CN101885754B - Method for continuously enriching and separating phosphoeptide at high throughput - Google Patents
Method for continuously enriching and separating phosphoeptide at high throughput Download PDFInfo
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Abstract
The invention relates to a method for continuously enriching and separating phosphoeptide at high throughput. The method comprises the following steps: assembling a microfluidic device; cleaning the microfluidic device for 1 to 3 minutes with two kinds of cleaning solution in the staying time of between 30 and 60 seconds in sequence by using a syringe pump as a fluid transportation power source, and feeding proteolysis products to the microchannel of the device; and enriching, cleaning and eluting to prepare the enriched product. The separation method has simple operation, and can regulate and control the stay time of the fluid in the microchannel by regulating the pushing speed of the syringe pump, namely automatically control the enriching condition by regulating the contact time of the fluid with adsorbing materials, thereby avoiding repeated manual operation required by the conventional methods, and realizing continuous, quick and high-throughput separation and enrichment of the phosphoeptide by means of inherent advantages of the microfluidic device.
Description
Technical field
The invention belongs to the enriching and separating phosphoeptide field, particularly a kind of method of continuously enriching and separating phosphoeptide at high throughput.
Background technology
Proteinic posttranslational modification (PTMs) is an important topic in the present proteomics research.Protein phosphorylation is modification mode behind the most general, most important a kind of protein translation (according to statistics, nearly 1/3rd protein is considered to phosphorylation modification in Mammals), and it is almost regulating the whole process of vital movement; Comprise cell signaling, the differentiation of cell, propagation, nervous activity; Muscle contraction; Metabolism is transcribed and is translated, protein degradation etc.The vital role of phosphorylated protein in cell activities makes it become the focus of proteomics research.
The biological mass spectrometry technology has become the main tool of identifying phosphorylated protein and peptide section at present.Yet; Because the abundance of protein phosphorylation is low and the phospho-peptide Ionization Efficiency is low; The mass signal of phospho-peptide is often covered by the mass signal of non-phosphorylating peptide, therefore before mass spectroscopy is identified, phospho-peptide is carried out enrichment; Improve the relative content of phospho-peptide, become the important content in the phosphorylation proteomics research.The technical development of existing in the past few decades multiple separation and evaluation phosphorylated protein is got up.
Solid metallic ion affinity chromatography (Immobilized metal affinity chromatography, IMAC) method is a kind of method for separating and concentrating commonly used of present phospho-peptide, it is that metals ion through positively charged is (like Fe
3+, Ga
3+) combine with electronegative phosphate group generation electrostatic interaction, and the bonding force of in the damping fluid of high pH value or phosphoric acid salt existence, destroying between metals ion and phosphate group discharges phosphated peptide section.Yet the specificity of this method is not high, and some tart non-phosphorylating peptide sections are also often come out with the phospho-peptide enrichment.
(metal oxide affinity chromatography is the method for the another kind of enrichment acid peptide of rising in recent years MOAC) to the MOX affinity chromatography, and it has higher selectivity than the IMAC method.MOX such as ZrO
2, TiO
2, SnO
2Deng being successfully used to the selective enrichment phospho-peptide.Some investigators are improving TiO in recent years
2Done a large amount of research on the efficient of enrichment acid peptide and the selectivity.As in load liquid, adding 2,5-resorcylic acid (DHB) strengthens TiO
2Selectivity (Molecular & Cellular Proteomics 2005,4 (7), 873-886); With nano-TiO
2Fast enriching that utilizes pulsed laser deposition (PLD) directly to be coated on the MALDI target plate to realize phospho-peptide and detection (Journal of ProteomeResearch 2009,8 (4), 1932-1942); With nano-TiO
2Inject phospho-peptide enricher (Journal of Chromatography A 2007,1165 (1-2), the 128-135 of porous polymer support design suction pipe hair style; Proteomics 2008,8 (21), 4593-4602) etc.Yet; These methods of the phospho-peptide enrichment that is useful at present all need repeated manual operation; Time and effort consuming not only, and be difficult to realize phospho-peptide continuously, high-throughput separates and enrichment, from big volume complex clinical analytic sample, separating and the enrichment acid peptide seems powerless.In addition,, sometimes also be difficult to reach the ideal selectivity, depend on solvent used in load liquid and the scavenging solution and additive toward the contact meeting from complex sample separation and enrichment acid peptide even utilize said new method.So, research be used to realize phospho-peptide fast and automatically, high-throughput separates and novel method, the new unit of enrichment still are very important, especially (Point-of-Care Testing POCT) uses at clinical real-time test.
