CN101054406A - Method of separating and enriching phosphated peptide section by metal oxide magnetic micro-sphere - Google Patents
Method of separating and enriching phosphated peptide section by metal oxide magnetic micro-sphere Download PDFInfo
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- CN101054406A CN101054406A CN 200710041168 CN200710041168A CN101054406A CN 101054406 A CN101054406 A CN 101054406A CN 200710041168 CN200710041168 CN 200710041168 CN 200710041168 A CN200710041168 A CN 200710041168A CN 101054406 A CN101054406 A CN 101054406A
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Abstract
The invention relates to inorganic material and biochemical analysis technology field, more specificly a method of separating and enriching phosphorylated peptide adopting metal oxide magnetic microshpere with core-shell structure. micro sorbent magnetic microshpere is directly added in to mixed solution containing phosphorylated peptide in the present invention for enrich phosphorylated peptide under certain condition. The method is conveniently operated, doesn't need centrifugation, avoid the coprecipitation effect during centrifugation and hole effect of tradition fixed metal ion affinity material and possesses higher chemical inertia and stability. The present method has good selective enriching function for phosphorylated peptide. The enriching efficiency can upgrade a order of magnitude. The enriched sample can be directly analyzed by matrix-assisted laser desorption Ionization-flying time mass spectrogarphy without elution or after elution. The method has practical value in proteome, protein translation-modification field.
Description
Technical field
The invention belongs to inorganic materials and biochemical analysis technical field, the metal oxide coated ferroferric oxide magnetic microballoon with nucleocapsid structure that is specifically related to a kind of superparamagnetism is applied to the method for separating and enriching phosphated peptide section.
Background technology
Proteinic phosphorylation plays an important role in regulating vital process, and the phosphorylated protein of therefore studying in the COMPLEX MIXED matter sample is very significant for the regulation mechanism that discloses vital process.Biological mass spectrometry is the powerful measure of resolving the phosphorylated peptide segment structure, but because the Ionization Efficiency of phosphated peptide section is relatively poor relatively, so non-phosphorylating peptide segment signal has seriously disturbed the detection of phosphated peptide section in the sample.Therefore, before mass spectroscopy phosphated peptide section being carried out the pre-separation enrichment is very important.At present, fixing metal ions affinity chromatography (IMAC) is the separation and concentration phosphorylated protein that widely adopts and the effective ways of peptide section.Yet the body material that this technology adopts is a macroporous resin, and the phosphated peptide section that is enriched in the hole can't be arrived by mass spectrometric detection, promptly so-called " hole effect ".But metal ion is to be sequestered on the body material in the fixing metal ions affinity chromatography material, and is higher relatively to using system condition to require.
In recent years, people hanker after the research of new IMAC body material.Along with the fast development of phosphated peptide section isolation and identification method, investigators find the optionally phosphated peptide section in the separation and concentration complex sample of a lot of metal oxides.Metal oxide with its highly selective and preferably as a result circulation ratio become the another powerful measure of separating and enriching phosphated peptide section.Metal oxide can adopt and load into pillar or the method for directly centrifugation is used for separating and enriching phosphated peptide section.The dress post uses relative complex; Use high speed centrifugation, still have 2 deficiencies: operation is wasted time and energy; High speed centrifugation may cause " co-precipitation effect ", and promptly high-quality non-phosphorylating peptide section and phosphated peptide section co-precipitation are got off.And adopt magneticsubstance is matrix, utilizes the superparamagnetism of material just can solve the problem of above existence effectively.The selective binding of magnetic resolution and metal oxide is got up, can realize the fast simple enrichment of phosphated peptide section in the mixture.Therefore, composite structure closely and have a magneticsubstance of the coated metal oxide of complete nucleocapsid structure, utilize the separation performance of magnetic nuclear and metal oxide shell selection performance better, so that realize that better the selective enrichment for phosphated peptide section is necessary for phosphated peptide section.
Summary of the invention
The object of the present invention is to provide a kind of simple to operate, efficient is high, effective, can carry out the method for high-selectivity enrichment to the trace phosphated peptide section.
