CN104076114B - A kind of binary channels SPE post and the application at quantitative proteomics thereof - Google Patents
A kind of binary channels SPE post and the application at quantitative proteomics thereof Download PDFInfo
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Abstract
The present invention relates to a kind of novel Solid-Phase Extraction (SPE) device realizing Protein quantitative analysis pre-treatment overall process, this device utilizes dividing plate that SPE pipe is divided into two isopyknic chambers, after filling chromatograph packing material, can the sex change of in-situ accomplishes protein example, reduction, alkylation, enzymolysis, the overall process of sample preparation in the quantitative proteomics such as isotope labeling and mixing.This device is simple for production, and cost of manufacture is low, has that the recovery is high, easy to operate, quantitative result accuracy is good, precision advantages of higher during processing sample.
Description
Technical field
The present invention devises a kind of twin-channel SPE device, can realize the overall process of the quantitative protein sample preparation such as the sex change of protein example original position, reduction, alkylation, enzymolysis, mark and desalination mixing.
Background technology
Quantitative proteomics is the Hot Contents of systems biology research, it, for finding disease biomarkers, finds protein-protein interaction, and drug-protein interacts, explain disease genesis mechanism, explain that biological metabolism process etc. provides very important Data support.Protein is agent and the undertaker of vital movement, content is very abundant in vivo, participate in all metabolic processes of life entity, thus the change of protein content can cause physiological change, and for the mankind, then be presented as the generation of disease, the accurate quantitative analysis thus realizing protein is very important for understanding the pathogenetic mechanism of disease.
Existing quantivative approach is mainly based on liquid chromatography mass method for combined use, and this wherein uses particularly extensive, because the method is applicable to the protein example in any source based on the cold labeling method of chemical labeling.The sample pretreatment process of the method comprises Protein Extraction, urea-denatured, dithiothreitol (DTT) (DTT) reduces, iodoacetamide (IAA) alkylation, trypsin digestion and di-methylation mark, end mark and the anti-phase desalination of C18, the as easy as rolling off a log sample loss of so many operating process and pollution.Existing solution filters auxiliary sample preparation (FASP) (NatMethods, 2009.6:359), but the method sample recovery rate is low and cannot in the analysis of chemical labeling sample; Heck etc. realize di-methylation mark (NatureProtocols, 2009.4:484) in SPE, but the method ignores the front sample loss of mark.
Summary of the invention
In order to solve the problem, the object of the invention is to develop a kind of twin-channel SPE post, all processes of in-situ accomplishes protein example process, to parallel processing two kinds of samples, reduce sample loss, realize efficient and easy sample pretreatment and obtain the quantitative result of pin-point accuracy and high precision.
For achieving the above object, the technical solution used in the present invention is:
A kind of binary channels SPE post,
Comprise cylinder or toot that the airtight cross-sectional diameter in upper end open, lower end is 50 μm of-5cm, be provided with plunger or sieve plate in the sealed end place of internal tank; Be axially arranged with a dividing plate along internal tank, the internal cavities of container is separated into two identical equal portions of left and right volume by dividing plate, forms two passages; Seal the chromatograph packing material of the matter equivalent such as the interior filling of backward two passages; Be provided with efflux outlet in sealed end, the central axis of efflux outlet is on dividing plate.
Container comprises: 20-5000 μ L pipettor gun head, and 1-20ml Solid-Phase Extraction (SPE) is managed, the one in 1-20ml syringe needle tube.
Described plunger can be the integral post plunger of fabricated in situ in column jecket; Sieve plate is which is provided with the sieve plate that aperture is 3nm-20 μm of through hole.
Filler in two passages of same SPE can be reverse phase filler or ion-exchange packing;
Reverse phase filler comprises: the one in C18 reverse phase filler, C8 reverse phase filler, C4 reverse phase filler, and particle diameter is 1.7 μm of .-100 μm; Ion-exchange packing comprises: the one in SCX filler and WCX filler; Filler form comprises: particle type materials or integral material.
To be provided with the pipe of hollow away from vessel side in efflux outlet, pipe by efflux export respectively with two mutually not communicating passage be connected, dividing plate and plunger or sieve plate is airtight is connected, dividing plate is connected with container inner wall is airtight; Described dividing plate is the plastic foil of teflon paillon foil or the anti-organic solvent of other waterproof.
