CN103864967B - Pharmalyte modify polymer beads and apply in protein example pre-treatment - Google Patents
Pharmalyte modify polymer beads and apply in protein example pre-treatment Download PDFInfo
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- CN103864967B CN103864967B CN201210531884.4A CN201210531884A CN103864967B CN 103864967 B CN103864967 B CN 103864967B CN 201210531884 A CN201210531884 A CN 201210531884A CN 103864967 B CN103864967 B CN 103864967B
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Abstract
The present invention relates to the preparation of polymer beads and the application in protein example pre-treatment thereof of the modification of a kind of pharmalyte, belong to analysis technical field.Described particle is surperficial in polymer microballoon by pharmalyte molecular linkage by chemical connecting arm, and this particle can be used for the abundance reducing high-abundance proteins matter in protein example.The method is a kind of novel protein example pretreatment process, the abundance difference between high-abundance proteins matter and low abundance proteins in protein example can be effectively reduced, compared to traditional protein pretreatment process, there is the features such as convenient and easy, with low cost.This invention has good application prospect and practical value in proteomics.
Description
Technical field
The present invention relates to protein example pre-treatment, the preparation of polymer beads of specifically a kind of pharmalyte modification and the application in protein example pre-treatment thereof.
Background technology
The composition of protein is extremely complicated, the dynamicrange of a lot of protein example internal protein is wide especially, concentration difference large (abundance difference is large), in such as plasma sample, front 10 kinds of high-abundance proteins matter account for 90% of total protein number, front 22 kinds of protein account for 99%, remaining several thousand kinds of albumen only account for 1%, and there is potential biological significance in these low abundance proteinses, important biomolecule function (J.ProteomeRes. may be played in the discovery and treatment of disease marker, 2011,10,5 – 16).Therefore reduce the difference between high abundance and low abundance proteins, can detect that low abundance proteins becomes one of pretreated key problem of protein easily by existing detection technique.
The method of the high low abundance proteins difference of current reduction mainly contains ultracentrifugation filtration method, pre-stage division, immune removal method etc.Ultracentrifugation realizes being separated according to varying in size of protein molecular weight, thus remove part high-abundance proteins matter, but can remove insufficient to high-abundance proteins matter, and low abundance proteins (the Molecular & CellularProteomics of high molecular may be removed simultaneously, 2003,2,1096 – 1103).Pre-stage division is that the difference such as solubleness, molecular weight, iso-electric point, structure according to protein example carries out classification to protein, thus reduce protein example complicacy, be easy to low abundance proteins be detected, the method of general use electrophoresis or chromatogram realizes (J.ProteomeRes., 2009,8,1143 – 1155; Electrophoresis, 2010,31,3580 – 3585; J.ProteomeRes., 2009,8,1143 – 1155; J.ProteomeRes., 2010,9,1902-1912), but this methods experiment step is more loaded down with trivial details, and need corresponding plant and instrument.Immunity minimizing technology is the antibody using high-abundance proteins matter, removes the high-abundance proteins matter in sample, thus reduces dynamicrange (MolCellProteomics2008,7, the 1963-1973 of sample internal protein; JProteomeRes2010,9 (10), 4982-4991), but this method also has a lot of inevitable shortcoming at present, as, low abundance proteins easily with part high-abundance proteins matter non-specific binding, phenomenon (Electrophoresis2010 is removed in generation altogether, 31 (3), 471-482); The antibody type found few (20 kinds); The finite volume of processing sample; Sample after removal is diluted; Different albumen removal efficiency has different etc.
Summary of the invention
For the deficiency that above method exists, the object of this invention is to provide a kind of method of simple and effective to reduce the abundance difference of high low abundance proteins in protein example.
For achieving the above object, the technical solution used in the present invention is:
Adopt the polyacrylic ester particle disposal protein example that pharmalyte is modified, and adopt sodium chloride solution, glycine solution, the method for acetonitrile solution classification wash-out reduces the abundance difference of high-abundance proteins matter and low abundance proteins in protein example.
The concrete steps that described pharmalyte modifies the preparation of polyacrylic ester particle and protein example process are as follows,
(1) polymer beads activation: particle is respectively through after organic solvent and phosphoric acid buffer washing, and employing quality-volume percent (per-cent of Solute mass (g) and liquor capacity (ml)) concentration is the glutaraldehyde activated 3-9 hour of 10-15%.
