CN105866317B - The protein group reactor and its application of integrated protein pre-treatment and the classification of polypeptide high ph-values reverse phase - Google Patents
The protein group reactor and its application of integrated protein pre-treatment and the classification of polypeptide high ph-values reverse phase Download PDFInfo
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Abstract
The protein group reactor being classified the present invention relates to integrated protein pre-treatment and polypeptide high ph-values reverse phase and its application, the protein group reactor include liquid-transfering gun pipette tips (1), strong cation-exchanging resin filler (2) and solid-phase extraction membrane (3)；Solid-phase extraction membrane (3) is filled in the lower end of liquid-transfering gun pipette tips (1), and strong cation-exchanging resin filler (2) is filled in liquid-transfering gun pipette tips (1) lower end and on solid-phase extraction membrane (3).Protein group reactor provided by the invention is integrated with the operations such as preenrichment, reduction, alkylation, enzymolysis, the desalination of polypeptide, elution and the classification of high ph-values reverse phase of protein, available in the protein Large scale identification and quantitative proteomics of a small amount of cell or tissue sample；In addition, protein group reactor technology provided by the invention can realize the automation mechanized operation based on automated fluid processing platform (such as Agilent Bravo platforms etc.), sample processing throughput and quantitative analysis accuracy are improved.
Description
Technical field
The present invention relates to qualitative and quantitative proteomics technical field more particularly to a kind of integrated protein pre-treatment and
The protein group reactor of polypeptide high ph-values reverse phase classification and its application.
Background technology
In proteomics research, by by the protein digestion in sample into after polypeptide, for liquid chromatography-mass spectrography point
To obtain protein information, which is current most popular proteomics research means for analysis.
Proteomics sample pre-treatments step is mainly including the preenrichment of protein, reduction, alkylation, enzymolysis and more
The desalination of peptide, classification.Conventional method is related to multiple sample transfer when handling protein sample, be easy to cause sample pollution and damage
It loses.Above-mentioned steps are effectively integrated by the protein group reactor developed in recent years, substantially increase a small amount of protein sample
Treatment effeciency.Rare cell protein group reactor (Rare Cell Proteomic Reactor, RCPR) based on it is strong sun from
Sub-exchange resin (Strong Cation Exchange, SCX) capillary monolithic column, realize the preenrichment of protein, reduction,
The classification of alkylation, enzymolysis and polypeptide, identifies 409 and 2,281 protein respectively from 5,000 and 50,000 cells
(Mol.Cell.Proteomics 2011,10,M110.000679).The protein group reactor (Centrifugal of centrifugation
Proteomic Reactor) pre-treatment step of protein is completed in centrifuge using centrifuge tube and SCX fillers, significantly
Ground improves the identification quantity (Mol.Cell.Proteomics 2011,10, O111.008425) of memebrane protein.Narrow closing is empty
Between protein example pre-treating method (in-StageTip method) sample of protein is realized in the tubule of closing
Pre-treatment, and pass through the realizations such as the SCX films of tubule lower end, strong anion exchange (Strong Anion Exchange, SAX) film
The classification of polypeptide identifies more than 7,000 protein (Nat.Methods 2014,11,319) from 20 μ g protein samples.
However, proteolysis and the polypeptide classification due to RCPR are all completed on SCX resins, the effect of classification is affected；
Also, classification can also influence mass spectrographic detection efficiency online based on salinity.The protein group reactor of centrifugation 1.5mL from
It is operated in heart pipe, operational volume is big, easily causes the loss of micro-example.In-StageTip method using SCX films into
The salinity that row polypeptide uses when being classified is higher, influences Mass Spectrometer Method；Other desalination is wanted when being classified using SAX, sample is caused to damage
It loses；And using C18The high ph-values reverse phase classification of polypeptide is not carried out during film.In addition, without surface-active in its lysate for using
Agent is unfavorable for the dissolving and extraction of hydrophobic protein.
Therefore a kind of integrated protein pre-treatment and reverse phase classification, high-throughput and easy-operating protein how to be developed
Group reactor has become current urgent problem to be solved.
Invention content
In view of problems of the prior art, the present invention provides a kind of integrated protein pre-treatment and polypeptide high ph-values
The protein group reactor of reverse phase classification and its application, can be in situ by using protein group reactor provided by the invention
Realize the overall process that preenrichment, reduction, alkylation, enzymolysis, the desalination of polypeptide, elution and the high ph-values reverse phase of protein are classified,
And the efficiency for improving enzymolysis and classification is, it can be achieved that the protein Large scale identification of a small amount of cell sample, improve reproducibility and
Quantitative analysis accuracy.In addition, protein group reactor provided by the invention can realize automation mechanized operation.
For this purpose, the present invention uses following technical scheme：
In a first aspect, the present invention provides a kind of protein group reactor, protein pre-treatment and polypeptide can be integrated
High ph-values reverse phase is classified；The protein group reactor includes liquid-transfering gun pipette tips 1, strong cation-exchanging resin filler 2 and solid phase
Spe membrane 3；Solid-phase extraction membrane 3 is filled in the lower end of liquid-transfering gun pipette tips 1, and strong cation-exchanging resin filler 2 is filled in liquid-transfering gun
1 lower end of pipette tips is simultaneously located on solid-phase extraction membrane 3.
The heretofore described preenrichment of " protein example pre-treatment " including protein, reduction, alkylation, enzymolysis,
The operations such as the desalination of polypeptide and elution, the protease used in each operating process, reducing agent, alkylating reagent and buffering
The reagent that salting liquid etc. is all known in the art, typical but non-limiting example are：Protein can be the group of biological sample
It knits, the extracting solution of protein or standard protein of cell or body fluid；Protease can be coagulated selected from alkali protease such as trypsase, pancreas
Galactase or elastoser etc.；Reducing agent can be selected from dithiothreitol (DTT), trichloroethyl phosphate, beta -mercaptoethanol etc., alkane
Base reagent can be iodo acetic acid or iodoacetamide etc..
