CN101481402B - Method for separating and purifying glycopeptide - Google Patents
Method for separating and purifying glycopeptide Download PDFInfo
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- CN101481402B CN101481402B CN2008100173861A CN200810017386A CN101481402B CN 101481402 B CN101481402 B CN 101481402B CN 2008100173861 A CN2008100173861 A CN 2008100173861A CN 200810017386 A CN200810017386 A CN 200810017386A CN 101481402 B CN101481402 B CN 101481402B
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Abstract
The invention discloses a method for separating and purifying glycopeptide by magnetic particles. The realization steps of the method are as follows: putting water-washed epoxy magnetic particles into a flask, putting the flask containing suspension on a magnetic separator for magnetic separation and removing supernatant without the magnetic particles; adding 50ml of hydrazine hydrate with the mass fraction of 5%, putting the flask into a water bath, inserting a stirrer into the flask for fully mixing and reacting, cleaning with absolute ethyl alcohol and water, performing magnetic separation to remove the supernatant without the magnetic particles; dissolving or diluting a protein sample in coupling buffer solution, adding sodium periodate to the final concentration of the solution, then reacting at normal temperature without light; removing the sodium periodate which does not react by a G-25 desalination column; and coupling with glycoprotein and obtaining the glycopeptide by enzymolysis. The method optimizes a coupling condition of the glycopeptide and hydrazine functionalized magnetic particles and hydrazide functionalized magnetic particles, and has quick and high-efficiency separation and purification.
Description
Technical field
The present invention relates to a kind of method of separation and purification glycopeptide, relate in particular to a kind of method with magnetic particle separation and purification glycopeptide.
Background technology
Proteinic glycosylation modified be one of the most common, most important protein post-translational modification, in the present known Mammals protein over half be take place glycosylated.Glycosylation plays an important role to proteinic 26S Proteasome Structure and Function, and the degree of Protein Glycosylation Overview and the ANOMALOUS VARIATIONS of sugar chain structure usually are the signs that cancer and other diseases take place.
The variation of Protein Glycosylation Overview degree and glycosylation site can detect by the variation of glycosylated polypeptides (glycopeptide).In recent years, the method for glycopeptide mainly contained lectin method and hydrazine chemical method in the separation and purification complex sample.Complex sample such as serum and cytolemma total protein etc.
The lectin method is the glycoprotein of using earlier in the lectin extraction separation purification of samples, in conjunction with bidimensional electrophoretic separation, extracts glycopeptide with lectin once more behind enzymolysis or the direct enzymolysis again, and mass spectrum is identified afterwards.The main drawback of this method is: the specificity binding characteristic of lectin itself can cause the selectivity of sample separation purifying, the spy that different lectins has in conjunction with dissimilar glycoprotein gives birth to, thereby single lectin can not one time whole glycoprotein in the extraction separation purification of samples, and during the two-dimensional electrophoresis working expenditure, effort.
The hydrazine chemical method is to extract glycoprotein with hydrazine chemical separation purifying, can be disposable glycoprotein/the glycopeptide of the nearly all type of separation and purification selectively not, then with diverse ways wash-out successively.Such as: discharge N-glycoprotein/glycopeptide with glycopeptidase (PNGase F), β-null method discharges O-glycoprotein/glycopeptide, but the main drawback of this method is: at present the general solid carrier that uses is the hydrazine gel column during hydrazine chemical separation purifying glycopeptide, velocity of separation is slow, time-consuming, effort, some in addition also need separation equipment such as whizzer, secondly, the specific surface area of hydrazine gel column is less, so the loading capacity of glycoprotein molecule is just less, the catalytic chance of sample is less, has increased the coupling time of glycoprotein and hydrazine gel column.
Summary of the invention
The object of the present invention is to provide a kind of method of separation and purification glycopeptide,, it has solved hydrazine chemical process separation and purification glycopeptide program complexity in the background technology, the technical problem of separation and purification long reaction time.
Magnetic particle is called for short the magnetic grain, carrier as a kind of new bio-molecular separation purifying, has bigger specific surface than non-magnetic carrier, fast to speed of response such as albumen, loading capacity is big, magnetic resolution is simply quick, is applied to biochemical technology fields such as cell, nucleic acid, proteinic separation, affinity chromatography and radioimmunity the sixth of the twelve Earthly Branches gradually.