In the recent two decades in the past, chip lab (1ab-on-a-chip) device has been simplified the biochemical analysis operating process greatly through integrated fluidic components (microfluidic device).Be miniaturized as design fast, low-cost, easy-operating mini POCT device provides an approach.On the internal surface of microchannel, constructing specific micro is very useful for the micro-fluidic device of designing specific functionization; As the cell separator of having designed (Nature 2007; 450 (7173); 1235-U10) (Analytical Chemistry 2004,76 (1), 15-22) etc. with the DNA separator.Monodimension nanometer material has huge specific surface area and is prone to modification property with the surface, it is integrated into will help in the micro-fluidic device realizing the high-throughput separation of biomolecules and detecting.In view of the avidity between phospho-peptide and the MOX; Be necessary on the basis of nano metal oxide materials, to design and have more strong affinity and the more novel micro-fluidic device of highly selective, the novel method of searching continuously enriching and separating phosphoeptide at high throughput from complex biological sample with high-specific surface area and high activity surface.
Summary of the invention
Technical problem to be solved by this invention provides a kind of method of continuously enriching and separating phosphoeptide at high throughput; This method is simple to operate; Can through regulate micro-injection pump push away the residence time of velocity modulation control fluid in the microchannel also promptly with control enrichment condition the duration of contact of sorbing material automatically; Avoided the required repeated manual operation of ordinary method, by the inherent advantage of micro-fluidic device realized to phospho-peptide continuously, fast, high-throughput separates and enrichment.
The method of a kind of continuously enriching and separating phosphoeptide at high throughput of the present invention comprises:
(1) assembling of micro-fluidic device
With the microfluidic channel of quartz capillary as the enrichment acid peptide, vertical-growth TiO is gone up on the surface within it
2Clading ZnO (TiO
2/ ZnO) nanometer stick array is as the enrichment sorbing material of phospho-peptide; Choose three tetrafluoroethylene microtubules; With two microtubules wherein as fluid supplying passage; The 3rd microtubule carries microtubule to be sealed to an end of quartz capillary fluid as the fluid reception channel with epoxy resin glue, and fluid receives microtubule and is sealed to the other end; Carry microtubule to place the syringe on the micro-injection pump to link to each other with two respectively two fluids,, promptly obtained required micro-fluidic device receiving microtubule place placement fluid receiver;
(2) the continuous high-throughput enrichment of phospho-peptide in micro-fluidic device
With above-mentioned micro-fluidic device with pre-washing liquid pre-washing 1～3min under the residence time of 30～60s; Carry with the proteolysis product after the dilution in 1: 5 by volume of load liquid to microfluidic channel then; Behind enrichment, secondary cleaning, wash-out, obtain enriched product, and it is carried out MALDI-TOF MS analyze.
Quartz capillary internal diameter in the said step (1) is 530～550 μ m, and external diameter is 660～700 μ m, and length is 8～12cm.The enrichment sorbing material of the phospho-peptide in the said step (1) is TiO
2/ ZnO nanometer stick array; At first will wait the zinc solution and the amine aqueous solution of volumetric molar concentration to be respectively charged into two syringes that place on the micro-injection pump; Pushing away speed is under 10～25 μ L/min; Two solution are transported to simultaneously the kapillary microchannel that places 90 ℃ of baking ovens; Control fluid time of delivery is 2～5h, and internal surface prepares orthotropic ZnO nanometer stick array in the microchannel; Subsequently syringe pump push away speed be 5 μ L/min, oven temperature be under 70～80 ℃ in microfluidic channel transportation concentration be the TiO of 65 μ g/mL
2Colloidal sol, control fluid time of delivery is 12h, obtains orthotropic TiO
2/ ZnO nanometer stick array.
Preferred zinc salt is a zinc acetate, and amine aqueous solution is a vulkacit H solution.
Tetrafluoroethylene microtubule in the said step (1), wherein fluid conveying microtubule diameter is 300～400 μ m, and length is 20～40cm, and it is 800～900 μ m that fluid receives microtubule diameter, and length is 10～20cm.