Heretofore described " classification of polypeptide high ph-values reverse phase " refers to that the principle based on reversed-phase liquid chromatography realizes polypeptide
Classification.The characteristics of wherein reversed-phase liquid chromatography (reverse-phase liquid chromatography, RPLC) is stationary phase
Polarity than weak mobile phase, due to the hydrophobicity of RPLC stationary phase carriers, it can be according to separated object matter molecule in mobile phase
It is hydrophobic different and interaction of different strengths and weaknesses occurs, so as to which different molecular be made to be separated from each other in reversed-phase column, hydrophobicity
Weaker sample molecule and fixed alternate interaction are weaker, can just be flowed out when organic solvent content is relatively low in mobile phase；
Conversely, the relatively stronger molecule of hydrophobicity and fixed alternate there are stronger interaction, the organic solvent content in mobile phase
It could be flowed out when higher, it is thus achieved that the classification of polypeptide.
In protein group reactor of the present invention, the strong cation-exchanging resin filler is sulfonic group strong cation
Exchanger resin filler.
Preferably, the solid-phase extraction membrane is C18Film.
Protein group reactor of the present invention can be used in the pre-treatment of in-situ accomplishes protein example and the high pH of polypeptide
It is worth reverse phase classification, specifically includes：Preenrichment, reduction, alkylation, enzymolysis, the desalination of polypeptide, elution and the high ph-values of protein
The overall process of reverse phase classification, wherein, high ph-values refer to pH value higher than 8, such as pH value is 8,9,9.2,9.5 or 10 etc..
When in use, concrete operations are as follows for protein reactor of the present invention：As shown in (B) in Fig. 1, by supporting block
4 are placed in 5 upper end of collecting pipe, and protein group reactor is placed in by supporting block 4 on collecting pipe 5, collecting pipe 5 is put into centrifugation
In machine 6, protein solution or reagent can be made to flow through protein group reactor by centrifugal action, complete the pre- richness of protein
The operations such as collection, enzymolysis, the elution of polypeptide and the classification of high ph-values reverse phase.In addition, protein group reactor provided by the invention can be real
Existing automation mechanized operation；For example, the protein group can be used on the automatic fluid processing platform Bravo of agilent company
Reactor technology automation handles multiple samples simultaneously with high throughput.
Second aspect, the present invention also provides a kind of automation reaction system of protein, the automation reaction system
Including protein group reactor as described in relation to the first aspect.
Preferably, the automation reaction system further includes automatic fluid processing platform, such as can be Agilent
Bravo platforms.
The third aspect, the present invention also provides protein group reactor as described in relation to the first aspect in cell or tissue sample
Application in the protein identification and quantitative proteomics of product, the protein in particular for a small amount of cell or tissue sample are big
Application in scale identification and quantification proteomics.
In specific application, mainly by the protein group reactor for the pre-treatment of sample in biological sample and more
Peptide high ph-values reverse phase is classified, and the protein example in biological sample is digested on strong cation-exchanging resin filler, enzyme
The polypeptide of generation is transferred on solid-phase extraction membrane after the completion of solution, then carries out high ph-values reverse phase classification, with realize improve enzymolysis and
The technology requirement of classification efficiency.
In the present invention, in the protein that the protein group reactor described in first aspect is used for cell or tissue sample
When in identification and quantification proteomics, concrete operations include the following steps：
(1) cell or tissue sample are added to preactivated good protein group reaction after lysate cracks and is acidified
In device, made on protein-enriched to strong cation-exchanging resin filler by centrifugation；
(2) surface being attached on solid-phase extraction membrane is washed using the solution containing organic solvent or pure organic solvent
Activating agent sequentially adds corresponding reagent and enzyme, completes the reduction, alkylation and enzymolysis of protein；
(3) polypeptide of generation is transferred to from strong cation-exchanging resin filler on solid-phase extraction membrane using salting liquid；
(4) after desalination, using the solution containing different proportion organic solvent of high ph-values in ratio from low to high successively
Polypeptide is eluted, carries out high ph-values reverse phase classification.
Preferably, during step (4) described classification should be higher than that 8 using the pH value of solution.
Surfactant in step (1) described lysate is that the classification of high ph-values reverse phase and liquid chromatography-mass spectrography are compatible with,
Preferably dodecyl-β-D-Maltose glycosides (n-dodecyl β-D-maltoside, DDM), cholesteryl hemisuccinate three
Any one or two kinds in hydroxymethyl aminomethane salt (Cholesteryl hemisuccinate tris salt, CHS)
Mixture.
Present invention employs the lysates of compatibility, i.e. surfactant in lysate is the classification of high ph-values reverse phase and liquid
Phase chromatography-mass spectroscopy compatibility, as dodecyl-β-D-Maltose glycosides (n-dodecyl β-D-maltoside, DDM), courage are solid
Alcohol monomester succinate trishydroxymethylaminomethane salt (Cholesteryl hemisuccinate tris salt, CHS) etc., from
And be integrated into the high ph-values reverse phase classification technique of polypeptide in the protein group reactor of the present invention, improve the identification number of protein
Amount.
The solution containing organic solvent is selected from the potassium citrate aqueous solution containing acetonitrile and/or methanol described in step (2),
In, the volume content of acetonitrile and/or methanol in the solution is 20%, and potassium citrate is a concentration of in the solution
8mmol/L。
Preferably, step (2) the pure organic solvent is acetonitrile and/or methanol.
