.Remove deimpurity key problem in technology also is to reduce PD and protein modified as far as possible.This shows, so far, the preparation method of the protein example general technology of neither one still in the proteome analysis, need through a large amount of experiments grope with experience accumulation just can guarantee experimental result credible accurately.In the process of research albumen, both needed rigorous techniqueflow but can not stick to experience.Therefore, improve experiment flow and sample preparation technology, reduce PD, degraded (or destruction) and reduce the artificial modification of protein or modified, minimizing protein and the macromolecular interaction of other biological, and make protein be in complete denatured state.The efficient separation that realizes low abundance target protein point has great importance.
(3) summary of the invention
The present invention proposes a kind of method of catching cutting by tissue in situ collection and micro-amplification, can greatly reduce foreign protein content and degraded loses, reduce the artificial modification of albumen, effect is very good in the efficient separation of the low abundance target protein point of two dimensional gel electrophore-sis (2-DE) realization animal tissue.
The technical solution used in the present invention is:
A kind of preparation method of the animal protein quality sample for proteome analysis, described method comprises:
(1) tissue specimen obtains: peel off the epidermal tissue of earthworm, clean, suck unnecessary liquid with filter paper, the gained tissue specimen moves to-80 ℃ of Refrigerator stores after the quick-frozen in liquid nitrogen; Biological selection and tissue are determined: soil invertebrate---earthworm, determine to be organized as epidermal tissue.
(2) frozen section: after tissue specimen takes out from-80 ℃ of refrigerators, with-30 ℃ freezing microtome section, slice thickness 10 μ m, the 1st section of every routine sample dyeed with HE, and brazilwood extract dyeing is only done in later every section.The identification location that the 1st section HE dyeing is mainly used in organizing, the brazilwood extract dyeing of later every section is also to be in complete denatured state in order to ensure target protein in dyeing;
(3) micro-ly catch cutting: the section that will prepare is placed on the microscopical objective table, the selected zone that will cut of the lower comparative observation of direct-view, by maximum capture rate setup parameter, be specially: beam diameter (beam diameter) 60 μ m, beam energy (beam power) 80mV, laser transfer pulse (transfer pulses) is 1.6ms, catches cutting and obtains 40000～50000 cells, by the cell count of software to obtaining;
(4) sample preparation: the cell that cuts down adds to be organized in the lysis buffer, ice bath vibration 30～40min, again after 1～5 ℃ of lower ultrasonic processing 2～5 times, centrifugal (20000 * g, 1h), get supernatant, namely get the earthworm protein quality sample, it is for subsequent use to be stored in-80 ℃ of refrigerators, simultaneously with the protein concentration in the Coomassie brilliant blue method mensuration extract; The described lysis buffer of organizing is composed as follows: 8mol/L urea, 2mol/L thiocarbamide, 4%CHAPS, 65mmol/L DTT, 0.2% ampholyte (Ampholyte electrophoresis level, pH3.0～9.5, Amresco company), 10ml/L cocktail.
Preferably, step (4) tissue lysis buffer is composed as follows: 8mol/L urea, 2mol/L thiocarbamide, 4%CHAPS, 65mmol/L DTT, 0.2% ampholyte, 10ml/L cocktail.
In soil animal biomarker screening process, can adopt said method to obtain protein example, be further analyzed by the following method again:
(A) two dimensional gel electrophore-sis: get above-mentioned protein example 100 μ g and add heavily swelling liquid to final volume 350 μ l mixings.Sample is held in the glue groove even the adding, and glue faces down and puts into 17cm IPG immobilized ph gradient strip, drips mineral oil, places on the isoelectric focusing instrument, and heavily swelling is spent the night, and isoelectric focusing is carried out under 19 ℃ automatically.After the isoelectric focusing, the IPG adhesive tape is each balance 15min in equilibrium liquid A (containing 20g/L DTT) and equilibrium liquid B (containing the 25g/L iodoacetamide) respectively, IPG adhesive tape after the balance placed 12% even sds page top, the adhesive tape end drips the protein marker of wide region, the sealing of LMP agarose, the electrophoresis parameter: 5mA/ glue 1h, when treating that the bromophenol blue forward position moves into SDS glue, with 30mA/ glue 6h until the bromophenol blue forward position when arriving at apart from the about 0.5cm of glue feather edge till.