Technical scheme of the present invention is as follows:
A kind of method of separation and purification glycopeptide, its special character is: comprise the steps
(1) preparation hydrazine functionalized magnetic particles
A cleans
Get 200 milligrams of epoxidation magnetic grains and place flask, water cleans, and the flask that will fill suspension after the cleaning is put into and carries out magnetic resolution on the magnetic separator, removes the clear liquid that the upper strata does not contain the magnetic grain;
B reacts modification
Add 50 milliliters of massfractions and be 5% hydrazine hydrate in flask, flask is placed water-bath, insert agitator in flask, turn on agitator stirs and makes magnetic grain and hydrazine hydrate thorough mixing, reaction again, and epoxidation magnetic grain is modified into the hydrazine functionalized magnetic particles;
(2) separation and purification of glycopeptide
A cleans
After reaction is finished, the hydrazine functionalized magnetic particles is cleaned respectively with dehydrated alcohol and water, carry out magnetic resolution, remove the clear liquid that the upper strata does not contain the magnetic grain; Use 40 ml waters resuspended again, suspension is preserved after resuspended standby;
The oxidation of b glycoprotein
With the dissolving of a certain amount of protein sample or be diluted in the coupling buffer, add sodium periodate to its ultimate density 10～15 mmoles, reacting at normal temperature without light 0.5～1.5 hour;
C removes sodium periodate
Remove salt plug with G-25 and remove unreacted sodium periodate;
The coupling of d glycoprotein
Protein solution behind the desalination is joined in the pretreated hydrazine functionalized magnetic particles of coupling buffer, room temperature vibration coupling 4～24 hours, magnetic resolution after the coupling is removed supernatant; Wash the magnetic grain 3～6 times with scavenging solution, to remove not covalently bound albumen;
The e enzymolysis obtains glycopeptide
Glycoprotein on the magnetic grain is that 7.8 three propyloic phosphorus were dissolved in 0.1 mole the solution of ammonium hydrogencarbonate the room temperature reduction more than 30 minutes with 5 mmole pH; Adding iodo-acetamide to its ultimate density then is 8～15mM, and room temperature continued reaction more than 30 minutes, and magnetic resolution is removed supernatant; With pH is 7.8 the resuspended magnetic grain of ammonium bicarbonate solution, adds trypsinase to its ultimate density 20 mg/litre, and 37 ℃ of vibration enzymolysis 4 hours are added the trypsinase of same amount, continue enzymolysis more than 10 hours;
F cleans
The magnetic grain cleans respectively earlier with 2 mol sodium chloride solutions and dehydrated alcohol, is that 7.8 0.1 mol ammonium bicarbonate solution is washed with pH again, removes fully until non-glycosylated polypeptide;
E discharges glycopeptide
With pH is 7.8 the resuspended magnetic grain of 0.1 mol ammonium bicarbonate solution, adds the glycopeptidase F of 0.5 microlitre, and 37 ℃ of vibration enzymolysis 4 hours add the glycopeptidase F of same amount, continue reaction more than 4 hours, finish the release of glycopeptide;
A kind of method of separation and purification glycopeptide, its special character is: comprise the steps
(1) preparation hydrazides functionalized magnetic particles
A cleans
Get 200 milligrams of carboxylated magnetic grains and place flask, place flask, water cleans, add 0.1 mole of pH and be 4.75 2-(N-morpholine) ethyl sulfonic acid damping fluid pre-treatment, the flask that will fill suspension after the cleaning is put into and carries out magnetic resolution on the magnetic separator, removes the clear liquid that the upper strata does not contain the magnetic grain;
B reacts modification
In flask, add 50 milliliters of 2-(N-morpholine) ethyl sulfonic acid damping fluids that contain 1 gram adipic dihydrazide flask is put into water-bath, insert agitator, turn on agitator adds 0.5 gram carbodiimide in stirring and make carboxylated magnetic grain and adipic dihydrazide thorough mixing, stirring again, continue stirring reaction more than 3 hours, carboxylated magnetic grain is modified into the hydrazides functionalized magnetic particles;
(2) separation and purification of glycopeptide
A cleans
After reaction is finished, the hydrazides functionalized magnetic particles is cleaned respectively with dehydrated alcohol, 1.5 mol sodium chloride solutions and water, magnetic resolution is removed the clear liquid that the upper strata does not contain the magnetic grain; Use 40 ml waters resuspended again, suspension is preserved after resuspended standby.Here generally clean 3 effects and get final product, hydromining is preferable with ultrapure water free from foreign meter or distilled water effect, and normal temperature condition is preserved down and got final product, and 4 ℃ of preservation effects are better.