Preferably, step (3) described salting liquid is volatile salts solution, preferably ammonium formate and/or ammonium hydrogen carbonate.
Compared with prior art, the present invention at least has the advantages that：
(1) protein group reactor of the invention be integrated in a liquid-transfering gun pipette tips preenrichment of protein, reduction,
Alkylation, enzymolysis, the desalination of polypeptide, elution and high ph-values reverse phase classification etc. operations, realize that the protein of a small amount of cell sample is big
Scale is identified, improves reproducibility and quantitative analysis accuracy, and can realize the enzymolysis that protein is efficiently completed in 15min；
In addition, the protein group reactor of the present invention can realize automation mechanized operation.
(2) the invention further relates to the lysates of compatibility, i.e. surfactant in lysate is the classification of high ph-values reverse phase
With liquid chromatography-mass spectrography compatibility, such as dodecyl-β-D-Maltose glycosides (n-dodecyl β-D-maltoside, DDM) and
Cholesteryl hemisuccinate trishydroxymethylaminomethane salt (Cholesteryl hemisuccinate tris salt, CHS),
So as to which the high ph-values reverse phase classification technique of polypeptide is integrated into the protein group reactor of the present invention, the identification of protein is improved
Quantity.
(3) the invention further relates to cleaning C18The operation of surfactant on film, i.e., by protein-enriched to strong cation
After on exchanger resin filler, wash using the solution containing organic solvent or pure organic solvent and be attached to C18Table on film
Face activating agent.
(4) protein group reactor of the invention carries out the enzymolysis of protein and point of polypeptide on different materials respectively
Grade, i.e., protein is digested on strong cation-exchanging resin filler, and the polypeptide of generation is transferred to C after the completion of enzymolysis18Film
On, then high ph-values reverse phase classification is carried out, be conducive to improve the efficiency of enzymolysis and classification.
Description of the drawings
Structure diagram (B) when Fig. 1 is protein group reactor (A) of the present invention and concrete operations.
In figure：1- liquid-transfering gun pipette tips, 2- strong cation-exchanging resin fillers, 3-C18Film, 4- supporting blocks, 5- collecting pipes, 6-
Centrifuge.
Fig. 2 be surfactant be 1% (w/v) DDM or 1% (v/v) Triton X-100 when (A) lysate albumen
Matter extraction efficiency compares；(B) comparison of polypeptide chromatogram, the peak containing DDM are marked with " I ", the peak containing Triton X-100
It is marked with " * ".
Fig. 3 is the high ph-values reverse phase classification of the present invention and the proteins and peptides quantitative comparison identified when not being classified.
Fig. 4 is the label-free analytical performance evaluation of the protein group reactor of the present invention.Wherein, (A)-(C) is to appoint
The linear fit result of the meaning label-free intensity of experimental identification protein twice；(D)-(F) is arbitrary experimental identification twice
The distribution of the label-free intensity rate of protein.R1, R2 and R3 represent experiment 1,2 and 3 and identify the nonstandard of protein respectively
Remember quantitative intensities.
Fig. 5 is influence of the enzymolysis time to protein identification quantity.
The present invention is described in more detail below.But following examples is only the simple example of the present invention, not generation
Table or limitation the scope of the present invention, protection scope of the present invention are subject to claims.
Specific embodiment
Technical solution to further illustrate the present invention below with reference to the accompanying drawings and specific embodiments.
For the present invention is better described, technical scheme of the present invention is easy to understand, of the invention is typical but non-limiting
Embodiment is as follows：
First, the present invention provides a kind of lysates of compatibility.Rare cell protein group reactor (RCPR) uses
Lysate be very suitable for the cracking of a small amount of cell, ingredient is 10mmol/L HEPES, pH7.4,150mmol/L NaCl,
2mmol/L CaCl2, 2mmol/L MgCl2, 600mmol/L guanidine HCl, 1%Triton X-100 and protease support
Preparation.But the surfactant Triton X-100 in lysate are not that liquid chromatography-mass spectrography is compatible with, such as (B) in Fig. 2
It is shown, occur much influencing polypeptide inspection with the relevant strong peaks of Triton X-100 in the polypeptide chromatogram for using the lysate
It surveys.Therefore, we change 1%Triton X-100 into 1%DDM.As shown in (A) in Fig. 2, the protein of the lysate containing 1%DDM
Extraction efficiency is suitable with the lysate effect of original RCPR.Moreover, it can see in polypeptide chromatogram ((B) in Fig. 2), with DDM
Relevant peak just comes out in final time section, does not influence polypeptide detection, therefore, the lysate of the invention containing DDM is liquid phase
Chromatography-mass spectroscopy compatibility.
The protein group reactor of the present invention is integrated with the high ph-values reverse phase progressive operation of polypeptide, so as to increase polypeptide and
The identification number of protein.As shown in figure 3, during 50,000 HEK 293T cell of analysis, can be identified after the classification of high ph-values reverse phase
57,008 polypeptides and 6,821 protein are 2.2 times when not being classified and 1.7 times respectively.
The protein group reactor of the present invention has higher sensitivity.As shown in table 1.6 μ g of similary processing come from HEK
The protein example of 293T cells, does not do the high ph-values reverse phase classification of polypeptide, and protein group reactor of the invention can reflect
Determine to 19,493 polypeptides and 3,693 protein, be the protein group reactor (Centrifugal of centrifugation respectively
Proteomic Reactor) 2.8 times and 1.7 times.
