CN104076114A - Double-channel SPE column and application of double-channel SPE column in quantitative proteomics - Google Patents

Abstract

The invention relates to a novel solid phase extraction (SPE) device capable of achieving the whole process of quantitative analysis pretreatment of protein. According to the device, an SPE tube is partitioned into two chambers with the same capacity through a partition plate, and after the chambers are filled with chromatographic packing, the whole process of sample treatment in quantitative proteomics can be achieved in situ, wherein the treatment comprises denaturation, reduction, alkylation, enzymolysis, isotope labeling and mixing of protein samples. The device is easy and convenient to manufacture and low in manufacturing cost, and has the advantages that the recovery rate is high, operation is easy, the accuracy of quantitative results is good and the precision is high when the samples are treated.

Description

A kind of binary channels SPE post and in the application of quantitative proteomics

Technical field

The present invention has designed 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 biomarker, finds protein-protein interaction, medicine-protein-interacting, explain disease genesis mechanism, explanation biology metabolic 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, thereby the variation of protein content can cause physiological variation, and for the mankind, be presented as the generation of disease, thereby it is very important for understanding the pathogenetic mechanism of disease to realize the accurate quantitative analysis of protein.

Existing quantivative approach is mainly based on liquid chromatography mass method for combined use, and wherein the use of the cold labeling method based on chemical labeling is particularly extensive for this, because the method is applicable to the protein example in any source.The sample pretreatment process of the method comprises Protein Extraction, urea-denatured, dithiothreitol (DTT) (DTT) reduction, iodoacetamide (IAA) alkylation, trypsin digestion and dimethyl mark, the anti-phase desalination of end mark and C18, the as easy as rolling off a log sample loss of so many operating process and pollution.Existing solution is to filter auxiliary sample preparation (FASP) (Nat Methods, 2009.6:359), but the method sample recovery rate is low and cannot be in the analysis of chemical labeling sample; Heck etc. realize dimethyl mark (Nature Protocols, 2009.4:484) in SPE, but the method has been ignored the front sample loss of mark.

Summary of the invention

In order to address the above problem, the object of the invention is to develop a kind of twin-channel SPE post, all processes that in-situ accomplishes protein example is processed, to two kinds of samples of parallel processing, reduce sample loss, realize efficient and easy sample pretreatment and obtain pin-point accuracy and the quantitative result of high precision.

For achieving the above object, the technical solution used in the present invention is:

A binary channels SPE post,

Comprise that upper end open, the airtight cross-sectional diameter in lower end are cylinder or the toot of 50 μ m-5cm, in the sealed end place of internal tank, be provided with plunger or sieve plate; Along internal tank, be axially arranged with a dividing plate, dividing plate is separated into by the internal cavities of container two equal portions that left and right volume is identical, forms two passages; Seal the chromatograph packing material of the matter equivalent such as the interior filling of backward two passages; In sealed end, be provided with efflux outlet, the central axis of efflux outlet is on dividing plate.

Container comprises: 20-5000 μ L pipettor gun head, 1-20ml Solid-Phase Extraction (SPE) pipe, a kind of in 1-20ml syringe needle tube.

Described plunger can be the synthetic integral post plunger of original position in column jecket; Sieve plate is to which is provided with the sieve plate that aperture is 3nm-20 μ m through hole.

Filler in two passages of same SPE can be reverse phase filler or ion-exchange packing;

Reverse phase filler comprises: a kind of in C18 reverse phase filler, C8 reverse phase filler, C4 reverse phase filler, and particle diameter is 1.7 μ m.-100 μ m; Ion-exchange packing comprises: a kind of in SCX filler and WCX filler; Filler form comprises: granular pattern material or integral material.

In efflux outlet, away from vessel side, be provided with the pipe of hollow, pipe by efflux export respectively with two mutually not communicating passage be connected, dividing plate is with 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, add respectively 100 μ L denaturant solution (as 8M urea, 6M guanidine hydrochloride or 1%SDS etc.) protein of the 1-1000 μ g that dissolves, adding subsequently reductive agent to make final concentration is 10mM(dithiothreitol (DTT) (DTT), trichloroethyl phosphate (TCEP), or beta-mercaptoethanol etc.) carry out the reduction of protein, adding subsequently alkylating reagent to make final concentration is 55mM(iodo acetic acid or iodoacetamide) alkylation of carrying out protein processes, with buffer salt solution by after 10 times of denaturant concentration dilutions, 37 ℃ of water enzyme digestions that add proteinase to carry out protein according to albumen: enzyme=50:1 (w/w) spend the night, existence due to dividing plate, can in two passages, join respectively above reagent, after enzymolysis completes, first with playing level pad (equalizing and buffering is the aqueous solution of volumetric concentration 0.1%TFA), rinse, 2) then in two passages, add respectively labelled reagent, after mark completes, first with playing level pad (equalizing and buffering is the aqueous solution of volumetric concentration 0.1%TFA), rinse, then use elution buffer (when filler is reverse phase filler, elution buffer is the volumetric concentration 80%CAN aqueous solution that contains volumetric concentration 0.1% trifluoroacetic acid, or when filler is SCX chromatograph packing material, elution buffer is pH3.0 containing the phosphorylation damping fluid of the 10-20mM of 1M NaCl) 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: a kind of in phosphate buffer, ammonium bicarbonate buffers, borate buffer solution;

Labelled reagent described in step (2) and corresponding solvent 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 that is dissolved in the 0.01-1M of volumetric concentration 70% ethanol water, the addition of labelled reagent is iTRAQ or the mTRAQ reagent of every 100 μ g protein requirement 100 μ L0.01-1M.