The oxidation of b glycoprotein
With the dissolving of a certain amount of protein sample or be diluted in and add sodium periodate in the coupling buffer to its ultimate density 10～15 mmoles, reacting at normal temperature without light 0.5～1.5 hour;
C removes sodium periodate
Remove salt plug with G-25 and remove unreacted sodium periodate;
The coupling of d glycoprotein
Protein solution behind the desalination is joined in the pretreated hydrazides functionalized magnetic particles of coupling buffer, room temperature vibration coupling 4～24 hours, magnetic resolution after the coupling is removed supernatant; Wash the magnetic grain 3～6 times with scavenging solution, to remove not covalently bound albumen.
The e enzymolysis obtains glycopeptide
Glycoprotein on the magnetic grain is that 7.8 three propyloic phosphorus were dissolved in 0.1 mole the solution of ammonium hydrogencarbonate the room temperature reductase 12 more than 0 minute with 5 mmole pH; Adding iodo-acetamide to its ultimate density then is 8～15 mmoles, and room temperature continued reaction more than 30 minutes, and magnetic resolution is removed supernatant; With pH is 7.8 the resuspended magnetic grain of ammonium bicarbonate solution, adds trypsinase to its ultimate density 20 mg/litre, and 37 ℃ of vibration enzymolysis 4 hours are added the trypsinase of same amount, continue enzymolysis more than 10 hours;
F cleans
The magnetic grain cleans respectively earlier with 2 mol sodium-chlor and dehydrated alcohol, is that 7.8 0.1 mol ammonium bicarbonate solution is washed with pH again, removes fully until non-glycosylated polypeptide;
E discharges glycopeptide
With pH is 7.8 the resuspended magnetic grain of 0.1 mol ammonium bicarbonate solution, adds the glycopeptidase F of 0.5 microlitre, and 37 ℃ of vibration enzymolysis 4 hours add the glycopeptidase F of same amount, continue reaction more than 4 hours, finish the release of glycopeptide;
Can comprise before above-mentioned (2) purification procedures
1) quantitatively goes out the content of suspension hydrazine functionalized magnetic particles
After getting the oven dry of 2ml suspension, weigh, calculate the hydrazine functionalized magnetic particles content in the suspension;
2) measure the content that diazanyl is rolled into a ball on the hydrazine functionalized magnetic particles
With 0.1 milliliter of massfraction be 80% hydrazine hydrate respectively water do the different multiples dilution, as standard hydrazine sample;
With 0.01 milliliter of each concentration standard hydrazine sample, 0.05 milligram of hydrazine magnetic grain, 0.05 milligram of epoxidation magnetic grain, the latter two volumes are 0.01 milliliter, with 0.2 milliliter of dicinchonine acid work reagent mix, and 37 ℃ of oscillatory reactions 0.5 hour, magnetic resolution;
Make blank with dicinchonine acid work reagent, ultraviolet-visible pectrophotometer is surveyed supernatant 562 nanometers light absorption values in each pipe; It is for referencial use to draw the hydrazine typical curve, calculates diazanyl mass contg on the magnetic grain with the difference of 562 nanometers light absorption values of supernatant behind hydrazine magnetic grain and epoxy magnetic grain and the dicinchonine acid reagent react.
Also can comprise before above-mentioned (2) purification procedures
1) quantitatively goes out the content of suspension hydrazides functionalized magnetic particles
After getting the oven dry of 2ml suspension, weigh, calculate the hydrazides functionalized magnetic particles content in the suspension;
2) content of hydrazides group on the mensuration hydrazides functionalized magnetic particles
With adipic dihydrazide respectively water do different multiples dilution, as standard hydrazides sample;
With 0.01 milliliter of each concentration standard hydrazides sample and 0.05 milligram of hydrazides magnetic grain, 0.05 milligram of carboxylated magnetic grain, the latter two volumes are 0.01 milliliter, with 0.2 milliliter of dicinchonine acid work reagent mix, and 37 ℃ of oscillatory reactions 0.5 hour, magnetic resolution;
Make blank with dicinchonine acid work reagent, ultraviolet-visible pectrophotometer is surveyed supernatant 562 nanometers light absorption values in each pipe; It is for referencial use to draw the hydrazine typical curve, calculates diazanyl content on the magnetic grain with the difference of 562 nanometers light absorption values of supernatant behind hydrazine magnetic grain and epoxy magnetic grain and the dicinchonine acid work reagent react.