(B) cma staining: 40% ethanol+10% glacial acetic acid is 60min → 6.8% sodium acetate+30% ethanol+0.2%Na fixedly
2S
2O
3Sensitization 30min → rinsed with deionized water 5min * 3 time → 0.2% cma staining 20min → rinsed with deionized water 1min * 2 time → 2.5% natrium carbonicum calcinatum+0.04% formaldehyde colour developing: 5% glacial acetic acid stops 10min → 10% glycerine and preserves.
(C) gel images collection and analysis: application scanning instrument MICROTEK 4850II gathers gel images, under the same conditions, the total protein sample of two kinds of cells is carried out respectively 3 dielectrophoresis and graphical analysis, in homocellular protein electrophorese collection of illustrative plates, select protein spots clearly image as reference, mate with the image of other 2 electrophoresis, then the protein spots collection of illustrative plates with 3 electrophoresis gained creates average glue in PDQuest2Dversion 7.4 softwares, average glue to 2 kinds of cells mates, choose the point of expression (relative volume) increase more than 2 times and 2 times, draw the differential expression point.
(D) Mass Spectrometric Identification: choose differential protein particle on the glue, downcut along the burl dyeing edge with blade, place the Eppendof pipe, carry out digestion in the protein adhesive, then carry out the MALDI-TOF Mass Spectrometric Identification.By the protein database search program NCBInr on the internet (http://prospector.ucs.f edu).
(E) screening of biomarker is with definite: analyze the histiocytic protein profiling of epidermis, the feature of Study on Protein collection of illustrative plates and with the kind of pollutant in soil and the relation of content, determine to characterize the differential expression point of pollutant, find out corresponding mass spectrometric data, biomarker is determined in screening.
The screening of above-mentioned geobiont label can be useful in the early diagnosis and ecological risk assessment of POPs single and binary-combined contamination soil ecology toxicity
Beneficial effect of the present invention is mainly reflected in: fix by organizing freezing realization albumen original position, the micro-cutting of catching is carried out in the target area, the cell that cuts down is split the extraction total protein by tissue, experiment flow and sample preparation technology have been improved, reduced widely PD, degraded (or destroy) and to the artificial modification of protein, reduced the macromolecular interaction of protein and other biological, and can make protein be in complete denatured state, fine for the efficient identification separating effect of realizing low abundance target protein point, be applicable to the screening of protein biomarker with definite.
(4) description of drawings
Fig. 1 is the luxuriant and rich with fragrance Pollution exposure of earthworm 7 days and 14 days albumen two dimensional gel electrophore-sis figure of epidermal tissue, and Control is blank; Treatment is Pollution exposure group (Phe 12.5mgkg
-1The differential protein spot that the arrow indication is possible);
The peptide mass fingerprinting spectrum of Fig. 2 differential protein spot 971 is cut glue to differential protein spot 971, trypsin digestion, and MALDI-TOF/TOF analyzes; A, MS analysis chart, 1222.608 ion asterisk marks; B, 1222.608 ion MS/MS analysis charts.
Fig. 3 is that earthworm is luxuriant and rich with fragrance, the pyrene combined pollution exposes 14 days albumen two dimensional gel electrophore-sis figure of epidermal tissue, (Phe20.0mgkg
-1+ Pyr 300.0mgkg
-1The differential protein spot that the arrow indication is possible);
Fig. 4 is the peptide mass fingerprinting spectrum of differential protein spot 29, and differential protein spot 29 is cut glue, trypsin digestion, and MALDI-TOF/TOF analyzes.
(5) embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Embodiment 1: the screening of phenanthrene-polluted soil biomarker
(1) chronic toxicity test of earthworm
Adopt the soil of the luxuriant and rich with fragrance Single Pollution of artificial preparation, control group and experimental group are set, the concentration of experimental group phenanthrene is 12.5mgkg
-1Phe establishes 3 parallel group.From each experimental group and control group, get at random 3 individualities of various experimental animals respectively at 7d and 14d after the cultivation beginning.