Micro-injection pump in the said step (1) is adjustable two channels micro syringe pump.
Pre-washing liquid in the said step (2) is followed successively by 100% acetonitrile and the 50vol% acetonitrile solution that contains the 0.1vol% trifluoroacetic acid.
Load liquid in the said step (2) is the 50～80vol% acetonitrile solution that contains 0.1vol% trifluoroacetic acid or formic acid.
Proteolysis product in the said step (2) is single proteolysis product or mixed protein enzymolysis product; Wherein single proteolysis product is α casein or β rennase hydrolysis products, and the mixed protein enzymolysis product is alpha-casein, beta-casein and 1: 1: 1 in molar ratio mixed enzymolysis product of bovine serum albumin BSA enzymolysis product.
The enrichment residence time in the said step (2) is 10～30s, and enrichment time is 1～3min; The secondary cleaning residence time is 30～60s, and scavenging period is 1～3min; The wash-out residence time is 30～60s, and elution time is 1～3min;
The used scavenging solution of secondary cleaning in the said step (2) is the 50vol% acetonitrile solution that contains the 0.1vol% trifluoroacetic acid; The used elutriant of wash-out is 5vol%NH
3H
2O, the residence time is 30～300s.
Beneficial effect
(1) utilize the ZnO nanometer stick array of vertical-growth on the microfluidic channel internal surface as load TiO
2The support of nano particle has increased TiO when increasing the internal surface of microchannel
2With the contact area of phospho-peptide, by TiO
2And the specificity between the phospho-peptide interacts, and has realized the efficient selective enrichment to phospho-peptide.
(2) this method is simple to operate; Can through regulate micro-injection pump push away the residence time of velocity modulation control fluid in the microchannel also promptly with control enrichment condition the duration of contact of sorbing material automatically; Avoided the required repeated manual operation of ordinary method, by the inherent advantage of micro-fluidic device realized to phospho-peptide continuously, fast, high-throughput separates and enrichment.
(3) microchannel after nanometer-material-modified still has very high selectivity to the enrichment acid peptide in this method after reusing repeatedly continuously.
Description of drawings
The TiO of Fig. 1 for growing on the internal surface of microchannel
2The field emission scanning electron microscope photo of the ZnO nanometer stick array that coats.
Fig. 2 is the synoptic diagram that is used for the micro-fluidic device of phospho-peptide enrichment; Wherein, 1) micro-injection pump, 2) tetrafluoroethylene conveying microtubule, 3) the quartz capillary microchannel, 4) fluid receiver, 5) the long microchannel amplifier section that nanometer stick array is arranged, 6) tetrafluoroethylene reception microtubule, 7) syringe.
Fig. 3 is the MALDI-TOF MS spectrogram of 500fmol alpha-casein enzymolysis product,
number be the characteristic peak of phospho-peptide.
(the enrichment residence time is that 10s continues 1min to Fig. 4 for the MALDI-TOF MS spectrogram after the enrichment of 500fmol alpha-casein enzymolysis product; Experimental technique is according to embodiment 1),
number is the characteristic peak of phospho-peptide.
(the enrichment residence time is that 30s continues 3min to Fig. 5 for the MALDI-TOF MS spectrogram after the enrichment of 500fmol alpha-casein enzymolysis product; Experimental technique is according to embodiment 2),
number is the characteristic peak of phospho-peptide.
Fig. 6 is the MALDI-TOF MS spectrogram of 500fmol beta-casein enzymolysis product, and " ◆ " number is the characteristic peak of phospho-peptide.
Fig. 7 is the MALDI-TOF MS spectrogram after the enrichment of 500fmol beta-casein enzymolysis product (the wash-out residence time is that 30s continues 1min, and experimental technique is according to embodiment 3), and " ◆ " number is the characteristic peak of phospho-peptide.
Fig. 8 is the MALDI-TOF MS spectrogram after the enrichment of 500fmol beta-casein enzymolysis product (the wash-out residence time is that 1min continues 2min, and experimental technique is according to embodiment 4), and " ◆ " number is the characteristic peak of phospho-peptide.
Fig. 9 is the MALDI-TOF MS spectrogram after the enrichment of 500fmol beta-casein enzymolysis product (the wash-out residence time is that 5min continues 10min, and experimental technique is according to embodiment 5), and " ◆ " number is the characteristic peak of phospho-peptide.