(2) pharmalyte is modified: after polymer beads step (1) activated adopts phosphoric acid buffer to wash several times, the pharmalyte being 30-45% with massfraction reacts 12-24 hour.
(3) sealing of particle surface and reduction: after particle and pharmalyte react, reacts 6-9h with the sodium cyanoborohydride solution of the quality-volume percent per-cent of liquor capacity (ml) (Solute mass (g) with) concentration to be 10-15% glycine and the quality-volume percent per-cent of liquor capacity (ml) (Solute mass (g) with) concentration be 5-10%.
(4) process protein example: the particle of preparation adopt phosphate buffered saline buffer washing several all over after, hatch 1-4 hour at ambient temperature with protein example.
(5) Protein elution: particle is centrifugal, removes supernatant liquor.Adopt phosphate buffered saline buffer washing granule, remove supernatant liquor.Adopt sodium chloride solution (NaCl), glycine solution (Gly-HCl), acetonitrile solution (ACN, containing 0.1% trifluoroacetic acid (TFA)) washs respectively, collects elutriant.Wherein the concentration of sodium chloride solution is 0.5-2M, and the concentration of glycine solution is 50-300mM, pH is 1-3.5, and in acetonitrile solution, the volume ratio of acetonitrile and water is 0.2-0.3, pH=2-4;
Particle is polyacrylate polymers particle, and modify pharmalyte molecule by the functional group on surface, the pharmalyte iso-electric point of modification is between 2-14, and grain diameter is 3-8 μm, and particle aperture is
Quality-concentration of volume percent described in the present invention refers to the per-cent of Solute mass (g) and liquor capacity (ml).
Tool of the present invention has the following advantages:
(1) application of the polymer microballoon that the pharmalyte that prepared by the present invention is modified and three kinds of elution reagents effectively can reduce the abundance difference of high-abundance proteins matter and low abundance proteins in protein example; (2) polymeric microspheres stabilize prepared, preparation method's favorable reproducibility; (3) sample treatment is easy and simple to handle, and cost is low, only needs compact centrifuge to realize, without the need to being equipped with the supplementary instruments such as liquid phase instrument, electrophoresis apparatus, without the need to using antibody column etc.
Accompanying drawing explanation
Fig. 1 gel electrophoresis analysis protein example.
(serum, HS) front ten kinds of high-abundance proteins matter Plantago fengdouensis in Fig. 2 protein example.Front ten kinds of high-abundance proteins matter are respectively: 1, serum albumin (Albumin); 2, α-2-macroglobulin matter (Alpha-2-macroglobulin); 3, immunoglobulin G (IgG); 4, cellulose protein former (Fibrinogen); 5, serum complement PROTEIN C 3 (ComplementC3); 6, Transferrins,iron complexes matter (Serotransferrin); 7, immunoglobulin M (IgM); 8, α-1-antitrypsin (Alpha-1-antitrypsin); 9, immunoglobulin A (IgA); 10, haptoglobin (HPRHaptoglobin).
One of (serum, HS) protein abundance changing conditions in Fig. 3 (a) protein example;
(serum, HS) protein abundance changing conditions two in Fig. 3 (b) protein example.
Embodiment
Specific embodiment is adopted to be described further technical scheme of the present invention below.
Pharmalyte (has another name called: carrier ampholyte, carrier ampholyte; No. CAS is: 37348-94-0; English trade name: Ampholine (Sigma-Aldrich company))
Embodiment 1
1. the pharmalyte preparation of polymer microballoon of modifying and Zeta electric potential measure
Take 30mg polyacrylic ester particle, and adopt ethanol, phosphoric acid buffer (pH8.0) cleans three times respectively, after reacting 6h at ambient temperature, cleans particle respectively with water and phosphoric acid buffer with 1mL10% glutaraldehyde solution.
Measure the 400 μ L pharmalytes (mixture of a kind of aliphatic polyamine Quito carboxyl, be made up of isomer and homologue, pI=3-10) liquid, dissolves with the mixed solution of 8mg sodium cyanoborohydride and 600 μ L phosphoric acid buffers, by this liquid and above-mentioned particle room temperature reaction 24h.Abandon supernatant, adopt phosphate buffered saline buffer (pH7.4) to clean 7 times, be i.e. the polyacrylic acid ester microsphere of obtained pharmalyte modification.