(2) screening of biomarker is with definite:
1) preparation of protein sample: peel off the animal cuticle tissue that is extracted, clean up in brine ice (containing 0.1mmol/L PMSF), filter paper sucks more than liquid, places immediately the liquid nitrogen quick-frozen, then moves to-80 ℃ of Refrigerator stores.After tissue specimen takes out from-80 ℃ of refrigerators, move into immediately in the freezing microtome that temperature setting is set to-30 ℃, slice thickness 10 μ m, the 1st section of every routine sample dyeed with HE, and brazilwood extract dyeing is only done in later every section.The section for preparing is placed on the microscopical objective table, and lower observation of direct-view selected the zone that will cut, by maximum capture rate setup parameter.Be specially: beam diameter (beam diameter) 60 μ m, beam energy (beam power) 80mV, laser transfer pulse (transfer pulses) is 1.6ms, obtains 40000～50000 cells by this method, and by the cell count of software to obtaining.The cell that cuts down is by organizing lysis buffer (8mol/L urea, the 2mol/L thiocarbamide, 4%CHAPS, 65mmol/LDTT, 0.2% ampholyte (Ampholyte electrophoresis level, pH3.0～9.5, Amresco company), 10ml/Lcocktail) extract total protein, after adding lysate, ice bath vibration 30min is again behind the ultrasonic several of low temperature, give 4 ℃, the centrifugal 1h of 20 000 * g gets supernatant, packing, be stored in-80 ℃ of refrigerators, simultaneously with the protein concentration in the Coomassie brilliant blue method mensuration extract.
2) two dimensional gel electrophore-sis: get above-mentioned protein example 100 μ g and add heavily swelling liquid to final volume 350 μ l mixings.Sample is held in the glue groove even the adding, and glue faces down and puts into 17cm IPG immobilized ph gradient strip, drips mineral oil, places on the isoelectric focusing instrument, and heavily swelling is spent the night, and isoelectric focusing is carried out under 19 ℃ automatically.After the isoelectric focusing, the IPG adhesive tape is each balance 15min in equilibrium liquid A (containing 20g/L DTT) and equilibrium liquid B (containing the 25g/L iodoacetamide) respectively, IPG adhesive tape after the balance placed 12% even sds page top, the adhesive tape end drips the protein marker of wide region, the sealing of LMP agarose, the electrophoresis parameter: 5mA/ glue 1h, when treating that the bromophenol blue forward position moves into SDS glue, with 30mA/ glue 6h until the bromophenol blue forward position when arriving at apart from the about 0.5cm of glue feather edge till.
3) cma staining: 40% ethanol+10% glacial acetic acid is 60min → 6.8% sodium acetate+30% ethanol+0.2%Na fixedly
2S
2O
3Sensitization 30min → rinsed with deionized water 5min * 3 time → 0.2% cma staining 20min → rinsed with deionized water 1min * 2 time → 2.5% natrium carbonicum calcinatum+0.04% formaldehyde colour developing: 5% glacial acetic acid stops 10min → 10% glycerine and preserves.
4) gel images collection and analysis: application scanning instrument MICROTEK 4850II gathers gel images, under the same conditions, the total protein sample of two kinds of cells is carried out respectively 3 dielectrophoresis and graphical analysis, in homocellular protein electrophorese collection of illustrative plates, select protein spots clearly image as reference, mate with the image of other 2 electrophoresis, then the protein spots collection of illustrative plates with 3 electrophoresis gained creates average glue in PDQuest2Dversion 7.4 softwares, average glue to 2 kinds of cells mates, choose the point of expression (relative volume) increase more than 2 times and 2 times, draw the differential expression point.
5) Mass Spectrometric Identification: choose differential protein particle on the glue, downcut along the burl dyeing edge with blade, place the Eppendof pipe, carry out digestion in the protein adhesive, then carry out the MALDI-TOF Mass Spectrometric Identification.By the protein database search program NCBInr on the internet (http://prospector.ucs.f edu).
6) screening of biomarker is with definite:
Analyze the histiocytic protein profiling of epidermis, the feature of Study on Protein collection of illustrative plates and with the kind of pollutant in soil and the relation of content, determine to characterize the differential expression point of pollutant, find out corresponding mass spectrometric data, the definite biomarker of screening.