As shown in table 2, protein group reactor of the invention is from 2,000,5,000,20,000,50,000 and 100,000
1,270,2,566,5,749,6,821 and 7,826 protein are identified in a HEK 293T cells respectively.And RCPR is from 5,
409 and 2,281 protein are identified in 000 and 50,000 cell respectively.In the case of same cell amount, egg of the invention
The sensitivity of white matter group reactor is 6.3 times and 3.0 times of RCPR.
Table 1
| Technology | Identify polypeptide quantity | Identify protein quantity |
| The protein group reactor of the present invention | 19,493 | 3,693 |
| The protein group reactor of centrifugation | 6,888 | 2,145 |
Table 2
The protein group reactor of the present invention is applied to 100,000 people deciduous teeth pulp matrix stem cell (stem of processing
Cells from human exfoliated deciduous teeth, SHED) sample.The result tested three times such as 3 institute of table
Show, experiment every time can identify more than 7,000 protein, test identify 120,456 polypeptides and 9 altogether three times, 078
Protein, this is the maximum protein data collection of SHED cells so far.
Table 3
| Experiment number | Identify polypeptide quantity | Identify protein quantity |
| Experiment 1 | 87,150 | 7,765 |
| Experiment 2 | 78,211 | 7,257 |
| Experiment 3 | 77,650 | 7,364 |
| Amalgamation result | 120,456 | 9,078 |
The label-free intensity of the protein of experimental identification three times is obtained using MaxQuant softwares, is arbitrarily tested twice
Linear fit the results are shown in Figure 4, coefficient R be more than 0.98.The label-free of arbitrary experimental identification protein twice
The distribution of intensity rate is as shown in figure 4, the ratio variation of 97% protein is less than 2.The result shows that protein group of the invention is anti-
Sensitivity and the label-free analysis ability for answering device are suitable with in-StageTip method.
Since traditional free solution enzyme solution needs enzymolysis overnight.And the protein group reactor of the present invention has enzyme
Solve time short, efficient advantage.As shown in figure 5, use described 20, the 000 HEK 293T of protein group reactor for treatment
Cell can identify more than 2,900 protein when not being classified.Also, when enzymolysis time was dropped to 15 minutes by 120 minutes,
The protein amounts of identification are not reduced.Therefore, protein group reactor of the invention can be efficiently complete in 15 minutes
Into the enzymolysis of protein.
Embodiment 1
As shown in (A) and (B) in Fig. 1, the albumen of a kind of integrated protein pre-treatment and the classification of polypeptide high ph-values reverse phase
Matter group reactor, including liquid-transfering gun pipette tips 1, strong cation-exchanging resin filler 2 and C18Film 3.Wherein, liquid-transfering gun pipette tips 1 are mark
200 accurate μ L pipette tips, C18Film 3 (3M Empore, USA) is filled in 1 lower end of liquid-transfering gun pipette tips, the strong sun of length about 3mm, 1.2mg from
Sub-exchange resin filler (sulfonic group strong cation-exchanging resin filler) 2 (Applied Biosystems, USA) is filled in liquid relief
1 lower end of rifle pipette tips and C18On film 3.
Supporting block 4 is placed in 5 upper end of 1.5mL collecting pipes, protein group reactor is placed in collecting pipe 5 by supporting block 4
On, collecting pipe 5 is put into centrifuge 6, protein solution can be made by centrifugal action or reagent to flow through protein group anti-
Device is answered, the operations such as preenrichment, enzymolysis, the desalination of polypeptide and the classification of high ph-values reverse phase of protein is completed, is as follows：
50,000 cell sample is taken, adds in 25 μ L compatibility lysates, lysate ingredient is 10mmol/L HEPES, pH
7.4,150mmol/L NaCl, 2mmol/L CaCl2, 2mmol/L MgCl2, 600mmol/L guanidine HCl, 1%DDM
With protease inhibitor, add in trifluoroacetic acid after the completion of cracking and sample solution is acidified to pH2.Protein group reactor divides first
Do not pass through the 10mmol/L potassium citrate aqueous solutions of the methanol of 20 μ L, the 100mmol/L potassium citrates aqueous solution of 20 μ L and 20 μ L
Activation.After activation, sample is added in protein reactor, by the centrifuge 6 centrifugation make protein-enriched to it is strong it is positive from
On sub-exchange resin filler 2；Then, fall to be attached to C using the 8mmol/L potassium citrates aqueous cleaning containing 20% acetonitrile18
Surfactant D DM on film 3；Then, (2- carboxyethyls) the phosphonium salt hydrochlorates of 10mmol/L tri- (Tris (2- are added in
Carboxyethyl) phosphine hydrochloride, TCEP) solution, reacts 15 minutes, completes protein at room temperature
Reduction.Then, 20 μ L ultra-pure waters are added in and washes away TCEP, it is molten to add the 10mmol/L iodacetyl ammoniums containing 4 μ g trypsase
Liquid reacts 60 minutes under room temperature and dark environment, completes alkylation and the enzymolysis of protein.Then, using 20 μ L's
The polypeptide of generation is transferred to C by 200mmol/L formic acid aqueous ammonium from strong cation-exchanging resin filler 218On film 3；Then, add
The 5mmol/L formic acid aqueous ammonium for entering 20 μ L carries out desalination.Finally, using pH value be 10 respectively containing 3%, 6%, 9%,
15%th, the 5mmol/L ammonium formate solutions of 80% acetonitrile successively elute polypeptide, that is, carry out high ph-values reverse phase classification.Elution
The polypeptide to get off is redissolved after freeze dryer freeze-drying in 0.1% aqueous formic acid, you can is analyzed with liquid chromatography-mass spectrography.
