CN103134852A - Detection method for protein with nucleic acid aptamer target gathering and laser enzymolysis - Google Patents
Detection method for protein with nucleic acid aptamer target gathering and laser enzymolysis Download PDFInfo
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- CN103134852A CN103134852A CN2013100421947A CN201310042194A CN103134852A CN 103134852 A CN103134852 A CN 103134852A CN 2013100421947 A CN2013100421947 A CN 2013100421947A CN 201310042194 A CN201310042194 A CN 201310042194A CN 103134852 A CN103134852 A CN 103134852A
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Abstract
The invention belongs to the field of chemicobiology and relates to a detection method for protein with nucleic acid aptamer target gathering and laser enzymolysis. Firstly, nanogold with the diameter of 13nm and a target board are in epoxy dripping mode, the anogold is dryed and calcined, and even and thick gold films are formed on the target board. With the method, the gold films which are with a loose hole-type structure are formed on the surface finish of the target board, large specific surface area is provided, excellent stability and biocompatibility are achieved, and the method for protein with nucleic acid aptamer target gathering and laser enzymolysis can be applied to various functionalized modifications. Secondly, nucleic acid aptamers of mercapto modification is fixed on the target board. Thirdly, target protein is gathered and target quick laser enzymolysis is supplemented, and samples are sent to matrix-assisted laser desorption ionization (MALDI)-time-off-flight mass spectrometer (TOFMS) to accept qualitative and quantitative analysis. The whole experiment process is simple, quick, accurate and can be widely applied into high-throughput qualitative and quantitative analysis of important protein.
Description
Technical field
The invention belongs to the chemicobiology field, be specifically related to a kind ofly utilize on the aptamers target enrichment target protein and be aided with the laser enzymolysis, and MALDI-TOF MS analyzes, be used for simple, fast, accurately the target protein molecule is carried out the method for qualitative and quantitative analysis.
Background technology
The protein molecular marker thing is the important biomacromolecule of a class, can provide disease to occur, the information of development.Can promote the early diagnosis of disease to the detection of protein molecular marker thing, the progress of monitoring disease promotes follow-up treatment etc., and is therefore extremely important.Yet many protein molecular marker things with clinical practice often are in very low concentration level in body fluid at present, detect very difficult.Traditional method is the immunization method that utilizes based on antibody, as ELISA etc., because antibody has affinity and the specificity of height to substrate molecule, target protein can be extracted from the background of complexity.Aptamer development in the last few years is widely used in diagnosis and detection rapidly, is a kind of very promising antibody surrogate thing.Aptamer is a kind of oligomerization single-chain nucleic acid (ssDNA or RNA), obtains by being called " the aglucon phyletic evolution technology of index concentration " in-vitro screening method (SELEX).Compare with antibody, aptamer has following advantage: (1) can be synthetic in batches with the method for chemistry, has a cost low, and sequence is characteristics accurately; (2) substrate is wider, and is little of ion, little molecule, can become the target of its combination to albumen, cell greatly; (3) aptamers is more stable, and is easier of chemical modification.Many aptamers detection methods based on fluorescence, galvanochemistry, colorimetric have been arranged at present.
MALDI-TOF MS technology is used in the analysis of biomolecule widely.MALDI-TOF MS has high flux, operates relatively simply, and analysis speed is fast, and resolution is high, highly sensitive advantage.Utilize MALDI-TOF MS to compare with additive method as detecting unit and have several advantages: the identity of (1) substrate can further be confirmed by quality signal or tandem mass spectrometry signal; (2) the rear modification situation of substrate, perhaps the isomery situation can be analyzed by mass spectrum; (3) MALID-TOF MS has impayable express-analysis ability and mass resolution ability, detects when can realize a plurality of substrate molecule, and can carry out the high throughput analysis of hundreds of samples.
Therefore, provide on a kind of aptamer target enrichment and laser enzymolysis to detect method of protein imperative.
Summary of the invention
The object of the invention is to provide enrichment and laser enzymolysis detection method of protein on a kind of aptamer target.