Described binary channels SPE post is in the application of quantitative proteomics.
For protein example processing procedure, 1) in two passages of SPE post, 100 μ L denaturant solution are added respectively (as 8M urea, 6M guanidine hydrochloride or 1%SDS etc.) protein of 1-1000 μ g that dissolves, adding reductive agent subsequently makes final concentration be 10mM(dithiothreitol (DTT) (DTT), trichloroethyl phosphate (TCEP), or beta-mercaptoethanol etc.) carry out the reduction of protein, adding alkylating reagent subsequently makes final concentration be 55mM(iodo acetic acid or iodoacetamide) carry out the alkylation process of protein, after denaturant concentration being diluted 10 times with buffer salt solution, according to albumen: enzyme=50:1 (w/w) adds 37 DEG C of water enzyme digestions that proteinase carries out protein and spends the night, due to the existence of dividing plate, above reagent can be joined respectively in two passages, after enzymolysis completes, first rinse with playing level pad (equalizing and buffering is the aqueous solution of volumetric concentration 0.1%TFA), 2) then in two passages, labelled reagent is added respectively, after having marked, first rinse with playing level pad (equalizing and buffering is the aqueous solution of volumetric concentration 0.1%TFA), then use elution buffer (when filler is reverse phase filler, elution buffer is the volumetric concentration 80%CAN aqueous solution containing volumetric concentration 0.1% trifluoroacetic acid, or when filler is SCX chromatograph packing material, elution buffer is the Phosphorylation Buffer of the 10-20mM containing 1MNaCl of pH3.0) carry out wash-out,
When filler is SCX or WCX, also need to carry out desalination (with C18 trapping column desalination); Evaporate to dryness, for mass spectrophotometry.
Buffer salt solution described in step (1) is: the one in phosphate buffer, ammonium bicarbonate buffers, borate buffer solution;
The solvent of the labelled reagent described in step (2) and correspondence can be:
A: formaldehyde and sodium cyanoborohydride, deuterated formaldehyde and sodium cyanoborohydride or
13the deuterated formaldehyde of C-and cyano group boron deuterate sodium, solvent can be phosphate buffer, borate buffer solution or triethylamine-carbonic acid buffer (TEAB damping fluid), the addition of labelled reagent be the formaldehyde of every 100 μ g protein 16 μ L body volumetric concentration 0.01-40%, deuterated formaldehyde or
13the sodium cyanoborohydride of the deuterated formaldehyde of C-and 16 μ L0.006-6M or cyano group boron deuterate sodium;
Or b:iTRAQ or mTRAQ reagent, dissolution system is the TEAB solution of the 0.01-1M being dissolved in volumetric concentration 70% ethanol water, and the addition of labelled reagent is iTRAQ or the mTRAQ reagent that every 100 μ g protein need 100 μ L0.01-1M.
Protein is extracting solution of protein or the standard protein of the tissue of biological sample, cell or body fluid; Proteinase can select alkaline protein as one or two or more kinds in trypsase, endopeptidase Arg-C, endopeptidase Lys-C, chymotrypsin or elastoser, and denaturant is the one in urea, guanidine hydrochloride, SDS; Reductive agent is the one in dithiothreitol (DTT) (DTT), trichloroethyl phosphate (TCEP), beta-mercaptoethanol; Alkylating reagent is the one in iodo acetic acid, iodoacetamide.
1, the manufacturing process of binary channels SPE: SPE pipe be separated into two equal portions with paper knife or be directly moulded two equal portions, upper sieve plate is added in the bottom of passage, using the thin slice of non-watertight anti-organic solvent as division board in the middle of passage, packing chromatography filler after sealing, as shown in Figure 1.
2, protein example processing procedure: respectively add the protein being dissolved in denaturant and add the DTT that final concentration is 10mM in two passages, at room temperature reaction 1h, then the IAA lucifuge reaction 30min of DTT stoichiometry 2 times is added, then after diluting 10 times with damping fluid, according to 1:20 (enzyme/albumen, w/w) proteinase is added, room temperature reaction 1-3h, labelled reagent is added respectively in two passages, SPE is flow through by Action of Gravity Field, use Equilibration buffer wash two SPE passages in mobile phase subsequently, finally collect cut with the elution buffer wash-out in mobile phase.