Protein is extracting solution of protein or the standard protein of tissue, cell or the body fluid of biological sample; 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 a kind of in urea, guanidine hydrochloride, SDS; Reductive agent is a kind of in dithiothreitol (DTT) (DTT), trichloroethyl phosphate (TCEP), beta-mercaptoethanol; Alkylating reagent is a kind of in iodo acetic acid, iodoacetamide.

1, the manufacturing process of binary channels SPE: SPE pipe is separated into two equal portions or is directly moulded two equal portions with paper knife, bottom at passage adds upper sieve plate, the thin slice of non-watertight anti-organic solvent using in the middle of passage as division board, packing chromatography filler after sealing, as shown in Figure 1.

2, protein example processing procedure: respectively add in two passages that to be dissolved in the protein of denaturant and to add final concentration be the DTT of 10mM, at room temperature reaction 1h, then the IAA lucifuge reaction 30min that adds 2 times of DTT stoichiometries, then with after 10 times of damping fluid dilutions, according to 1:20 (enzyme/albumen, w/w) add proteinase, room temperature reaction 1-3h, in two passages, add respectively labelled reagent, by Action of Gravity Field, flow through SPE, with level pad in mobile phase, rinse two SPE passages subsequently, finally with the elution buffer wash-out in mobile phase and collect cut.

Tool of the present invention has the following advantages:

1. the preparation of binary channels SPE post is simple.SPE is separated into two equal portions, adds upper spacer sealing.

2. easy and simple to handle.The overall process of sample pretreatment, only need to add albumen and reaction reagent to two passages of SPE.

3. high-recovery.Adopt chromatograph packing material, can guarantee protein example reservation on chromatograph packing material in different courses of reaction, avoid causing in processing procedure sample loss.In addition, whole sample pretreatment process all in SPE original position carry out, the loss of having avoided transfer, inboard wall of test tube absorption etc. to cause, the recovery high (Fig. 2), without mixing, takes out volume and does not wait and cause quantitative result and theoretical value not to be inconsistent while avoiding sample to shift.

4. pin-point accuracy and high precision are quantitative.Realized the quantitative test of complex sample, due to former bit manipulation, sample parallel processing and loss are few, and 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; 1: the central dividing plate of binary channels SPE, 2: two passages of binary channels SPE, 3: chromatograph packing material, 4: sieve plate, 5: efflux outlet;

Fig. 2 binary channels SPE labeling effciency is investigated;

Fig. 3 SPE recovery is investigated;

Fig. 4 actual sample is processed 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: the effective paper knife of 1mL SPE left and right is separated into two equal portions, bottom at passage adds upper sieve plate, in the middle of passage, using polytetrafluoroethylene film as division board, and with plastic cement rod, dividing plate and SPE column jecket are sealed, fill respectively C18 reverse-phase chromatography filler (40 μ m particle diameter).

2, protein example processing procedure: the BSA that is dissolved in 8M urea that respectively adds 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 add 2 μ L0.1M iodoacetamide lucifuge reaction 30min, then after using 50mM phosphate buffer (pH8.0) to dilute 10 times, according to 1:20 (enzyme/albumen, w/w) add trypsase, room temperature reaction 3h adds respectively 8 μ L to contain 0.4% (v/v) formalin (CH in two passages ₂o) and 0.06mmol/LNaCNBH ₃(sodium cyanoborohydride) mixed solution, and 8 μ L contain 0.4% deuterated formaldehyde (CD ₂o) and 0.06mmol/L NaCNBH ₃solution, and flow through SPE by Action of Gravity Field.Then with containing 0.1%TFA1mL aqueous solution, rinse two SPE passages, finally with containing 0.1%TFA1mL ACN eluant solution, collect cut.With MALDI-TOF (Bruker, Germany), analyze the peptide section obtaining, result as shown in Figure 2.Can see and use this device mark peptide section, labeling effciency can approach 100%.

Then, to the 0.1mol/LDTT room temperature reaction 1h that adds 10 μ L in the BSA that is dissolved in 8M urea of 100 μ L1 μ g/ μ L, then add 20 μ L0.1M IAA lucifuge reaction 30min, then after using 50mM phosphate buffer (pH8.0) to dilute 10 times, according to 1:20 (enzyme/albumen, w/w) add trypsase, 37 ℃ reaction 3h, add 16 μ L contain 4% deuterated formaldehyde ( ¹³cD ₂o) and 16 μ L0.6mmol/LNaCNBD ₃solution.Get eluent that 100 these solution of μ L process with above-mentioned SPE and mix to investigate the recovery of this device, result as shown in Figure 3, can find out that the recovery of SPE approaches 100%, even surpasses the recovery under free solution condition.