Content of the present invention provides a kind of method that has the golden film of loose and porous structure in the target plate finishing, and concrete steps are as follows:
(1) with the gold size solution point of 50-100nmol/L on the surface of target plate, after naturally drying 10-12 hour, target plate was calcined 1-3 hour under 150-200 ℃, obtain the target plate of finishing gold film;
(2) be that the aptamer covalent modification of sulfydryl modification is on the target plate surface of finishing gold film with an end;
The target protein that (3) will need to analyze drops on the target plate of step (2) gained, at wet box (English name: after placing 1 hour Humidity Chamber), be that 0.1% polysorbas20 and deionized water rinse successively with volume ratio, dry;
(4) with the surperficial trypsin solution that drips 10-30 ng/ μ L of the target plate of step (3) gained, use Ear Mucosa Treated by He Ne Laser Irradiation 10-40 second, dry;
(5) drip matrix alpha-cyano-4-hydroxycinnamic acid (being CHCA) on the target plate surface of step (4) gained, send into mass spectrophotometry.
In the present invention, described in step (1), gold size is of a size of 13 nm, and synthesis step is:
(a) add 1mmol/L gold chloride (HAuCl in being connected with the three-neck flask of reflux condensing tube
4) solution and boiling;
(b) add the 38.8mmol/L citric acid three sodium solution, backflow 15min;
(c) cooling.Resulting red solution by 0.22 μ m membrane filtration, is namely got the nanometer gold size that is of a size of 13nm.In the present invention, the concentration of the described gold size solution of step (1) is 92nmol/L.
In the present invention, the described optical maser wavelength of step (4) is 808 nm.
Beneficial effect of the present invention is: the method for the target plate finishing gold film that provides is simple, and formed golden film has loose porous structure, has larger specific surface area and stability, is conducive to next step modification and application.Simultaneously, enrichment on the aptamers target, laser enzymolysis and MALDI-TOF MS technology can carry out simply target protein, quantitative and qualitative analysis fast and accurately.
Description of drawings
Fig. 1 is the schematic flow sheet that on a kind of aptamer target, enrichment and laser enzymolysis detect method of protein;
Fig. 2 is the photo of the target plate scanning electron microscope of finishing gold film in embodiment 1;
Fig. 3 is 5000 ng/mL lysozyme mass spectrum mass spectrograms in embodiment 2, and signal peptide section (m/z=1400.6) is wherein the strongest peak;
Fig. 4 is the standard solution neutral line curve that utilizes lysozyme signal peptide section (m/z=1400.6) to set up in embodiment 2;
Fig. 5 is 1000 ng/mL lysozyme soln mass spectrograms in embodiment 2;
Fig. 6 is 100 ng/mL lysozyme soln mass spectrograms in embodiment 2;
Fig. 7 is 14 ng/mL lysozyme soln mass spectrograms in embodiment 2;
Fig. 8 be in embodiment 2 on the aptamers target enrichment and laser enzymolysis detect selectivity schematic diagram to cromoci;
Fig. 9 be in embodiment 2 on the aptamers target enrichment and laser enzymolysis detect selectivity schematic diagram to human serum albumins;
Figure 10 be in embodiment 2 on the aptamers target enrichment and laser enzymolysis detect selectivity schematic diagram to transferrins;
Figure 11 be in embodiment 2 on the aptamers target enrichment and laser enzymolysis detect selectivity schematic diagram to fibrin ferment;
Figure 12 be in embodiment 2 on the aptamers target enrichment and laser enzymolysis detect selectivity schematic diagram to myoglobins;
Figure 13 be in embodiment 2 on the aptamers target enrichment and laser enzymolysis detect selectivity schematic diagram to lysozyme;
Figure 14 adds the typical curve of antalzyme protein in people's urine sample in embodiment 3;
Figure 15 is the testing result of lysozyme in urine under 5000 ng/mL lysozyme concentration in embodiment 3;
Figure 16 is the testing result of lysozyme in urine under 500 ng/mL lysozyme concentration in embodiment 3;
Figure 17 is the testing result of lysozyme in urine under 100 ng/mL lysozyme concentration in embodiment 3;
Figure 18 is the testing result of lysozyme in embodiment 3 empty urines;
Figure 19 be in embodiment 3 in people's urine sample peptide section (m/z=1400.6) the tandem mass spectrometry figure of endogenous lysozyme and with (m/z=1400.6) tandem mass spectrometry figure of standard lysozyme signal peptide section contrast (figure a is standard antalzyme protein signal peptide section tandem mass spectrometry figure, and figure b is the tandem mass spectrometry figure of endogenous bacteriolyze BPTI section in people's urine sample).
Number in the figure, 1 is gold size, 2 is target plate, 3 target plates for finishing gold film, 4 be aptamers, and 5 be protein, and 6 is laser, and asterisk represents trypsase from the degradation peak, triangle represents that lysozyme peak, circle represent the internal standard peptide peak.