Tool of the present invention has the following advantages:
1. binary channels SPE Column preparation is simple.SPE is separated into two equal portions, adds upper spacer and seal.
2. easy and simple to handle.The overall process of sample pretreatment, only needs two passages to SPE to add albumen and reaction reagent.
3. high-recovery.Adopt chromatograph packing material, the reservation of protein example in different courses of reaction on chromatograph packing material can be ensured, avoid causing sample loss in processing procedure.In addition, whole sample pretreatment process all in SPE original position carry out, avoid the loss that transfer, inboard wall of test tube absorption etc. cause, the recovery high (Fig. 2), without the need to mixing, when avoiding sample shift, taking-up volume not etc. does not cause quantitative result and theoretical value not to be inconsistent.
4. pin-point accuracy and high precision quantitative.Achieve the quantitative test of complex sample, due to manipulation in situ, sample parallel process and loss is few, quantification of protein result and theoretical value meet better, and the reappearance of analysis is good.
Accompanying drawing explanation
Fig. 1 binary channels SPE structural representation; The central dividing plate of 1: binary channels SPE, two passages of 2: binary channels SPE, 3: chromatograph packing material, 4: sieve plate, 5: efflux exports;
Fig. 2 binary channels SPE labeling effciency is investigated;
Fig. 3 SPE recovery is investigated;
Fig. 4 actual sample carries out process quantification of protein distribution of results figure through binary channels SPE.
Embodiment
In order to investigate the effect of binary channels SPE to quantification of protein, use this device to process protein example, concrete steps are as follows:
1, the manufacturing process of binary channels SPE: be separated into two equal portions by about effective for 1mLSPE paper knife, upper sieve plate is added in the bottom of passage, using polytetrafluoroethylene film as division board in the middle of passage, and with plastic cement rod, dividing plate and SPE column jecket are sealed, fill C18 reverse-phase chromatography filler (40 μm of particle diameters) respectively.
2, protein example processing procedure: the BSA being dissolved in 8M urea respectively adding 100 μ L0.1 μ g/ μ L in two passages, and add the 0.1mol/L dithiothreitol (DTT) room temperature reaction 1h of 1 μ L, then 2 μ L0.1M iodoacetamide lucifuge reaction 30min are added, then after using 50mM phosphate buffer (pH8.0) to dilute 10 times, according to 1:20 (enzyme/albumen, w/w) trypsase is added, room temperature reaction 3h, adds 8 μ L respectively and contains 0.4% (v/v) formalin (CH in two passages
2and 0.06mmol/LNaCNBH O)
3(sodium cyanoborohydride) mixed solution, and 8 μ L contain 0.4% deuterated formaldehyde (CD
2and 0.06mmol/LNaCNBH O)
3solution, and flow through SPE by Action of Gravity Field.Then rinse two SPE passages with containing 0.1%TFA1mL aqueous solution, finally with containing 0.1%TFA1mLACN eluant solution, collect cut.Analyze the peptide section obtained with MALDI-TOF (Bruker, Germany), result as shown in Figure 2.Can see and use this device mark peptide section, labeling effciency can close to 100%.
Then, the 0.1mol/LDTT room temperature reaction 1h adding 10 μ L is dissolved in the BSA of 8M urea to 100 μ L1 μ g/ μ L, then 20 μ L0.1MIAA lucifuge reaction 30min are added, then after using 50mM phosphate buffer (pH8.0) to dilute 10 times, according to 1:20 (enzyme/albumen, w/w) add trypsase, 37 DEG C reaction 3h, add 16 μ L contain 4% deuterated formaldehyde (
13cD
2o) and 16 μ L0.6mmol/LNaCNBD
3solution.Get the recovery that 100 these solution of μ L and the eluent of above-mentioned SPE process mix to investigate this device, result as shown in Figure 3, can find out that the recovery of SPE is close to 100%, even exceedes the recovery under free solution condition.