It can be with using 20,000 HEK 293T cell of protein group reactor for treatment described in the present embodiment, when not being classified
It identifies more than 2,900 protein, also, when enzymolysis time was dropped to 15 minutes by 120 minutes, the protein amounts of identification are simultaneously
Do not reduce.Therefore, the protein group reactor of the present embodiment can efficiently complete the enzymolysis of protein in 15 minutes.
Embodiment 2
A kind of integrated protein pre-treatment and the protein group reactor of polypeptide high ph-values reverse phase classification, including liquid-transfering gun rifle
First 1, strong cation-exchanging resin filler 2 and C18Film 3.Wherein, 200 μ L pipette tips of the liquid-transfering gun pipette tips 1 for standard, C183 (3M of film
Empore, USA) 1 lower end of liquid-transfering gun pipette tips is filled in, length about 3mm, (sulfonic group is strong for 1.2mg strong cation-exchanging resins filler
Cation exchange resin filler) 2 (Applied Biosystems, USA) are filled in 1 lower end of liquid-transfering gun pipette tips and C18On film 3.
Supporting block 4 is placed in 5 upper end of 1.5mL collecting pipes, protein group reactor is placed in collecting pipe 5 by supporting block 4
On, collecting pipe 5 is put into centrifuge 6, protein solution can be made by centrifugal action or reagent to flow through protein group anti-
Device is answered, the operations such as preenrichment, enzymolysis, the desalination of polypeptide and the classification of high ph-values reverse phase of protein is completed, is as follows：
50,000 cell sample is taken, adds in 25 μ L compatibility lysates, lysate ingredient is 10mmol/L HEPES, pH
7.4,150mmol/L NaCl, 2mmol/L CaCl2, 2mmol/L MgCl2, 600mmol/L guanidine HCl, 1% courage consolidates
Alcohol monomester succinate trishydroxymethylaminomethane salt (Cholesteryl hemisuccinate tris salt, CHS) and albumen
Enzyme inhibitor adds in trifluoroacetic acid after the completion of cracking and sample solution is acidified to pH2.Protein group reactor passes through respectively first
The 10mmol/L potassium citrate activated in water solution of the methanol of 20 μ L, the 100mmol/L potassium citrates aqueous solution of 20 μ L and 20 μ L.It is living
After change, sample is added in protein reactor, makes protein-enriched to strong cation exchange tree by being centrifuged in centrifuge 6
On fat filler 2；Then, fall to be attached to C using the 8mmol/L potassium citrates aqueous cleaning containing 20% acetonitrile18Table on film 3
Face activating agent CHS；Then, 10mmol/L tri- (2- carboxyethyls) phosphonium salt hydrochlorate (Tris (2-carboxyethyl) is added in
Phosphine hydrochloride, TCEP) solution, reacts 15 minutes, completes the reduction of protein at room temperature.Then, add
Enter 20 μ L ultra-pure waters and wash away TCEP, the 10mmol/L iodacetyl ammonium salt solutions containing 4 μ g trypsase are added, in room temperature and dark
In the environment of react 60 minutes, complete alkylation and the enzymolysis of protein.Then, using the 200mmol/L ammonium formate water of 20 μ L
The polypeptide of generation is transferred to C by solution from strong cation-exchanging resin filler 218On film 3；Then, the 5mmol/L first of 20 μ L is added in
Sour aqueous ammonium carries out desalination.Finally, using pH value be 10 respectively containing 3%, 6%, 9%, 15%, 80% acetonitrile
5mmol/L ammonium formate solutions successively elute polypeptide, that is, carry out high ph-values reverse phase classification.The polypeptide eluted is being lyophilized
It is redissolved after machine freeze-drying in 0.1% aqueous formic acid, you can analyzed with liquid chromatography-mass spectrography.
It can be with using 20,000 HEK 293T cell of protein group reactor for treatment described in the present embodiment, when not being classified
It identifies more than 2,900 protein, also, when enzymolysis time was dropped to 16 minutes by 120 minutes, the protein amounts of identification are simultaneously
Do not reduce.Therefore, the protein group reactor of the present embodiment can efficiently complete the enzymolysis of protein in 16 minutes.
Embodiment 3
A kind of integrated protein pre-treatment and the protein group reactor of polypeptide high ph-values reverse phase classification, including liquid-transfering gun rifle
First 1, strong cation-exchanging resin filler 2 and C18Film 3.Wherein, 200 μ L pipette tips of the liquid-transfering gun pipette tips 1 for standard, C183 (3M of film
Empore, USA) 1 lower end of liquid-transfering gun pipette tips is filled in, length about 3mm, (sulfonic group is strong for 1.2mg strong cation-exchanging resins filler
Cation exchange resin filler) 2 (Applied Biosystems, USA) are filled in 1 lower end of liquid-transfering gun pipette tips and C18On film 3.