3, then utilize the low transfer cell line protein extract of morse ascites hepatoma clones with different metastatic ability lymphatic channel to investigate this device in the quantitative effect of complex sample, result as shown in Figure 4.Concrete steps are: in two passages, respectively add 100 μ L0.1 μ g/ μ L be dissolved in 8M urea low transfer cell line protein extract and add the 0.1mol/L DTT room temperature reaction 1h of 1 μ L, then add 2 μ L0.1M IAA lucifuge reaction 30min, then after using 50mM phosphate buffer (pH8.0) to dilute 10 times, according to 1:20 (enzyme/albumen, w/w) add trypsase, room temperature reaction 3h adds respectively 16 μ L to contain 0.4% formalin (CH in two passages ₂o) and 0.06mmol/L NaCNBH ₃(sodium cyanoborohydride) mixed solution, and 500 μ L contain 0.4% deuterated formaldehyde (CD ₂o) and 0.06mmol/L NaCNBH ₃solution, and flow through SPE by Action of Gravity Field.Then with containing 0.1%TFA1mL aqueous solution, rinse two SPE passages, finally with containing 0.1%TFA1mL ACN 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, embodied 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, the result that experiment obtains 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 (approximately 20%).To sum up, the quantitative sample pretreating method based on twin-channel SPE can be realized pin-point accuracy and high-accuracy quantitative result.

Claims (9)

1. a binary channels SPE post, is characterized in that:

Comprise that upper end open, the airtight cross-sectional diameter in lower end are cylinder or the toot of 50 μ m-5cm, in the sealed end place of internal tank, be provided with plunger or sieve plate; Along internal tank, be axially arranged with a dividing plate, dividing plate is separated into by the internal cavities of container two equal portions that left and right volume is identical, forms two passages; Seal the chromatograph packing material of the matter equivalent such as the interior filling of backward two passages; In sealed end, be provided with efflux outlet, 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, a kind of in 1-20ml syringe needle tube.

3. binary channels SPE post according to claim 1, is characterized in that: described plunger can be the synthetic integral post plunger of original position in column jecket; Sieve plate is to which is provided with the sieve plate that aperture is 3nm-20 μ m 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: a kind of in C18 reverse phase filler, C8 reverse phase filler, C4 reverse phase filler, and particle diameter is 1.7 μ m.-100 μ m; Ion-exchange packing comprises: a kind of in SCX filler and WCX filler; Filler form comprises: granular pattern material or integral material.

5. binary channels SPE post according to claim 1, is characterized in that:

In efflux outlet, away from vessel side, be provided with the pipe of hollow, pipe by efflux export respectively with two mutually not communicating passage be connected, dividing plate is with 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 in a claim 1 binary channels SPE post 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, add respectively 100 μ L denaturant solution (as 8M urea, 6M guanidine hydrochloride or 1%SDS etc.) protein of the 1-1000 μ g that dissolves, adding subsequently reductive agent to make final concentration is 10mM(dithiothreitol (DTT) (DTT), trichloroethyl phosphate (TCEP), or beta-mercaptoethanol etc.) carry out the reduction of protein, adding subsequently alkylating reagent to make final concentration is 55mM(iodo acetic acid or iodoacetamide) alkylation of carrying out protein processes, with buffer salt solution by after 10 times of denaturant concentration dilutions, 37 ℃ of water enzyme digestions that add proteinase to carry out protein according to albumen: enzyme=50:1 (w/w) spend the night, existence due to dividing plate, can in two passages, join respectively above reagent, after enzymolysis completes, first with playing level pad (equalizing and buffering is the aqueous solution of volumetric concentration 0.1%TFA), rinse, 2) then in two passages, add respectively labelled reagent, after mark completes, first with playing level pad (equalizing and buffering is the aqueous solution of volumetric concentration 0.1%TFA), rinse, then use elution buffer (when filler is reverse phase filler, elution buffer is the volumetric concentration 80%CAN aqueous solution that contains volumetric concentration 0.1% trifluoroacetic acid, or when filler is SCX chromatograph packing material, elution buffer is pH3.0 containing the phosphorylation damping fluid of the 10-20mM of 1M NaCl) 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.

8. application according to claim 6, is characterized in that: the buffer salt solution described in step (1) is: a kind of in phosphate buffer, ammonium bicarbonate buffers, borate buffer solution;

Labelled reagent described in step (2) and corresponding solvent 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 that is dissolved in the 0.01-1M of volumetric concentration 70% ethanol water, the addition of labelled reagent is iTRAQ or the mTRAQ reagent of every 100 μ g protein requirement 100 μ L0.01-1M.

9. application according to claim 6, is characterized in that:

Protein is extracting solution of protein or the standard protein of tissue, cell or the body fluid of biological sample; 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 a kind of in urea, guanidine hydrochloride, SDS; Reductive agent is a kind of in dithiothreitol (DTT) (DTT), trichloroethyl phosphate (TCEP), beta-mercaptoethanol; Alkylating reagent is a kind of in iodo acetic acid, iodoacetamide.