3, then utilize morse ascites hepatoma clones with different metastatic ability lymphatic channel low transfer cell line protein extract to investigate this device in the quantitative effect of complex sample, result as shown in Figure 4.Concrete steps are: respectively add in two passages 100 μ L0.1 μ g/ μ L be dissolved in 8M urea low transfer cell line protein extract and add the 0.1mol/LDTT room temperature reaction 1h of 1 μ L, then 2 μ L0.1MIAA lucifuge reaction 30min are added, then after using 50mM phosphate buffer (pH8.0) to dilute 10 times, according to 1:20 (enzyme/albumen, w/w) trypsase is added, room temperature reaction 3h, adds 16 μ L respectively and contains 0.4% formalin (CH in two passages
2and 0.06mmol/LNaCNBH O)
3(sodium cyanoborohydride) mixed solution, and 500 μ L contain 0.4% deuterated formaldehyde (CD
2and 0.06mmol/LNaCNBH O)
3solution, and flow through SPE by Action of Gravity Field.Then rinse two SPE passages with containing 0.1%TFA1mL aqueous solution, finally with containing 0.1%TFA1mLACN eluant solution, collect cut.As can be seen from Figure 4, only have a kind of log2 value of protein to be greater than 1, embody the accuracy of quantitative result.Owing to testing the low transitional cell protein extract that sample used is equivalent, so quantitative result theoretical value is 1, testing the result obtained is 1.009, conform to preferably with theoretical value, the RSD=16.47% of all protein quantification results simultaneously, this RSD lower than sample pretreatment in conventional soln (about 20%).To sum up, the quantitative sample pretreating method based on twin-channel SPE can realize pin-point accuracy and high-accuracy quantitative result.
Claims (9)
1. a binary channels SPE post, is characterized in that:
Comprise cylinder or toot that the airtight cross-sectional diameter in upper end open, lower end is 50 μm of-5cm, be provided with plunger or sieve plate in the sealed end place of internal tank; Be axially arranged with a dividing plate along internal tank, the internal cavities of container is separated into two identical equal portions of left and right volume by dividing plate, forms two passages; Seal the chromatograph packing material of the matter equivalent such as the interior filling of backward two passages; Be provided with efflux outlet in sealed end, the central axis of efflux outlet is on dividing plate.
2. binary channels SPE post according to claim 1, is characterized in that: container comprises: 20-5000 μ L pipettor gun head, 1-20ml Solid-Phase Extraction (SPE) pipe or 1-20ml syringe needle tube.
3. binary channels SPE post according to claim 1, is characterized in that: described plunger can be the integral post plunger of fabricated in situ in column jecket; Sieve plate is which is provided with the sieve plate that aperture is 3nm-20 μm of through hole.
4. binary channels SPE post according to claim 1, is characterized in that:
Filler in two passages of same SPE can be reverse phase filler or ion-exchange packing;
Reverse phase filler comprises: C18 reverse phase filler, C8 reverse phase filler or C4 reverse phase filler, and particle diameter is 1.7 μm of .-100 μm; Ion-exchange packing comprises: SCX filler or WCX filler; Filler form comprises: particle type materials or integral material.
5. binary channels SPE post according to claim 1, is characterized in that:
To be provided with the pipe of hollow away from vessel side in efflux outlet, pipe by efflux export respectively with two mutually not communicating passage be connected, dividing plate and plunger or sieve plate is airtight is connected, dividing plate is connected with container inner wall is airtight; Described dividing plate is the plastic foil of teflon paillon foil or the anti-organic solvent of other waterproof.
6. binary channels SPE post described in a claim 1 is in the application of quantitative proteomics.
7. application according to claim 6, is characterized in that: for protein example processing procedure, 1) in two passages of SPE post, 100 μ L8M urea are added respectively, the protein of the 1-1000 μ g that 6M guanidine hydrochloride or 1%SDS denaturant solution are dissolved, adding reductive agent subsequently makes final concentration be the reduction that 10mM carries out protein, add iodo acetic acid subsequently or iodoacetamide makes final concentration be the alkylation process that 55mM carries out protein, after denaturant concentration being diluted 10 times with buffer salt solution, according to albumen: enzyme=50:1 (w/w) adds 37 DEG C of water enzyme digestions that proteinase carries out protein and spends the night, due to the existence of dividing plate, above reagent is added respectively in two passages, after enzymolysis completes, first rinse by the aqueous solution containing volumetric concentration 0.1%TFA, 2) then in two passages, labelled reagent is added respectively, after having marked, first rinse by the aqueous solution containing volumetric concentration 0.1%TFA, then wash-out is carried out with elution buffer, when filler is reverse phase filler, elution buffer is the volumetric concentration 80%CAN aqueous solution containing volumetric concentration 0.1% trifluoroacetic acid, or when filler is SCX chromatograph packing material, elution buffer is the Phosphorylation Buffer of the 10-20mM containing 1MNaCl of pH3.0,
When filler is SCX or WCX, also need to carry out desalination by C18 trapping column; Evaporate to dryness, for mass spectrophotometry.