Embodiment
The following examples are to further illustrate of the present invention, rather than limit the scope of the invention.
Embodiment 1:
Embodiment 1:
The preparation of the target plate of finishing gold film, preparation flow be as shown in Figure 1:
(1) utilize classical Frens method, synthetic gold size under the sodium citrate protection, concrete steps are:
(a) add 1mmol/L gold chloride (HAuCl in being connected with the three-neck flask of reflux condensing tube
4) solution and boiling;
(b) add the 38.8mmol/L citric acid three sodium solution, backflow 15min;
(c) cooling.Resulting red solution by 0.22 μ m membrane filtration, is namely got the nanometer gold size that is of a size of 13nm.
(2) get 10 mL gold sizes, centrifugal 20min under 20000 rcf centrifugal force, supernatant discarded.According to the absorbance under 260nm, gold size concentration is adjusted to 92 nmol/L;
(3) with 2 μ L 92 nmol/L gold size points on clean target plate, naturally dry and spend the night;
(4) baking oven is adjusted to 200 ℃, put into target plate, calcining 2 h;
(5) naturally cooling, rinse gently with deionized water, dry standby.
The Electronic Speculum result of the finishing of gained gold film target plate as shown in Figure 2, the Electronic Speculum model is Philips XL30 (Holland), operating voltage 20 kV.Need not metal spraying, the target plate that directly will modify golden film sticks on by conducting resinl on the sample stage of Electronic Speculum, the sample introduction analysis.
Embodiment 2: take lysozyme as model proteins, utilize aptamer to carry out enrichment on target, laser enzymolysis and MALDI-TOF MS qualitative and quantitative analysis:
(1) the lysozyme aptamers of sulfydryl modification is fixing: sulfydryl modification lysozyme aptamers is processed 1h with 2.5 mM three (2-carboxyethyl) phosphines (TCEP), get 2 μ L points on target plate.Target plate is put in wet box, and lucifuge is spent the night;
(2) configuration of standard protein solution: antalzyme protein is diluted to respectively 5000 ng/mL, 1000 ng/mL, 100 ng/mL, 14 ng/mL with phosphate buffer (pH7.4 contains 30 μ g/mL human albumins for PBS, 10 mM);
(3) enrichment: draw on 2 μ L antalzyme protein solution and MALDI target plate, react 1h in wet box under room temperature.Use successively 0.1% polysorbas20 and deionized water rinsing 1min.Dry;
(4) laser enzymolysis: with 25 mmol/L ammonium bicarbonate buffers configuration 25 ng/ μ L trypsin solutions.Getting 2 μ L points is being enriched with on the target plate of lysozyme.Use Ear Mucosa Treated by He Ne Laser Irradiation 30s.Dry.Add internal standard peptide section SR-12;
(5) mass spectrophotometry: add CHCA(alpha-cyano-4-hydroxycinnamic acid) matrix is sent into MALDI-TOF MS and is analyzed.Utilize bacteriolyze BPTI section m/z=1400.6 and interior mark SR-12 to carry out qualitative and quantitative analysis.Wherein internal standard peptide SR-12 and bacteriolyze BPTI section m/z=1400.6 only differ an amino acid, and molecular weight differs 14 Da.
The Mass Spectrometer Method result is as shown in Fig. 3-13, Fig. 8-13rd, on the aptamers target, enrichment and laser enzymolysis detect the selectivity schematic diagram of lysozyme method, can draw other 5 kinds of albumen (human serum albumins (Human Serum Albumin), myoglobins (Myoglobin), cromoci (Cytochrome C), fibrin ferment (Thrombin), transferrins (Transferrin)) detection of lysozyme is not disturbed.
Embodiment 3: lysozyme is added in human urine, utilize aptamer to carry out enrichment on target, laser enzymolysis and MALDI-TOF MS qualitative and quantitative analysis:
(1) preparation of urine sample: collect the Healthy People urina sanguinis, centrifugal removal precipitation with 0.22 μ m membrane filtration, is stored in-20 ℃.Lysozyme is directly added urine sample respectively to 5000 ng/mL, 500 ng/mL, 100 ng/mL concentration;
(2) according to embodiment 2 steps (3), (4), (5) are described carries out enrichment to sample, laser enzymolysis and mass spectrophotometry.Lysozyme is carried out quantitative and qualitative analysis;
(3) detection of endogenous lysozyme in urine: do not add any albumen in urine sample, directly press embodiment 2 steps (3), (4), (5) are described carries out enrichment to blank urine sample, laser enzymolysis and mass spectrophotometry.Peptide section m/z=1400.6 is carried out MS/MS analyze, compare with the MS/MS spectrogram of standard.The concentration of utilizing typical curve to calculate the endogenous lysozyme in blank urine sample is 125ng/mL.