Figure 10 is the MALDI-TOF MS spectrogram of mixed protein enzymolysis product (respectively containing 500fmol).
Figure 11 is the MALDI-TOF MS spectrogram (experimental technique according to embodiment 6) after the enrichment of mixed protein enzymolysis product, and wherein
and " ◆ " number are respectively the characteristic peak of alpha-casein phospho-peptide and beta-casein phospho-peptide.
Figure 12 is the MALDI-TOF MS spectrogram (experimental technique according to embodiment 7) after the enrichment of 50fmol alpha-casein enzymolysis product, and " ◆ " number is the characteristic peak of phospho-peptide.
Figure 13 is for the MALDI-TOF MS spectrogram of alpha-casein enzymolysis product after reusing the enrichment of 5 round-robin micro-fluidic devices (experimental technique is according to embodiment 8),
number be the characteristic peak of phospho-peptide.
Figure 14 is the TiO in its microchannel after 5 circulations of the continuous repeated use of micro-fluidic device
2The field emission scanning electron microscope photo of/ZnO nanometer stick array.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.Should be understood that in addition those skilled in the art can do various changes or modification to the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Using ruler to measure length is 530 μ m as 8cm, internal diameter, and external diameter is 660 μ m quartz capillaries.0.05M zinc acetate and 0.05M vulkacit H solution are respectively charged into two syringes that place on the micro-injection pump; It is 25 μ L/min that setting pushes away speed; Two solution are transported to simultaneously the kapillary microchannel that places 90 ℃ of baking ovens; Control fluid time of delivery is 2h, and internal surface prepares orthotropic ZnO nanometer rod in the microchannel.Pushing away speed at syringe pump subsequently is that 5 μ L/min, oven temperature are the TiO that in the microchannel, carries 65 μ g/mL under 80 ℃
2Colloidal sol, control fluid time of delivery is 12h, obtains orthotropic TiO after the drying
2/ ZnO nanometer stick array.The TiO that on the internal surface of microchannel, grows that Fig. 1 obtains for present embodiment
2The field emission scanning electron microscope photo of/ZnO nanometer stick array can find out that the nanometer stick array vertical-growth is on the internal surface of microchannel.
Getting two internal diameters is 300 μ m, length be the tetrafluoroethylene microtubule of 20cm as fluid supplying passage, be sealed to an end of quartz capillary with epoxy resin glue; Getting an internal diameter again is 800 μ m, length be the tetrafluoroethylene microtubule of 10cm as the fluid reception channel, be sealed to the other end of quartz capillary with epoxy resin glue.
Two syringes that 100% acetonitrile is housed are placed on the micro-injection pump; Setting pushes away speed, and to make its residence time in the microchannel be 60s; After fluid was carried 1min, the liquid of changing in the syringe was the pre-washing of the same completion of 50vol% acetonitrile solution (containing the 0.1vol% trifluoroacetic acid) condition to micro-fluidic device.The fluid of changing in the syringe pump is the alpha-casein enzymolysis product after 50vol% acetonitrile solution (containing the 0.1vol% trifluoroacetic acid) 1: the 5 by volume dilution, and it is 10s and lasting 1min that the enrichment residence time is set; Then the fluid in the syringe being replaced by 50vol% acetonitrile solution (containing the 0.1vol% trifluoroacetic acid) as scavenging solution, is to clean 2min under the 30s in the residence time; Afterwards again with 5vol%NH
3H
2O as elutriant the wash-out residence time be under the 30s 1min enriched product is eluted and sample bottle that fluid packed in lyophilize.At last sample being carried out MALDI-TOF MS (4700 Proteomics Analyzer, Applied Biosystems) analyzes.Fig. 2 is the synoptic diagram of the micro-fluidic device that is used for the phospho-peptide enrichment that designed.Fig. 3 is the MALDI-TOF MS spectrogram of 500fmol alpha-casein enzymolysis product in this instance; Fig. 4 is the MALDI-TOF MS spectrogram after the enrichment of 500fmol alpha-casein enzymolysis product in this instance, and wherein
number is the characteristic peak of phospho-peptide.Relatively two figure can find out; A very characteristic peak (m/z 1660.79) of weak phospho-peptide only appears in the alpha-casein enzymolysis product in its spectrogram before enrichment; After the micro-fluidic device enrichment, can detect three characteristic peaks (m/z 1660.79,1832.70,1951.95) of alpha-casein phospho-peptide.