Supporting block 4 is placed in 5 upper end of 1.5mL collecting pipes, protein group reactor is placed in collecting pipe 5 by supporting block 4
On, collecting pipe 5 is put into centrifuge 6, protein solution can be made by centrifugal action or reagent to flow through protein group anti-
Device is answered, the operations such as preenrichment, enzymolysis, the desalination of polypeptide and the classification of high ph-values reverse phase of protein is completed, is as follows：
50,000 cell sample is taken, adds in 25 μ L compatibility lysates, lysate ingredient is 10mmol/L HEPES, pH
7.4,150mmol/L NaCl, 2mmol/L CaCl2, 2mmol/L MgCl2, 600mmol/L guanidine HCl, 0.5%
DDM, 0.5%CHS and protease inhibitor add in trifluoroacetic acid after the completion of cracking and sample solution are acidified to pH2.Protein group
Reactor passes through the 10mmol/L lemons of the methanol of 20 μ L, the 100mmol/L potassium citrates aqueous solution of 20 μ L and 20 μ L respectively first
Lemon acid aqueous solutions of potassium activates.After activation, sample is added in protein reactor, makes protein by being centrifuged in centrifuge 6
It is enriched on strong cation-exchanging resin filler 2；Then, it is clear using the 8mmol/L potassium citrate aqueous solutions containing 20% acetonitrile
It washes off and is attached to C18Surfactant D DM and CHS on film 3；Then, 10mmol/L tri- (2- carboxyethyls) phosphonium salt hydrochlorate is added in
(Tris (2-carboxyethyl) phosphine hydrochloride, TCEP) solution reacts 15 minutes at room temperature, complete
Into the reduction of protein.Then, 20 μ L ultra-pure waters are added in and washes away TCEP, add the 10mmol/L iodine containing 4 μ g trypsase
Acetyl ammonium salt solution reacts 60 minutes under room temperature and dark environment, completes alkylation and the enzymolysis of protein.Then, it uses
The polypeptide of generation is transferred to C by the 200mmol/L formic acid aqueous ammonium of 20 μ L from strong cation-exchanging resin filler 218On film 3；
Then, the 5mmol/L formic acid aqueous ammonium for adding in 20 μ L carries out desalination.Finally, using pH value be 10 respectively containing 3%,
6%th, the 5mmol/L ammonium formate solutions of 9%, 15%, 80% acetonitrile successively elute polypeptide, that is, carry out high ph-values reverse phase point
Grade.The polypeptide eluted is redissolved after freeze dryer freeze-drying in 0.1% aqueous formic acid, you can is carried out with liquid chromatography-mass spectrography
Analysis.
It can be with using 20,000 HEK 293T cell of protein group reactor for treatment described in the present embodiment, when not being classified
It identifies more than 2,900 protein, also, when enzymolysis time was dropped to 18 minutes by 120 minutes, the protein amounts of identification are simultaneously
Do not reduce.Therefore, the protein group reactor of the present embodiment can efficiently complete the enzymolysis of protein in 18 minutes.
Embodiment 4
A kind of integrated protein pre-treatment and the protein group reactor of polypeptide high ph-values reverse phase classification, including liquid-transfering gun rifle
First 1, strong cation-exchanging resin filler 2 and C18Film 3.Wherein, 200 μ L pipette tips of the liquid-transfering gun pipette tips 1 for standard, C183 (3M of film
Empore, USA) 1 lower end of liquid-transfering gun pipette tips is filled in, length about 3mm, (sulfonic group is strong for 1.2mg strong cation-exchanging resins filler
Cation exchange resin filler) 2 (Applied Biosystems, USA) are filled in 1 lower end of liquid-transfering gun pipette tips and C18On film 3.
Supporting block 4 is placed in 5 upper end of 1.5mL collecting pipes, protein group reactor is placed in collecting pipe 5 by supporting block 4
On, collecting pipe 5 is put into centrifuge 6, protein solution can be made by centrifugal action or reagent to flow through protein group anti-
Device is answered, the operations such as preenrichment, enzymolysis, the desalination of polypeptide and the classification of high ph-values reverse phase of protein is completed, is as follows：
50,000 cell sample is taken, adds in 25 μ L compatibility lysates, lysate ingredient is 10mmol/L HEPES, pH
7.4,150mmol/L NaCl, 2mmol/L CaCl2, 2mmol/L MgCl2, 600mmol/L guanidine HCl, 0.5%
DDM, 0.5%CHS and protease inhibitor add in trifluoroacetic acid after the completion of cracking and sample solution are acidified to pH2.Protein group
Reactor passes through the 10mmol/L lemons of the methanol of 20 μ L, the 100mmol/L potassium citrates aqueous solution of 20 μ L and 20 μ L respectively first
Lemon acid aqueous solutions of potassium activates.After activation, sample is added in protein reactor, makes protein by being centrifuged in centrifuge 6
It is enriched on strong cation-exchanging resin filler 2；Then, using the 8mmol/L citric acids containing 10% acetonitrile and 10% methanol
Aqueous solutions of potassium, which washes, is attached to C18Surfactant D DM and CHS on film 3；Then, 10mmol/L tri- (2- carboxyethyls) is added in
Phosphonium salt hydrochlorate (Tris (2-carboxyethyl) phosphine hydrochloride, TCEP) solution, reacts 15 at room temperature
Minute, complete the reduction of protein.Then, 20 μ L ultra-pure waters are added in and washes away TCEP, added containing 4 μ g trypsase
10mmol/L iodacetyl ammonium salt solutions react 60 minutes under room temperature and dark environment, complete alkylation and the enzymolysis of protein.
Then, the polypeptide of generation is transferred to from strong cation-exchanging resin filler 2 using the 200mmol/L formic acid aqueous ammonium of 20 μ L
C18On film 3；Then, the 5mmol/L formic acid aqueous ammonium for adding in 20 μ L carries out desalination.Finally, the use of pH value is 10 respectively to contain
The 5mmol/L ammonium formate solutions for having 3%, 6%, 9%, 15%, 80% acetonitrile successively elute polypeptide, that is, carry out high ph-values
Reverse phase is classified.The polypeptide eluted is redissolved after freeze dryer freeze-drying in 0.1% aqueous formic acid, you can with liquid chromatogram-matter
Spectrum is analyzed.