The Mass Spectrometer Method result is as shown in Figure 14-19.
Claims (5)
1. on an aptamer target, enrichment and laser enzymolysis detect method of protein, it is characterized in that concrete steps are as follows:
(1) with the gold size solution point of 50-100nmol/L on the surface of target plate, after naturally drying 10-12 hour, target plate was calcined 1-3 hour under 150-200 ℃, obtain the target plate of finishing gold film;
(2) be that the aptamer covalent modification of sulfydryl modification is on the target plate surface of finishing gold film with an end;
The target protein that (3) will need to analyze drops on the target plate of step (2) gained, place 1 hour in wet box after, is that 0.1% polysorbas20 and deionized water rinse successively with volume ratio, dries;
(4) with the surperficial trypsin solution that drips 10-30 ng/ μ L of the target plate of step (3) gained, use Ear Mucosa Treated by He Ne Laser Irradiation 10-40 second, dry;
(5) drip matrix alpha-cyano-4-hydroxycinnamic acid on the target plate surface of step (4) gained, carry out mass spectrophotometry.
2. on a kind of aptamer target according to claim 1, enrichment and laser enzymolysis detect method of protein, it is characterized in that described in step (1), gold size is of a size of 13 nm, and its synthesis step is:
(a) add 1 mmol/L chlorauric acid solution and boiling in being connected with the three-neck flask of reflux condensing tube;
(b) add the 38.8mmol/L citric acid three sodium solution, 15 min reflux;
(c) cooling, resulting red solution by membrane filtration, is namely got required gold size.
3. on a kind of aptamer target according to claim 1, enrichment and laser enzymolysis detect method of protein, it is characterized in that the concentration of the described gold size solution of step (1) is 92 nmol/L.
4. on a kind of aptamer target according to claim 1, enrichment and laser enzymolysis detect method of protein, it is characterized in that the described optical maser wavelength of step (4) is 808 nm.
5. on a kind of aptamer target according to claim 1, enrichment and laser enzymolysis detect method of protein, it is characterized in that the described trypsin solution concentration of step (4) is 25 ng/ μ L.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104764796A (en) * | 2015-04-01 | 2015-07-08 | 复旦大学 | Method for detecting content of glycosylated hemoglobin in blood based on MALDI-ToF MS |
| CN105301091A (en) * | 2015-11-18 | 2016-02-03 | 复旦大学 | Method for promoting protein N-terminal peptide fragment identification by using laser-assisted enzymolysis |
| CN107543850A (en) * | 2017-07-28 | 2018-01-05 | 苏州健雄职业技术学院 | Detect biology sensor of trypsase and its preparation method and application |
| CN110168376A (en) * | 2018-06-08 | 2019-08-23 | 廖世奇 | The method and kit that the more target molecules of magnetic bead-aptamer-detect simultaneously |
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| CN101470052A (en) * | 2007-12-28 | 2009-07-01 | 中国科学院大连化学物理研究所 | Protein collection and enzymolysis integrated sample pretreatment apparatus and its production |
| CN102713626A (en) * | 2009-10-20 | 2012-10-03 | 丹麦达科有限公司 | Immunochemical detection of single target entities |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104764796A (en) * | 2015-04-01 | 2015-07-08 | 复旦大学 | Method for detecting content of glycosylated hemoglobin in blood based on MALDI-ToF MS |
| CN105301091A (en) * | 2015-11-18 | 2016-02-03 | 复旦大学 | Method for promoting protein N-terminal peptide fragment identification by using laser-assisted enzymolysis |
| CN107543850A (en) * | 2017-07-28 | 2018-01-05 | 苏州健雄职业技术学院 | Detect biology sensor of trypsase and its preparation method and application |
| CN107543850B (en) * | 2017-07-28 | 2019-12-13 | 苏州健雄职业技术学院 | Biosensor for detecting trypsin, and preparation method and application thereof |
| CN110168376A (en) * | 2018-06-08 | 2019-08-23 | 廖世奇 | The method and kit that the more target molecules of magnetic bead-aptamer-detect simultaneously |
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Application publication date: 20130605 |