The metal oxide magnetic micro-sphere with nucleocapsid structure that the present invention relates to is at superparamagnetism Fe
3O
4The outside carbon-coating that coats of microballoon is as template, and then coated metal oxide, and calcination is removed the method preparation of carbon-coating and got then, and structure is tight.Its nuclear is Fe
3O
4, particle diameter is 100-300nm, and shell is a metal oxide layer, and thickness is 30-50nm.Here metal oxide comprises TiO
2, Al
2O
3, Ga
2O
3, ZrO
2, In
2O
3Or Ce
2O
3Deng.
The preparation method of the metal oxide magnetic micro-sphere with nucleocapsid structure that adopts among the present invention is: at Fe
3O
4The outside carbon-coating that coats of microballoon is as template, and then coated metal oxide, and carbon-coating is removed in calcination then.Its synthetic route as shown in Figure 1.
The method of the separating and enriching phosphated peptide section that the present invention proposes is that above-mentioned metal oxide magnetic micro-sphere with nucleocapsid structure is directly put into the complicated peptide section mixture that contains phosphorylated peptide, carries out trace phosphorylated peptide selective enrichment, need not special processing; After enrichment is good, adopt magnetic field to the phosphorylated peptide of enrichment and separating of other samples, need not centrifugal, so can overcome the common centrifugation problem of traditional centrifugal non-phosphorylating peptide that causes; Then enriched sample is carried out wash-out, also wash-out is not directly analyzed, and has overcome the sample loss problem that the sample elution process causes.And there is not traditional material " hole effect " in this material, can be directly used in ground substance assistant laser and resolve the ionization massspectrum analysis, and method is simple and practical effectively.
Among the present invention, the pH value of above-mentioned enrichment system is 1-6, the phosphorylated peptide sample concentration is 0.05-5ng/ μ L, superparamagnetism microballoon amount is a 10-1000 μ g/1mL sample, enrichment time was at 30 seconds-90 minutes, the enrichment temperature is 20-45 ℃, and sample eluent system pH value is 10-14, and elution time is 5-60 minute.The consumption of magnetic microsphere is a 10-1000 μ g/L sample.The present invention can have the metal oxide magnetic micro-sphere of nucleocapsid structure with any synthetic, and its solution dispersiveness is very good, and system is uniform and stable, helps the enrichment of phosphorylated peptide on material in the solution.
It is of the present invention that phosphorylated peptide is carried out the method for effective selectivity enrichment is simple effectively and have a good magnetic field induction; Enrichment process need not centrifugation, adopts the action of a magnetic field just can realize separating of material and sample; Resolve ionization massspectrum with ground substance assistant laser good consistency is arranged, being had sample after the metal oxide magnetic micro-sphere absorption of nucleocapsid structure also can need not the sample elution step and can directly carry out ground substance assistant laser desorption ionization-flying time mass spectrum analysis, the sample loss of having avoided elution process to cause; Method is simply effective.The present invention can realize high-selectivity enrichment to the phosphorylated peptide in the complicated peptide section mixture that is low to moderate 2fmol/ μ L level, and bioaccumulation efficiency improves one more than the order of magnitude.This phosphorylated peptide selective enrichment method has good practical value and application prospect in fields such as proteomics posttranslational modification researchs.
Description of drawings
Fig. 1 is the synthetic route chart of the metal oxide magnetic micro-sphere with nucleocapsid structure that adopts in the inventive method.
Fig. 2 is the transmission electron microscope picture of the metal oxide magnetic micro-sphere with nucleocapsid structure that adopts in the inventive method.As seen magnetic ball outside has evenly coated the layer of metal oxide compound.
Fig. 3 is the metal oxide magnetic micro-sphere enrichment 2 * 10 that 10 μ g have nucleocapsid structure
-8M (a and b) and 2 * 10
-9The MALDI-TOF MS spectrogram of the front and back of the trypsin digestion mixed peptide section of M (d and e) β-casein.A and b figure relatively, c and d figure, visible phosphorylation peptide gathering efficient is all above one more than the order of magnitude.