The adjustment enrichment residence time is 30s and continues 3min, and other conditions are with embodiment 1.Fig. 5 is the MALDI-TOF MS spectrogram after the enrichment of 500fmol alpha-casein enzymolysis product in this instance, and wherein
number is the characteristic peak of phospho-peptide.Be not difficult to find that the signal of three of the alpha-casein phospho-peptide characteristic peaks in spectrogram strengthens when the enrichment residence time is 30s, the signal of non-phosphorylating peptide section reduces, and shows that this micro-fluidic device can carry out fast and effectively enrichment to phospho-peptide.
Using ruler to measure length is 530 μ m as 12cm, internal diameter, and external diameter is 660 μ m quartz capillaries.0.05M zinc acetate and 0.05M vulkacit H solution are respectively charged into two syringes that place on the micro-injection pump; It is 10 μ L/min that setting pushes away speed; Two solution are transported to simultaneously the kapillary microchannel that places 90 ℃ of baking ovens; Control fluid time of delivery is 5h, and internal surface prepares orthotropic ZnO nanometer rod in the microchannel.Pushing away speed at syringe pump subsequently is that 5 μ L/min, oven temperature are the TiO that in the microchannel, carries 65 μ g/mL under 70 ℃
2Colloidal sol, control fluid time of delivery is 12h, obtains orthotropic TiO after the drying
2/ ZnO nanometer stick array.
Getting two internal diameters is 300 μ m, length be the tetrafluoroethylene microtubule of 40cm as fluid supplying passage, be glued onto an end of quartz capillary with epoxy resin; Getting an internal diameter again is 800 μ m, length be the tetrafluoroethylene microtubule of 20cm as the fluid reception channel, be sealed to the other end of quartz capillary with epoxy resin glue.
Two syringes that 100% acetonitrile is housed are placed on the micro-injection pump; Setting pushes away speed, and to make its residence time in the microchannel be 40s; After fluid was carried 2min, the liquid of changing in the syringe was the pre-washing of the same completion of 50vol% acetonitrile solution (containing the 0.1vol% trifluoroacetic acid) condition to micro-fluidic device.The fluid of changing in the syringe pump is the beta-casein enzymolysis product after 50vol% acetonitrile solution (containing the 0.1vol% trifluoroacetic acid) 1: the 5 by volume dilution, and it is 30s and lasting 1.5min that the enrichment residence time is set; Then the fluid in the syringe being replaced by 50vol% acetonitrile solution (containing the 0.1vol% trifluoroacetic acid) as scavenging solution, is to clean 80s under the 30s in the residence time; Afterwards again with 5vol%NH
3H
2O as elutriant the wash-out residence time be under the 30s 1min enriched product is eluted and sample bottle that fluid packed in lyophilize.At last sample being carried out MALDI-TOF MS (4700 Proteomics Analyzer, Applied Biosystems) analyzes.Fig. 6 is the MALDI-TOF MS spectrogram of 500fmol beta-casein enzymolysis product in this instance, and Fig. 7 is the MALDI-TOF MS spectrogram after the enrichment of 500fmol beta-casein enzymolysis product in this instance, and wherein " ◆ " number is the characteristic peak of phospho-peptide.Relatively two figure can find; A very characteristic peak (m/z 2061.83) of weak phospho-peptide only appears in the beta-casein enzymolysis product in its spectrogram before enrichment; After the micro-fluidic device enrichment, can detect three characteristic peaks (m/z2061.84,1832.70,1951.95) of beta-casein phospho-peptide.
The adjustment wash-out residence time is 1min and continues 2min, and other conditions are with embodiment 3.Fig. 8 is the MALDI-TOF MS spectrogram after the enrichment of 500fmol beta-casein enzymolysis product in this instance, and wherein " ◆ " number is the characteristic peak of phospho-peptide.When the wash-out residence time is 1min; The signal of three characteristic peaks in spectrogram of beta-casein phospho-peptide has obvious enhancing during than 30s; The signal of non-phosphorylating peptide section significantly reduces, and this shows when the wash-out residence time is for 1min and can the phospho-peptide of micro-fluidic device institute enrichment fully be eluted.