8. application according to claim 7, is characterized in that: the buffer salt solution described in step (1) is: the one in phosphate buffer, ammonium bicarbonate buffers, borate buffer solution;
The solvent of the labelled reagent described in step (2) and correspondence is:
A: formaldehyde and sodium cyanoborohydride, deuterated formaldehyde and sodium cyanoborohydride or
13the deuterated formaldehyde of C-and cyano group boron deuterate sodium, solvent is phosphate buffer, borate buffer solution or triethylamine-carbonic acid buffer (TEAB damping fluid), the addition of labelled reagent be the formaldehyde of every 100 μ g protein 16 μ L volumetric concentration 0.01-40%, deuterated formaldehyde or
13the sodium cyanoborohydride of the deuterated formaldehyde of C-and 16 μ L0.006-6M or cyano group boron deuterate sodium;
Or b:iTRAQ or mTRAQ reagent, dissolution system is the TEAB solution of the 0.01-1M being dissolved in volumetric concentration 70% ethanol water, and the addition of labelled reagent is iTRAQ or the mTRAQ reagent that every 100 μ g protein need 100 μ L0.01-1M.
9. application according to claim 7, is characterized in that:
Protein is extracting solution of protein or the standard protein of the tissue of biological sample, cell or body fluid; Proteinase is one or two or more kinds in trypsase, endopeptidase Arg-C, endopeptidase Lys-C, chymotrypsin or elastoser, and denaturant is the one in urea, guanidine hydrochloride, SDS; Reductive agent is the one in dithiothreitol (DTT) (DTT), trichloroethyl phosphate (TCEP), beta-mercaptoethanol; Alkylating reagent is the one in iodo acetic acid, iodoacetamide.
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| CN106146607A (en) * | 2015-04-09 | 2016-11-23 | 深圳华大基因研究院 | One peptide species method for extraction and purification and test kit |
| CN106399432B (en) * | 2015-07-31 | 2020-05-26 | 中国科学院大连化学物理研究所 | Method for preparing N-linked glycopeptide from monoclonal antibody and N-linked glycopeptide |
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| CN105866317B (en) * | 2016-03-31 | 2018-06-29 | 南方科技大学 | The protein group reactor and its application of integrated protein pre-treatment and the classification of polypeptide high ph-values reverse phase |
| CN108088727B (en) * | 2016-11-21 | 2020-07-07 | 中国科学院大连化学物理研究所 | Pretreatment method of protein in-vitro body fluid |
| CN108226317B (en) * | 2016-12-21 | 2020-10-20 | 复旦大学 | Method for performing whole proteomics analysis based on cell level |
| CN109839469A (en) * | 2017-11-28 | 2019-06-04 | 中国科学院大连化学物理研究所 | A kind of protein analysis method based on hydrophobic amino acid specific protease |
| CN110609103A (en) * | 2018-06-15 | 2019-12-24 | 中国科学院大连化学物理研究所 | Protein and material interaction structure analysis method based on chemical labeling and mass spectrometry |
| CN108982787A (en) * | 2018-07-30 | 2018-12-11 | 绿城农科检测技术有限公司 | A method of for detecting melamine in meat |
| CN114544846B (en) * | 2022-02-24 | 2023-01-31 | 河海大学 | Resistance gene research method under influence of tide in coastal region and solid phase extraction instrument |
| CN115184526A (en) * | 2022-05-31 | 2022-10-14 | 南方科技大学 | Mixed-mode-filler-based small-amount cell proteomics reactor and application thereof |
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