Fig. 4 is the metal oxide magnetic micro-sphere enrichment 2 * 10 that 10 μ g have nucleocapsid structure
-8MALDI-TOF MS spectrogram before and after the trypsin digestion mixed peptide section of M (a and b) β-casein.Be enriched to the ammoniacal liquor wash-out that phosphated peptide section on the material adopts 10 μ L 0.5%.The sample of wash-out is carried out mass spectroscopy.Comparison diagram 3b and Fig. 4 are as seen, the sample elution analysis is close with the phospho-peptide strength of signal of direct point sample method gained, do not show very big gap, illustrated and utilized the method for metal oxide magnetic micro-sphere enriching phosphated peptide section to have the higher rate of recovery, also demonstrated and use this method concentration and separation phosphorylated peptide on experimental implementation, to have stronger handiness with nucleocapsid structure.
Fig. 5 is the metal oxide magnetic micro-sphere enrichment 2 * 10 that 20 μ g have nucleocapsid structure
-7MALDI-TOF MS spectrogram (a and b) before and after the proteic trypsin digestion mixed peptide of the M casein section.Compare a and b, the phosphorylated peptide in the visible peptide section mixture has obtained selective enrichment.The phosphated peptide section that is enriched to is as shown in table 1.
Embodiment
By embodiment is that the metal oxide magnetic micro-sphere material with nucleocapsid structure to superparamagnetism provided by the present invention carries out further specifying of example enrichment and substance assistant laser desorpted ionization/mass spectrum direct analysis process.
The selective enrichment and the mass spectroscopy of phosphorylated peptide in the embodiment 1 complicated peptide section mixture
Getting 200 μ L concentration is 2 * 10
-8The peptide section mixture of M beta-casein trypsin digestion adds the 1mgmL of 10 μ L
-1The Fe with nucleocapsid structure
3O
4@TiO
2Magnetic microsphere is 2 with trifluoroacetic acid regulation system pH value, vibrates 5 minutes down at 37 degrees centigrade, and described peptide section is carried out enrichment; Under the action of a magnetic field, separation of supernatant and enrichment the material of phosphated peptide section.With the acetonitrile solution cleaning material twice of 50% (volume ratio), in precipitation, add the acetonitrile solution of 10 μ L 50% (volume ratio), vibration makes it to suspend.Suspension 0.4 μ L and equal-volume 30mg mL
-12, (50% acetonitrile solution is v/v) with 1% (v/v) H for 5-DHB
3PO
4The aqueous solution, 1: 1 (v/v) mixing point are analyzed on MALDI-TOF MS (4700 Proteomics Analyzer, Applied Biosystems) to the MALDI target plate; Laser apparatus is a Nd-YAG laser, wavelength 355nm, laser pulse frequency 200Hz; Acceleration voltage 20KV; Positive ion mode, reflective TOF detects.By Fig. 3 (b (and shown in, phosphated peptide section has obtained the effective selectivity enrichment.
Adjustment will be enriched to the ammoniacal liquor wash-out 5min that sample on the material adopts 10 μ L 0.5%, the sample that obtains behind the wash-out be carried out MALDI-TOF MS again analyze.The result as shown in Figure 4, the phosphated peptide section that has obtained selective enrichment is eluted and has been obtained effective detection from material.
Embodiment 3-5
Adjustment will be enriched to time of ammoniacal liquor wash-out that sample on the material adopts 10 μ L 0.5% for being respectively 5min, 15min, 60min, the sample that obtains behind the wash-out be carried out MALDI-TOF MS again analyze.Experimental result shows that the phosphated peptide section that is enriched on the material just can obtain wash-out substantially completely in 5 minutes.
Embodiment 6
The concentration of adjusting the peptide section mixture of beta-casein trypsin digestion is 2 * 10
-9M, other conditions are with embodiment 1.As seen result such as Fig. 3 (c) and 3 (d) contrast two figure, and phosphated peptide section has obtained the effective as selective enrichment after the enrichment.
Embodiment 7
Adjusting the mixed peptide section sample that adopts is that 200 μ L concentration are 2 * 10
-7The peptide section mixture of the proteic trypsin digestion of Casein of M, other conditions carry out selective enrichment and concentrate and the mass spectrum experiment with embodiment 1.Experimental result is shown in Fig. 5 (a) and Fig. 5 (b).Phosphated peptide section in the complicated peptide section mixture has obtained selective enrichment.