Above-mentioned coupling buffer is meant that pH is 5.5 100 mmoles/rise sodium-acetate and 150 mmoles/the rise solution of sodium-chlor.
Above-mentioned scavenging solution be meant pH be 83 8 mole of urea and 0.4 mole of ammonium hydrogencarbonate solution.
Above-mentioned water is ultrapure water or distilled water.
Above-mentioned magnetic resolution is all separated on magnetic separator.
The present invention has following advantage:
The present invention utilizes the characteristic of isolating simple characteristics fast of magnetic grain and the special modification oxosugar of hydrazine chemistry; make the hydrazine functionalized magnetic particles with hydrazine hydrate trimming loop oxidation magnetic grain; or modify carboxylated magnetic grain with 2-(N-morpholine) ethyl sulfonic acid; make the hydrazides functionalized magnetic particles; separation and purification glycopeptide again; optimized glycoprotein and hydrazine functionalized magnetic particles or hydrazides functionalized magnetic particles coupling condition, separation and purification reaction fast, efficient.
Description of drawings
Fig. 1 modifies the structural representation of epoxy magnetic grain for hydrazine hydrate of the present invention.
Fig. 2 adipic dihydrazide is modified the structural representation of carboxylated magnetic grain.
Fig. 3 a is the principle of hydrazine magnetic grain extraction separation purifying glycoprotein of the present invention.
Fig. 3 b is the schematic flow sheet of hydrazine magnetic grain extraction separation purifying glycoprotein of the present invention.
Fig. 4 measures the canonical plotting of hydrazine content for the present invention adopts the BCA method.
Fig. 5 detects the synoptic diagram of magnetic grain amount and coupling efficiency for the present invention.
Fig. 6 detects the synoptic diagram of coupling efficiency under differing temps, time conditions for the present invention.
Fig. 7 checks the synopsis of three kinds of albumen and hydrazine magnetic grain coupling situation for the present invention.
Fig. 8 dyes figure for SDS-PAGE electrophoresis detection hydrazine magnetic grain of the present invention extracts specific the examining of biased sample.
Fig. 9 is a RNase B glycopeptide mass spectrum of the present invention.
Embodiment
The present invention utilizes the characteristic of isolating simple characteristics fast of magnetic grain and the special modification oxosugar of hydrazine chemistry, with hydrazine hydrate trimming loop oxidation magnetic grain, makes the hydrazine functionalized magnetic particles, has optimized the coupling condition of glycoprotein and hydrazine functionalized magnetic particles.Extract the effect of glycoprotein with standard protein checking hydrazine functionalized magnetic particles, (MALDI-TOF-MS) identified desired polypeptides through substance assistant laser desorpted flight time mass spectrum, verified that the hydrazine functionalized magnetic particles is used for the feasibility of the evaluation of complex sample glycoprotein and glycosylation site.
The test instrument:
The magnetic separator of Shanxi North America Gene Co., Ltd; The ZHWY-2101C type constant temperature culture vibrator of Shanghai ZHICHENG Anaiytical Instrument Manufacturing Co., Ltd.; G-25 removes salt plug; The ultraviolet-visible pectrophotometer of the U.S. (Aglient); The substance assistant laser desorpted flight time mass spectrum of this analytical instrument company of Cray holder of Britain island Tianjin group.
More than each instrument all have sale in the market.