Zeta electric potential measurement result is as shown in table 1.Pharmalyte molecule is a kind of mixture of polyaminopolycarboxylic group, and its iso-electric point is distributed between 3-10.In different pH damping fluid, can there is different dissociative patterns in the amino on pharmalyte surface and carboxy functional group.In the phosphoric acid buffer of pH=5, surface potential is+3.63, illustrates that particle surface is positively charged; In the phosphoric acid buffer of pH=9, surface potential is-5.05, illustrates that particle surface is electronegative; In the phosphoric acid buffer of pH=7, surface potential is almost nil, illustrates that particle surface is charged hardly.Prove the existence of polymer particle surface pharmalyte.
Under table 1 condition of different pH, the polymer particle surface Zeta electric potential that pharmalyte is modified.
Embodiment 2
1. sample handling processes
Adopted by protein example (serum, HS) phosphoric acid buffer to dilute (PBS, pH=7.4) to 10mg/mL, above-mentioned sample (1mL) is added in the polymer beads of pharmalyte modification, incubated at room 2h.Centrifugal, take out supernatant and be labeled as FT.After PBS washing granule, centrifugal segregation supernatant liquor, particle adopts 2MNaCl respectively, and 200mMGly-HCl and 30%ACN (containing 0.1%TFA) washs, and often kind of reagent washs 3 times respectively, shakes 20min at every turn, collects each elutriant.
2. gel electrophoresis analysis (SDS-PAGE)
By above-mentioned sample effluent liquid FT, NaCl component sample, Gly-HCl component sample, ACN component sample and original serum sample carry out SDS-PAGE analysis.Concrete steps are as follows: above-mentioned sample is respectively taken out about 5 μ g, add the SDS-PAGE loading buffer of equal volume wherein, after boiling 5min, are added in gel duct in boiling water.Adopt the separation gel of 5% concentrated glue and 12%, be separated under 120V constant-pressure conditions.As shown in Figure 1, in original serum, high-abundance proteins matter band is more for result, NaCl component after process, and in Gly-HCl component and ACN component sample, high-abundance proteins matter band obviously weakens.The abundance of reduction high-abundance proteins matter can reduce the abundance difference between high low abundance proteins, describes the validity of the method in protein example pre-treatment.
3. protease solution preocess
Above-mentioned elutriant and original serum protein example are carried out enzymolysis respectively.Detailed process is as follows: by sample sex change 20min under 90 DEG C of conditions, add a certain amount of dithiothreitol (DTT) (DTT, every milligram of protein adds 8 μm of ol dithiothreitol (DTT) solution), 56 DEG C of reaction 1.5h, to realize the reduction process of protein, add iodoacetamide solution (IAA in proportion again, 1 μm of olDTT adds 2.5 μm of olIAA), under lucifuge condition, react 40min alkylation is carried out to protein, the mass ratio finally adding trypsin trypsinase and protein is 1:25), at 37 DEG C of reaction 16h.
4. ion exchange-reverse phase series connection liquid matter is analyzed and data processing
The liquid matter (SCX-RPLC-MS/MS) of above-mentioned protease hydrolysis products (HS, Gly-HCl, NaCl-ACN enzymolysis product) being carried out ion exchange-reverse phase series connection is analyzed.Adopt three kinds of moving phases: mobile phase A is 2% acetonitrile, 98% water, 0.1% formic acid; Mobile phase B is 98% acetonitrile, 2% water, 0.1% formic acid; Moving phase C is ammonium acetate, and volumetric molar concentration is 1mol/L.After sample is loaded in SCX trapping column, adopt 5% respectively, 10%, 15%, the moving phase C of 20%, 30% and 100% rinses 15min, adopts mobile phase A balance trapping column 15min after each flushing, adopt the following gradient that is separated to carry out reverse phase separation, 0-5%B again, be separated 5min; 5-35%B, is separated 95min; 35-80%B, is separated 5min, adopts 80% Mobile phase B to rinse 10min afterwards.LTQ-Orbitrap-Velos mass spectrum adopts positive ion scan pattern, and collision energy is 35%.The raw data obtained by mass spectrum adopts Mascot (2.4.0version) to retrieve, and database is IPI_human_v3.8.7 (91464sequence).Significance factors P<0.01 is adopted to process to Mascot result for retrieval the protein obtaining high confidence.
The protein information obtained is carried out statistical study.When under the condition that sample quality in mass spectrum is identical, the number of spectrogram number can represent the height of protein abundance, the relatively change of front ten kinds of high-abundance proteins mass spectrum numbers before and after the serum polymer beads process of modifying through pharmalyte, result as shown in Figure 2.Front ten kinds of protein spectrum map numbers have reduction in various degree after treatment, illustrate that the method plays vital role in reduction high-abundance proteins matter abundance.