The adjustment wash-out residence time is 5min and continues 10min, and other conditions are with embodiment 3.Fig. 9 is the MALDI-TOF MS spectrogram after the enrichment of 500fmol beta-casein enzymolysis product in this instance, and wherein " ◆ " number is the characteristic peak of phospho-peptide.When the wash-out residence time extends to 5min; The signal of three characteristic peaks in spectrogram of beta-casein phospho-peptide obviously do not strengthen during for 1min than the wash-out residence time, and also i.e. explanation continues time expand the phospho-peptide of enrichment in effective wash-out micro-fluidic device there is no need.
Using ruler to measure length is 550 μ m as 8cm, internal diameter, and external diameter is 700 μ m quartz capillaries.0.05M zinc acetate and 0.05M vulkacit H solution are respectively charged into two syringes that place on the micro-injection pump; It is 20 μ L/min that setting pushes away speed; Two solution are transported to simultaneously the kapillary microchannel that places 90 ℃ of baking ovens; Control fluid time of delivery is 2.5h, and internal surface prepares orthotropic ZnO nanometer rod in the microchannel.Pushing away speed at syringe pump subsequently is that 5 μ L/min, oven temperature are the TiO that in the microchannel, carries 65 μ g/mL under 75 ℃
2Colloidal sol, control fluid time of delivery is 12h, obtains orthotropic TiO after the drying
2/ ZnO nanometer stick array.
Getting two internal diameters is 300 μ m, length be the tetrafluoroethylene microtubule of 20cm as fluid supplying passage, be sealed to an end of quartz capillary with epoxy resin glue; Getting an internal diameter again is 800 μ m, length be the tetrafluoroethylene microtubule of 10cm as the fluid reception channel, be sealed to the other end of quartz capillary with epoxy resin glue.
Two syringes that 100% acetonitrile is housed are placed on the micro-injection pump; Setting pushes away speed, and to make its residence time in the microchannel be 50s; After fluid was carried 2min, the liquid of changing in the syringe was the pre-washing of the same completion of 50vol% acetonitrile solution (containing the 0.1vol% trifluoroacetic acid) condition to micro-fluidic device.Fluid in the replacing syringe pump is that alpha-casein, beta-casein and the bovine serum albumin (BSA) after 50vol% acetonitrile solution (containing the 0.1vol% trifluoroacetic acid) 1: the 5 by volume dilution is 1: 1: 1 mixed enzymolysis product (respectively containing 500fmol) by amount of substance, and it is 30s and lasting 3min that the enrichment residence time is set; Then the fluid in the syringe being replaced by 50vol% acetonitrile solution (containing the 0.1vol% trifluoroacetic acid) as scavenging solution, is to clean 2min under the 30s in the residence time; Afterwards again with 5vol%NH
3H
2O as elutriant the wash-out residence time be under the 1min 2min enriched product is eluted and sample bottle that fluid packed in lyophilize.At last sample being carried out MALDI-TOF MS (4700 Proteomics Analyzer, Applied Biosystems) analyzes.Figure 10 is the MALDI-TOF MS spectrogram of mixed protein enzymolysis product in this instance (respectively containing 500fmol); Figure 11 is the MALDI-TOF MS spectrogram after this mixed protein enzymolysis product enrichment in this instance, and wherein
and " ◆ " number are respectively the characteristic peak of alpha-casein phospho-peptide and beta-casein phospho-peptide.From two figure, can find out; Mixed protein enzymolysis product its spectrogram of covering owing to a large amount of non-phosphorylating peptide section ionization signals before enrichment detects the characteristic peak (Figure 10) less than any phospho-peptide; But the characteristic peak of alpha-casein phospho-peptide and beta-casein phospho-peptide is detected (Figure 11) as main mass signal after the micro-fluidic device enrichment, and this shows that this device has very high selectivity at enrichment acid peptide from complex sample.
Adjustment proteolysis product is the alpha-casein enzymolysis product of 100 times of concentration dilutions, and other conditions are with embodiment 6.Figure 12 is the MALDI-TOF MS spectrogram after the enrichment of 50fmol alpha-casein enzymolysis product in this instance, and wherein
number is the characteristic peak of phospho-peptide.As can be seen from the figure; When protein concentration reduces by 100 times; After the micro-fluidic device enrichment, still can show that this device has very high sensitivity on the selective enrichment phosphorylated peptide through two characteristic peaks of MALDI-TOF MS analyzing and testing to the alpha-casein phospho-peptide.