It can be with using 20,000 HEK 293T cell of protein group reactor for treatment described in the present embodiment, when not being classified
It identifies more than 2,900 protein, also, when enzymolysis time was dropped to 20 minutes by 120 minutes, the protein amounts of identification are simultaneously
Do not reduce.Therefore, the protein group reactor of the present embodiment can efficiently complete the enzymolysis of protein in 20 minutes.
Embodiment 5
A kind of integrated protein pre-treatment and the protein group reactor of polypeptide high ph-values reverse phase classification, including liquid-transfering gun rifle
First 1, strong cation-exchanging resin filler 2 and C18Film 3.Wherein, 200 μ L pipette tips of the liquid-transfering gun pipette tips 1 for standard, C183 (3M of film
Empore, USA) 1 lower end of liquid-transfering gun pipette tips is filled in, length about 3mm, (sulfonic group is strong for 1.2mg strong cation-exchanging resins filler
Cation exchange resin filler) 2 (Applied Biosystems, USA) are filled in 1 lower end of liquid-transfering gun pipette tips and C18On film 3.
Supporting block 4 is placed in 5 upper end of 1.5mL collecting pipes, protein group reactor is placed in collecting pipe 5 by supporting block 4
On, collecting pipe 5 is put into centrifuge 6, protein solution can be made by centrifugal action or reagent to flow through protein group anti-
Device is answered, the operations such as preenrichment, enzymolysis, the desalination of polypeptide and the classification of high ph-values reverse phase of protein is completed, is as follows：
50,000 cell sample is taken, adds in 25 μ L compatibility lysates, lysate ingredient is 10mmol/L HEPES, pH
7.4,150mmol/L NaCl, 2mmol/L CaCl2, 2mmol/L MgCl2, 600mmol/L guanidine HCl, 1%DDM
With protease inhibitor, add in trifluoroacetic acid after the completion of cracking and sample solution is acidified to pH2.Protein group reactor divides first
Do not pass through the 10mmol/L potassium citrate aqueous solutions of the methanol of 20 μ L, the 100mmol/L potassium citrates aqueous solution of 20 μ L and 20 μ L
Activation.After activation, sample is added in protein reactor, by the centrifuge 6 centrifugation make protein-enriched to it is strong it is positive from
On sub-exchange resin filler 2；Then, fall to be attached to C using the 8mmol/L potassium citrates aqueous cleaning containing 20% acetonitrile18
Surfactant D DM on film 3；Then, (2- carboxyethyls) the phosphonium salt hydrochlorates of 10mmol/L tri- (Tris (2- are added in
Carboxyethyl) phosphine hydrochloride, TCEP) solution, reacts 15 minutes, completes protein at room temperature
Reduction.Then, 20 μ L ultra-pure waters are added in and washes away TCEP, it is molten to add the 10mmol/L iodacetyl ammoniums containing 4 μ g trypsase
Liquid reacts 60 minutes under room temperature and dark environment, completes alkylation and the enzymolysis of protein.Then, using 20 μ L's
The polypeptide of generation is transferred to C by 200mmol/L formic acid aqueous ammonium from strong cation-exchanging resin filler 218On film 3；Then, add
The 5mmol/L formic acid aqueous ammonium for entering 20 μ L carries out desalination.Finally, using pH value be 10 respectively containing 3%, 6%, 9%,
15%th, the 5mmol/L ammonium formates of 80% acetonitrile and ammonium bicarbonate soln successively elute polypeptide, that is, carry out high ph-values reverse phase
Classification.The polypeptide eluted freeze dryer freeze-drying after redissolve in 0.1% aqueous formic acid, you can with liquid chromatography-mass spectrography into
Row analysis.
It can be with using 20,000 HEK 293T cell of protein group reactor for treatment described in the present embodiment, when not being classified
It identifies more than 2,900 protein, also, when enzymolysis time was dropped to 17 minutes by 120 minutes, the protein amounts of identification are simultaneously
Do not reduce.Therefore, the protein group reactor of the present embodiment can efficiently complete the enzymolysis of protein in 17 minutes.
Applicant states that the present invention illustrates the detailed construction feature of the present invention by above-described embodiment, but the present invention is simultaneously
Above-mentioned detailed construction feature is not limited to, that is, does not mean that the present invention has to rely on above-mentioned detailed construction feature and could implement.Institute
Belong to those skilled in the art it will be clearly understood that any improvement in the present invention, to the equivalence replacement of component selected by the present invention
And the increase of accessory, selection of concrete mode etc., it all falls within protection scope of the present invention and the open scope.
The preferred embodiment of the present invention has been described above in detail, still, during present invention is not limited to the embodiments described above
Detail, within the scope of the technical concept of the present invention, a variety of simple variants can be carried out to technical scheme of the present invention, this
A little simple variants all belong to the scope of protection of the present invention.
It is further to note that specific technical features described in the above specific embodiments, in not lance
In the case of shield, can be combined by any suitable means, in order to avoid unnecessary repetition, the present invention to it is various can
The combination of energy no longer separately illustrates.
In addition, various embodiments of the present invention can be combined randomly, as long as it is without prejudice to originally
The thought of invention, it should also be regarded as the disclosure of the present invention.