Because protein example after treatment, in each component, the complexity of sample is all different, therefore using the ratio (SAF) of protein spectrum map number and its length as a judgement criteria of all proteins Plantago fengdouensis.The relatively change of all proteins abundance in sample before and after the serum polymer beads process of modifying through pharmalyte, result as shown in Figure 3.In figure, transverse axis represents the ratio (SAF) of protein spectrum map number and protein length, and SAF numerical value larger expression protein abundance is higher.The longitudinal axis represents the protein number that Mass Spectrometric Identification arrives.In in serum, high-abundance proteins matter represents in (a) figure, and after process, each component (Gly-HCl, NaCl-ACN) protein is at identical SAF numerical value place, and qualification quantity is all lower than untreated original serum.In serum, low abundance proteins represents with (b) figure, and after process, each component (Gly-HCl, NaCl-ACN) protein is at identical SAF numerical value place, and qualification quantity compares with untreated original serum, has increase in various degree.After the polymer beads process of modifying through pharmalyte is described, high-abundance proteins matter qualification quantity reduces, low abundance proteins qualification quantity increases, and proves that the method can effectively reduce the abundance difference in protein example between high-abundance proteins matter and low abundance proteins.
Claims (7)
1. the polymer beads that pharmalyte is modified is used for the pretreated application of protein example, it is characterized in that:
(1) pharmalyte modify polymer beads and sample incubation: the polymer beads modified by pharmalyte adopts phosphoric acid buffer to wash, and hatches 1-4 hour at ambient temperature with protein example;
(2) after hatching, wash-out is carried out to the protein on particle: centrifugal pellet, remove supernatant liquor; Adopt phosphoric acid buffer washing granule, after removing supernatant liquor, adopt sodium chloride solution respectively, glycine solution, acetonitrile solution washs, and collects elutriant;
Particle is polyacrylate polymers particle, and modify pharmalyte molecule by the functional group on surface, the pharmalyte iso-electric point of modification is between 2-14, and grain diameter is 3-8 μm.
2. according to application according to claim 1, it is characterized in that: described protein example is serum or plasma sample.
3. according to application according to claim 1, it is characterized in that: the concentration of described sodium chloride solution is 0.5-2M, the concentration of glycine solution is 50-300mM, pH is 1-3.5, and in acetonitrile solution, the volume ratio of acetonitrile and water is 0.2-0.3, pH=2-4.
4. according to application according to claim 1, it is characterized in that: particle aperture is
described polyacrylate polymers is with methyl acrylate, ethyl propenoate, propyl acrylate, butyl acrylate wherein one or more polymkeric substance being monomer.
5. according to the application described in claim 1 or 4, it is characterized in that: particle surface contains amino-functional group, after glutaraldehyde activated, by pharmalyte molecular modification at particle surface, and sealing and reduction are carried out to particle surface.
6., according to application according to claim 5, the preparation process of the polymer beads that pharmalyte is modified comprises following concrete steps:
(1) polymer beads activation: particle is respectively through after organic solvent and phosphoric acid buffer washing, and employing quality-concentration of volume percent is the glutaraldehyde activated 3-9 hour of 5%-15%;
(2) the particle modification pharmalyte that activates of step (1): polymer beads step (1) activated reacts 12-24 hour with pharmalyte after adopting phosphoric acid buffer washing;
(3) after step (2) to particle surface sealing and reduction: after react with pharmalyte, the sodium cyanoborohydride solution of functional quality-concentration of volume percent to be the glycine of 10-15% and quality-concentration of volume percent be 5-10% reacts 6-9 hour respectively.
7. according to application according to claim 6, it is characterized in that: described pharmalyte is a kind of mixture of aliphatics polyaminopolycarboxylic group, is made up of isomer and homologue.
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| CN108088933A (en) * | 2016-11-21 | 2018-05-29 | 中国科学院大连化学物理研究所 | A kind of high throughput body fluid albumen quality sample pretreatment unit and its application |
| CN109520803B (en) * | 2018-12-29 | 2022-01-18 | 中翰盛泰生物技术股份有限公司 | Method for reducing nonspecific adsorption of microspheres and application |
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| CN102559648A (en) * | 2012-01-13 | 2012-07-11 | 南京工业大学 | Immobilized enzyme using modified epoxy resin as carrier and preparation method and application thereof |
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