Using ruler to measure length is 530 μ m as 8cm, internal diameter, and external diameter is 660 μ m quartz capillaries.0.05M zinc acetate and 0.05M vulkacit H solution are respectively charged into two syringes that place on the micro-injection pump; It is 25 μ L/min that setting pushes away speed; Two solution are transported to simultaneously the kapillary microchannel that places 90 ℃ of baking ovens; Control fluid time of delivery is 2h, and internal surface prepares orthotropic ZnO nanometer rod in the microchannel.Pushing away speed at syringe pump subsequently is that 5 μ L/min, oven temperature are the TiO that in the microchannel, carries 65 μ g/mL under 80 ℃
2Colloidal sol, control fluid time of delivery is 12h, obtains orthotropic TiO after the drying
2/ ZnO nanometer stick array.
Getting two internal diameters is 300 μ m, length be the tetrafluoroethylene microtubule of 20cm as fluid supplying passage, be sealed to an end of quartz capillary with epoxy resin glue; Getting an internal diameter again is 800 μ m, length be the tetrafluoroethylene microtubule of 10cm as the fluid reception channel, be sealed to the other end of quartz capillary with epoxy resin glue.
With placing on the micro-injection pump in two syringes that 100% acetonitrile is housed; Setting pushes away speed, and to make its residence time in the microchannel be 30s; After fluid was carried 3min, the liquid of changing in the syringe was the pre-washing of the same completion of 50vol% acetonitrile solution (containing the 0.1vol% trifluoroacetic acid) condition to micro-fluidic device.The fluid of changing in the syringe pump is the alpha-casein enzymolysis product after 50vol% acetonitrile solution (containing the 0.1vol% trifluoroacetic acid) 1: the 5 by volume dilution, and it is 30s and lasting 3min that the enrichment residence time is set; Then the fluid in the syringe being replaced by 50vol% acetonitrile solution (containing the 0.1vol% trifluoroacetic acid) as scavenging solution, is to clean 2min under the 30s in the residence time; Afterwards again with 5vol%NH
3H
2O as elutriant the wash-out residence time be under the 1min 2min enriched product is eluted and sample bottle that fluid packed in lyophilize.Utilize 0.5%NH
3H
2O, ultrapure water and absolute ethyl alcohol are successively to clean microchannel 2min under the 30s to make its internal surface activating and regenerating in the residence time, are used for the enrichment acid peptide once more.Micro-fluidic device is reused the sample that obtains after 5 circulations carry out MALDI-TOF MS (4700 Proteomics Analyzer, Applied Biosystems) analysis.Figure 13 is the MALDI-TOF MS spectrogram after the enrichment of alpha-casein enzymolysis product in this instance, and wherein
number is the characteristic peak of phospho-peptide.Can know by figure, after continuously reusing 5 circulations this micro-fluidic device still can be from the proteolysis product effective enrichment acid peptide, demonstrated its higher wearing quality.Figure 14 is the TiO in its microchannel after 5 circulations of the continuous repeated use of this micro-fluidic device
2The field emission scanning electron microscope photo of/ZnO nanometer stick array can find that the pattern of nanometer stick array is constant basically before use, has shown stability and abrasion resistance that it is high.