Claims (8)
1. a kind of method of biological sample pre-treatment and polypeptide high ph-values reverse phase classification based on protein group reactor, feature
It is, the protein group reactor includes liquid-transfering gun pipette tips（1）, strong cation-exchanging resin filler（2）And solid-phase extraction membrane
（3）；Solid-phase extraction membrane（3）It is filled in liquid-transfering gun pipette tips（1）Lower end, strong cation-exchanging resin filler（2）It is filled in liquid relief
Rifle pipette tips（1）Lower end is simultaneously located at solid-phase extraction membrane（3）On；
The protein group reactor for biological sample pre-treatment and polypeptide high ph-values reverse phase classification specific method be：It will
Protein example enrichment in biological sample is restored on strong cation-exchanging resin filler, is alkylated and digested, enzymolysis
The polypeptide of generation is transferred on solid-phase extraction membrane after the completion, then carries out high ph-values reverse phase classification, the high ph-values refer to pH value
Higher than 9；
The protein group reactor is used for the specific behaviour of the pre-treatment of sample and the classification of polypeptide high ph-values reverse phase in biological sample
It is as step：（1）Cell or tissue sample are added to preactivated good protein group reaction after lysate cracks and is acidified
In device, made on protein-enriched to strong cation-exchanging resin filler by centrifugation；
（2）The surface-active being attached on solid-phase extraction membrane is washed using the solution containing organic solvent or pure organic solvent
Agent sequentially adds corresponding reagent and enzyme, completes the reduction, alkylation and enzymolysis of protein；
（3）The polypeptide of generation is transferred to from strong cation-exchanging resin filler on solid-phase extraction membrane using salting liquid；
（4）The use of the solution containing different proportion organic solvent of high ph-values in ratio from low to high will be more successively after desalination
Peptide elutes, and carries out high ph-values reverse phase classification；
Wherein, step（1）Surfactant in the lysate is the classification of high ph-values reverse phase and liquid chromatography-mass spectrography compatibility
, the surfactant is dodecyl-β-D-Maltose glycosides, cholesteryl hemisuccinate trishydroxymethylaminomethane salt
In any one or two kinds mixture.
2. according to the method described in claim 1, it is characterized in that, the strong cation-exchanging resin filler is positive by force for sulfonic group
Ion exchange resin filler.
3. according to the method described in claim 1, it is characterized in that, the solid-phase extraction membrane is C18Film.
4. according to the method described in claim 1, it is characterized in that, step（2）The solution containing organic solvent, which is selected from, to be contained
The potassium citrate aqueous solution of acetonitrile and/or methanol, wherein, the volume content of acetonitrile and/or methanol in the solution is 20%,
A concentration of 8mmol/L of the potassium citrate in the solution.
5. according to the method described in claim 1, it is characterized in that, step（2）The pure organic solvent is acetonitrile and/or first
Alcohol.
6. according to the method described in claim 1, it is characterized in that, step（3）The salting liquid is volatile salts solution.
7. according to the method described in claim 6, it is characterized in that, the salting liquid is ammonium formate and/or ammonium hydrogen carbonate.
8. protein identification and quantitative proteomics are used for according to claim 1-7 any one of them method.
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| US16/089,503 US11959891B2 (en) | 2016-03-31 | 2017-01-05 | Proteomics reactor, protein chromatographic separation platform and use thereof |
| PCT/CN2017/070336 WO2017166897A1 (en) | 2016-03-31 | 2017-01-05 | Proteomic reactor, protein chromatographic separation platform and use thereof |
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| CN106770814B (en) * | 2016-11-18 | 2019-04-26 | 南方科技大学 | A kind of protein chromatography separation platform and its application |
| WO2017166897A1 (en) * | 2016-03-31 | 2017-10-05 | 南方科技大学 | Proteomic reactor, protein chromatographic separation platform and use thereof |
| CN108072555A (en) * | 2016-11-16 | 2018-05-25 | 中国科学院大连化学物理研究所 | A kind of analysis and processing method of protein methylation and its application |
| CN108226317B (en) * | 2016-12-21 | 2020-10-20 | 复旦大学 | Method for performing whole proteomics analysis based on cell level |
| CN107402269B (en) * | 2017-07-31 | 2021-02-09 | 南方科技大学 | Integrated proteomics sample pretreatment platform based on SCX/SAX mixed filler and application thereof |
| CN108387666A (en) * | 2018-03-01 | 2018-08-10 | 南方科技大学 | A kind of proteomics Mass Spectrometry detection method for urine specimen |
| CN108645934B (en) * | 2018-05-31 | 2021-03-30 | 中山大学孙逸仙纪念医院 | Mini-protein reactor applied to proteome sample preparation and application thereof |
| CN109142561A (en) * | 2018-07-17 | 2019-01-04 | 上海师范大学 | The method and its application of simultaneous quantitative protein abundance and cysteine oxidation level |
| CN109061016A (en) * | 2018-07-25 | 2018-12-21 | 大连工业大学 | A kind of preparation method and application of the solid-phase extraction column of enriched biological amine |
| CN109541223A (en) * | 2018-11-08 | 2019-03-29 | 新乡医学院 | The identification method of sample to be tested palmitoylation total protein |
| CN110922448B (en) * | 2019-12-11 | 2021-02-09 | 谱度众合(武汉)生命科技有限公司 | Phosphorylated peptide enrichment device and method |
| CN114577955A (en) * | 2020-11-30 | 2022-06-03 | 中国科学院大连化学物理研究所 | High-throughput automatic single-cell proteome sample processing method |
| CN114509521B (en) * | 2022-01-19 | 2023-12-19 | 南方科技大学 | Pretreatment device and method for glycoprotein proteomics sample |
| CN114749221A (en) * | 2022-03-25 | 2022-07-15 | 南方科技大学 | Proteomics sample pretreatment device and preparation method and application thereof |
| CN117169489A (en) * | 2023-07-18 | 2023-12-05 | 西湖实验室(生命科学和生物医学浙江省实验室) | Method for processing biological samples to identify proteins therein |
| CN117630249A (en) * | 2024-01-26 | 2024-03-01 | 华南生物医药研究院 | Extremely simple proteomics sample pretreatment method based on sample loading bottle |
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