Embodiment 8-10
Adjusting adsorption time is 30 seconds, and 15 minutes, 60 minutes, other conditions were carried out selective enrichment and concentrated and the mass spectrum experiment with embodiment 1.Experimental result shows that the magnetic microsphere with nucleocapsid structure just can realize the effective enrichment to phosphated peptide section when 30 seconds time.
Embodiment 11-12
Adjusting adsorption temp is 25,45 degree, and other conditions are carried out selective enrichment and concentrated and the mass spectrum experiment with embodiment 1.
Embodiment 13-14
Adjusting absorption system pH value is 4,6, and other conditions are carried out selective enrichment and concentrated and the mass spectrum experiment with embodiment 1.
Embodiment 15-19
Adjust its surface of metal oxide magnetic micro-sphere material with nucleocapsid structure of adopting and be respectively coating Al
2O
3, Ga
2O
3, ZrO
2, In
2O
3, Ce
2O
3, other conditions are carried out selective enrichment and are concentrated and the mass spectrum experiment with embodiment 1.
Embodiment 20-24
Adjust its surface of metal oxide magnetic micro-sphere material with nucleocapsid structure of adopting and be respectively coating Al
2O
3, Ga
2O
3, ZrO
2, In
2O
3, Ce
2O
3, other conditions are carried out selective enrichment and are concentrated and the mass spectrum experiment with embodiment 2.
The result of embodiment 4-21 and embodiment 1,2,3 is similar.
Phosphated peptide section in the detected casein albumen of the table 1 MALDI-TOF MS trypsin digestion peptide section mixture
| No. | Peptide section initiation site | Peptide section sequence | Mass-to- |
| 1 | α-S2/138-147 | TVDME[pS]TEVF | 1237.42 |
| 2 | α-S2/138-149 | TVDME[pS]TEVFTK | 1466.51 |
| 3 | α-S2/126-137 | EQL[pS]T[pS]EENSKK | 1562.04 * |
| 4 | α-S1/106-119 | VPQLEIVPN[pS]AEER | 1660.661 |
| 5 | α-S1/104-119 | YKVPQLEIVPN[pS]AEER | 1832.66 |
| 6 | α-S1/43-58 | DIG[pS]E[pS]TEDQAMETIK | 1927.49 |
| 7 | α-S1/104-119 | YKVPQLEIVPN[pS]AEER | 1951.76 |
| 8 | β/33-48 | FQ[pS]EEQQQTEDELQDK | 2061.64 |
| 9 | β/33-52 | FQ[pS]EEQQQTEDELQDKIHPF | 2555.86 |
| 10 | α-S2/2-21 | NTMEHV[pS][pS][pS]EESII[pS]QETYK | 2618.84 |
| 11 | α-S1/99-120 | LRLKKYKVPQLEIVPN[pS]AEERL | 2703.60 |
| 12 | α-S2/2-22 | NTMEHV[pS][pS][pS]EESII[pS]QETYKQ | 2746.59 |
| 13 | β/1-25 | RELEELNVPGEIVE[pS]L[pS][pS][pS]EESITR | 3121.70 |
Claims (1)
1, a kind of method that adopts the metal oxide magnetic micro-sphere separating and enriching phosphated peptide section, it is characterized in that directly metal oxide magnetic micro-sphere being added containing in the complicated peptide section mixture of phosphorylated peptide, carry out trace phosphorylated peptide selective enrichment, adopt magnetic field phosphorylated peptide and other sample separation then enrichment; At last enriched sample is carried out wash-out; Wherein, the enrichment system pH is 1-6, and sample concentration is 0.05-5ng/ μ L, and enrichment time is 30 seconds-90 minutes, and the enrichment temperature is 20-45 ℃; Sample eluent system pH value is 10-14, and elution time is 5-60 minute; The magnetic microsphere consumption is a 10-1000 μ g/mL sample;
Above-mentioned metal oxide microballoon is a nucleocapsid structure, and its nuclear is Fe
3O
4, particle diameter is 100-300nm, and shell is a metal oxide layer, and thickness is 30-50nm, and metal oxide is TiO here
2, Al
2O
3, Ga
2O
3, ZrO
2, In
2O
3Or Ce
2O
3
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| CN102818836A (en) * | 2012-08-06 | 2012-12-12 | 华中师范大学 | Sequential separation and mass spectrum identification method of multi-site phosphorylation peptide |
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