By the comparative analysis protein profiling, exposed in the 7th day as a result and find 48 differential protein spots, exposed in the 14th day and find 73 differential protein spots, by gene expression abundance analysis and Mass Spectrometric Identification, filter out 31 kinds of significant albumen, wherein 17 kinds of albumen are expressed at the earthworm epidermal cell and are obviously increased, and 14 kinds of protein expression downward modulations are the results detailed in Fig. 1, Fig. 2; Table 1, table 2.
31 kinds of significant albumen that filter out can be defined as the luxuriant and rich with fragrance contaminated soil biomarker of low concentration, can be used for early diagnosis and the ecological risk assessment of eco-toxicity.
Embodiment 2: the screening of phenanthrene, pyrene combined contamination soil biomarker
(1) chronic toxicity test of earthworm
Adopt the soil of phenanthrene, pyrene Single Pollution and the combined pollution of artificial preparation, according to the form below carries out the concentration gradient setting of pollutant, establishes 3 parallel group.From each experimental group and control group, get at random 3 individualities of various experimental animals respectively at 7d and 14d after the cultivation beginning.
Table 3: the concentration gradient setting of pollutant
(2) screening of biomarker is with definite:
Peel off the animal cuticle tissue that is extracted, clean up in brine ice (containing 0.1mmol/L PMSF), filter paper sucks more than liquid, places immediately the liquid nitrogen quick-frozen, then moves to-80 ℃ of Refrigerator stores.By embodiment 1 method the animal cuticle tissue of peeling off is carried out protein example preparation, two dimensional gel electrophore-sis, cma staining, gel images collection and the operations such as analysis and Mass Spectrometric Identification, comparative analysis protein profiling.Find out differential protein spot, by gene expression abundance analysis and Mass Spectrometric Identification, filter out significant albumen, determine the biomarker of phenanthrene, pyrene combined contamination soil, be used for early diagnosis and the ecological risk assessment of eco-toxicity.This embodiment is the results detailed in Fig. 3, Fig. 4.
Fig. 3 shows, through cma staining, can find that the collection of illustrative plates background of dielectrophoresis is lower, can detect above 600 comparatively obvious protein sites, they mainly are distributed between molecular weight 15～80kD, isoelectric point pI3～8.5, and the separating effect of protein site is more satisfactory.Take protein site 29 as example, can detect 22 obvious peptide Duan Feng (Fig. 4), the result of data retrieval, its sequential covering degree has reached 32%, must be divided into 114, error range is below 1%, and the isoelectric point by software prediction and molecular weight all relatively conform to demonstration in the collection of illustrative plates.
The luxuriant and rich with fragrance albumen that pollutes down-regulated expression in the earthworm epidermal tissue of table 1 soil
The luxuriant and rich with fragrance albumen that pollutes up-regulated in the earthworm epidermal tissue of table 2 soil
Claims (1)
1. preparation method who is used for the animal protein quality sample of proteome analysis, described method comprises:
(1) tissue specimen obtains: peel off the epidermal tissue of earthworm, clean, suck unnecessary liquid with filter paper, the gained tissue specimen moves to-80 ℃ of Refrigerator stores after the quick-frozen in liquid nitrogen;
(2) frozen section: after tissue specimen takes out from-80 ℃ of refrigerators, with-30 ℃ freezing microtome section, slice thickness 10 μ m, the 1st section of every routine sample dyeed with HE, and brazilwood extract dyeing is only done in later every section;
(3) micro-ly catch cutting: the stained for preparing is placed on the microscopical objective table, the selected zone that will cut of the lower comparative observation of direct-view, by maximum capture rate setup parameter, be specially: beam diameter 60 μ m, beam energy 80 mV, the laser transfer pulse is 1.6 ms, catches cutting and obtains 40 000 ~ 50 000 cells, by the cell count of software to obtaining;
(4) sample preparation: the cell that cuts down adds to be organized in the lysis buffer, and ice bath vibration 30 ~ 40 min are after 1 ~ 5 ℃ of lower ultrasonic processing 2 ~ 5 times, centrifugal again, get supernatant, namely get the earthworm protein quality sample, and it is for subsequent use to be stored in-80 ℃ of refrigerators; The described lysis buffer of organizing is composed as follows: 8 mol/L urea, 2 mol/L thiocarbamides, 4 % CHAPS, 65 mmol/L DTT, 0. 2% ampholyte, 10 ml/L cocktail.
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