Claims (6)
1. the method for a continuously enriching and separating phosphoeptide at high throughput comprises:
(1) assembling of micro-fluidic device
With the microfluidic channel of quartz capillary as the enrichment acid peptide, vertical-growth TiO is gone up on the surface within it
2Clading ZnO nano rod array is as the enrichment sorbing material of phospho-peptide; Choose three tetrafluoroethylene microtubules; With two microtubules wherein as fluid supplying passage; The 3rd microtubule carries microtubule to be sealed to an end of quartz capillary fluid as the fluid reception channel with epoxy resin glue, and fluid receives microtubule and is sealed to the other end; Carry microtubule to place the syringe on the micro-injection pump to link to each other with two respectively two fluids,, promptly obtained required micro-fluidic device receiving microtubule place placement fluid receiver;
Wherein, the enrichment sorbing material of phospho-peptide is TiO
2/ ZnO nanometer stick array; At first will wait the zinc solution and the amine aqueous solution of volumetric molar concentration to be respectively charged into two syringes that place on the micro-injection pump; Pushing away speed is under 10～25 μ L/min; Two solution are transported to simultaneously the kapillary microchannel that places 90 ℃ of baking ovens; Control fluid time of delivery is 2～5h, and internal surface prepares orthotropic ZnO nanometer stick array in the microchannel; Subsequently syringe pump push away speed be 5 μ L/min, oven temperature be under 70～80 ℃ in microfluidic channel transportation concentration be the TiO of 65 μ g/mL
2Colloidal sol, control fluid time of delivery is 12h, obtains orthotropic TiO
2/ ZnO nanometer stick array;
(2) the continuous high-throughput enrichment of phospho-peptide in micro-fluidic device
With above-mentioned micro-fluidic device with pre-washing liquid pre-washing 1～3min under the residence time of 30～60s; Carry with the proteolysis product after the dilution in 1: 5 by volume of load liquid to microfluidic channel then; Behind enrichment, secondary cleaning, wash-out, obtain enriched product, and it is carried out MALDI-TOF MS analyze;
Wherein, pre-washing liquid is followed successively by 100% acetonitrile and the 50vol% acetonitrile solution that contains the 0.1vol% trifluoroacetic acid;
Load liquid is the 50～80vol% acetonitrile solution that contains 0.1vol% trifluoroacetic acid or formic acid;
The used scavenging solution of secondary cleaning is the 50vol% acetonitrile solution that contains the 0.1vol% trifluoroacetic acid;
The used elutriant of wash-out is 5vol%NH
3H
2O.
2. the method for a kind of continuously enriching and separating phosphoeptide at high throughput according to claim 1, it is characterized in that: the quartz capillary internal diameter in the said step (1) is 530～550 μ m, and external diameter is 660～700 μ m, and length is 8～12cm.
3. the method for a kind of continuously enriching and separating phosphoeptide at high throughput according to claim 1; It is characterized in that: the tetrafluoroethylene microtubule in the said step (1); Wherein fluid conveying microtubule diameter is 300～400 μ m; Length is 20～40cm, and it is 800～900 μ m that fluid receives microtubule diameter, and length is 10～20cm.
4. the method for a kind of continuously enriching and separating phosphoeptide at high throughput according to claim 1, it is characterized in that: the micro-injection pump in the said step (1) is adjustable two channels micro syringe pump.
5. the method for a kind of continuously enriching and separating phosphoeptide at high throughput according to claim 1; It is characterized in that: the proteolysis product in the said step (2) is single proteolysis product or mixed protein enzymolysis product; Wherein single proteolysis product is α casein or β rennase hydrolysis products, and the mixed protein enzymolysis product is alpha-casein, beta-casein and 1: 1: 1 in molar ratio mixed enzymolysis product of bovine serum albumin BSA enzymolysis product.
6. the method for a kind of continuously enriching and separating phosphoeptide at high throughput according to claim 1, it is characterized in that: the enrichment residence time in the said step (2) is 10～30s, enrichment time is 1～3min; The secondary cleaning residence time is 30～60s, and scavenging period is 1～3min; The wash-out residence time is 30～60s, and elution time is 1～3min.
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| CN103864897B (en) * | 2014-04-03 | 2016-03-02 | 中国人民解放军防化学院 | A kind of purification process of phosphorylation eledoisin |
| CN106018862B (en) * | 2016-07-07 | 2017-11-17 | 中国科学院半导体研究所 | Chip type high flux biological detection freezes uniform sample injection method |
| CN109959648B (en) * | 2017-12-22 | 2022-05-13 | 深圳先进技术研究院 | Biological cold light detection device and detection method |
| CN110354920B (en) * | 2018-03-26 | 2021-08-17 | 首都师范大学 | Paper-based micro-fluidic chip and phosphorylated polypeptide enrichment detection method based on same |
| CN110343664B (en) * | 2018-04-03 | 2021-12-24 | 中国人民解放军军事科学院军事医学研究院 | Method for extracting exosome and exosome protein |
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Non-Patent Citations (2)
| Title |
|---|
| 周杏琴等.反相高效液相制备色谱分离酪蛋白磷酸肽.《分析仪器》.2002,(第04期),19-21. * |
| 隋少卉等.基于强阳离子交换色谱分离的磷酸化肽段富集策略.《色谱》.2008,第26卷(第02期),195-199. * |
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