Test reagent:
Epoxidation magnetic grain (Epoxy-Mag) is available from Shanxi North America Gene Co., Ltd; Carboxylated magnetic grain is available from Shanxi North America Gene Co., Ltd; Ribonuclease B (RNase B), ox Pp63 glycophosphoproteins (Fetuin), dicinchonine acid (BCA), iodo-acid amide and reductive agent three propyloic phosphorus (TCEP), matrix (name is called the matrix of CHCA), bovine serum albumin (BSA) are available from U.S. Sigma company; Bovine trypsin (Trypsin), glycopeptidase F (PNGaseF) are all available from the U.S.; 2-(N-morpholine) ethyl sulfonic acid (MES) is available from U.S. Amresco company; Carbodiimide (EDC) is available from Pierce company, and (ADH is available from Japanese Kasei Kogyo company for adipic dihydrazide; Sodium periodate (NaIO
4), sodium-acetate (NaAc), sodium-chlor (NaCL), ammonium hydrogencarbonate (NH
4HCO
3), methyl alcohol, urea, dehydrated alcohol and hydrazine hydrate etc. be in homemade Gneral analysis pure.Test water is the ultrapure water of handling through Ai Ke ultrapure water water manufacturing system.
More than each reagent also all have sale in the market.
Embodiment 1:
Referring to Fig. 1 and Fig. 3, the method for separation and purification glycopeptide
(1) separation and purification glycopeptide
(1-1) preparation hydrazine functionalized magnetic particles
A cleans
Get 200 milligrams of epoxidation magnetic grains and place flask, water cleans, and the flask that will fill suspension after the cleaning is put into and carries out magnetic resolution on the magnetic separator, removes the clear liquid that the upper strata does not contain the magnetic grain; Can be ortho-water, but use water better effects if free from foreign meter, as ultrapure water and distilled water (twice distillatory water).
B reacts (modification)
50 milliliters of massfractions of adding are 5% hydrazine hydrate in flask, flask is placed water-bath, insert agitator in flask, turn on agitator stirs and makes magnetic grain and hydrazine hydrate thorough mixing, reacted 3 hours again, and epoxidation magnetic grain is modified into the hydrazine functionalized magnetic particles; The temperature of water-bath is at 20 ℃-70 ℃, and the time of reaction also can surpass 3 hours, but generally should not be above 24 hours.
(1-2) separation and purification of glycopeptide
A cleans
After reaction is finished, the hydrazine functionalized magnetic particles is cleaned respectively with dehydrated alcohol and water, carry out magnetic resolution, remove the clear liquid that the upper strata does not contain the magnetic grain; Use 40 ml waters resuspended again, suspension is preserved after resuspended standby.Here generally clean and get final product for 3 times, hydromining is preferable with ultrapure water free from foreign meter or distilled water effect, and the suspension room temperature condition is preserved down and got final product, but 4 ℃ of shelf times are longer.
The oxidation of b glycoprotein
With the dissolving of a certain amount of protein sample or be diluted in the coupling buffer (100mM (mmole/liter) NaAc, 150mM NaCl pH5.5), adds sodium periodate (NaIO
4) to its ultimate density 10～15mM, reacting at normal temperature without light 0.5～1.5 hour;
C removes sodium periodate NaIO
4
Remove salt plug with G-25 and remove unreacted sodium periodate (NaIO
4);
The coupling of d glycoprotein
Protein solution behind the desalination is joined in the pretreated hydrazine functionalized magnetic particles of coupling buffer, room temperature vibration coupling 4～24 hours, magnetic resolution after the coupling is removed supernatant; With scavenging solution (8M urea/0.4M NH
4HCO
3, pH8.3) wash the magnetic grain 3～6 times, to remove not covalently bound albumen.
The e enzymolysis obtains glycopeptide
(5mM three propyloic phosphorus are dissolved in the NH of 0.1M to glycoprotein on the magnetic grain with TCEP
4In the HCO3 solution, pH7.8) the room temperature reductase 12 is more than 0 minute; Adding iodo-acetamide to its ultimate density then is 8～15mM, and room temperature continued reaction more than 30 minutes, and magnetic resolution is removed supernatant; NH with pH7.8
4HCO
3The resuspended magnetic grain of solution adds trypsinase to its ultimate density 20mg/L, and 37 ℃ of vibration enzymolysis 4h add the enzyme of same amount, continue more than the enzymolysis 10h;
F cleans
The magnetic grain cleans respectively earlier with 2mol/L sodium-chlor (NaCl) and dehydrated alcohol, uses the 0.1mol/L ammonium hydrogencarbonate (NH of pH7.8 again
4HCO
3) solution washes, remove fully until non-glycosylated polypeptide.General sodium-chlor (NaCL) and dehydrated alcohol clean 3～5 times respectively earlier and can remove fully;
E discharges glycopeptide
0.1mol/L ammonium hydrogencarbonate (NH with pH7.8
4HCO
3) the resuspended magnetic grain of solution, adding the glycopeptidase F (PNGase F) of 0.5 microlitre, 37 ℃ of vibration enzymolysis 4 hours add the same enzyme amount, continue reaction more than 4 hours, finish the release of glycopeptide;
Referring to Fig. 2 and Fig. 3, embodiment 2:
(1-1) preparation hydrazides functionalized magnetic particles
A cleans
Get 200 milligrams of carboxylated magnetic grains and place flask, place flask, water cleans, 2-(N-morpholine) ethyl sulfonic acid (MES) the damping fluid pre-treatment that adds 0.1 mole of pH4.75, the flask that will fill suspension after the cleaning is put into and carries out magnetic resolution on the magnetic separator, removes the clear liquid that the upper strata does not contain the magnetic grain; Can be ortho-water, but use water better effects if free from foreign meter, as ultrapure water and distilled water (twice distillatory water).
B reacts (modification)
In flask, add 50 milliliters of 2-(N-morpholine) ethyl sulfonic acid (MES) damping fluids that contain 1 gram adipic dihydrazide (ADH) flask is put into water-bath, insert agitator, turn on agitator stirs, add 0.5 gram carbodiimide (EDC) in the stirring, continue stirring reaction more than 3 hours, carboxylated magnetic grain is modified into hydrazides magnetic grain.It is good that the temperature of water-bath remains on 37 ℃ of effects, and the time of reaction also can surpass 3 hours, but generally should not be above 24 hours.
(1-2) separation and purification of glycopeptide
A cleans
After reaction is finished, the hydrazine functionalized magnetic particles is cleaned respectively with dehydrated alcohol (removal of impurity) and water, carry out magnetic resolution, remove the clear liquid that the upper strata does not contain the magnetic grain; Use 40 ml waters resuspended again, suspension is preserved after resuspended standby.Here generally clean and get final product for 3 times, hydromining is preferable with ultrapure water free from foreign meter or distilled water effect, and the suspension room temperature preservation gets final product, but 4 ℃ keep effects better.
The oxidation of b glycoprotein
With the dissolving of a certain amount of protein sample or be diluted in that (100mM NaAc, 150mM NaCl pH5.5), add sodium periodate (NaIO in the coupling buffer
4) to its ultimate density 10～15mM, reacting at normal temperature without light 0.5～1.5 hour;
C removes sodium periodate NaIO
4
Remove salt plug with G-25 and remove unreacted sodium periodate (NaIO
4);
The coupling of d glycoprotein
Protein solution behind the desalination is joined in the pretreated hydrazides functionalized magnetic particles of coupling buffer, room temperature vibration coupling 4～24 hours, magnetic resolution after the coupling is removed supernatant; With scavenging solution (8M urea and 0.4M ammonium hydrogencarbonate NH
4HCO
3, pH8.3) wash the magnetic grain 3～6 times, to remove not covalently bound albumen.
The e enzymolysis obtains glycopeptide
(5mM three propyloic phosphorus are dissolved in the ammonium hydrogencarbonate NH4HCO3 solution of 0.1M glycoprotein on the magnetic grain, and pH7.8) the room temperature reductase 12 is more than 0 minute with TCEP; Adding iodo-acetamide to its ultimate density then is 8～15mM, and room temperature continued reaction more than 30 minutes, and magnetic resolution is removed supernatant; Ammonium hydrogencarbonate NH with pH7.8
4HCO
3The resuspended magnetic grain of solution adds trypsinase to its ultimate density 20mg/L, and 37 ℃ of vibration enzymolysis 4h add the enzyme of same amount, continue more than the enzymolysis 10h;
F cleans
The magnetic grain cleans respectively earlier with 2mol/L sodium-chlor (NaCL) and dehydrated alcohol, uses the 0.1mol/L ammonium hydrogencarbonate (NH of pH7.8 again
4HCO
3) solution washes, remove fully until non-glycosylated polypeptide.General sodium-chlor (NaCL) and dehydrated alcohol clean 3～5 times respectively earlier and can remove fully;
E discharges glycopeptide
0.1mol/L ammonium hydrogencarbonate (NH with pH7.8
4HCO
3) the resuspended magnetic grain of solution, adding the glycopeptidase F (PNGase F) of 0.5 microlitre, 37 ℃ of vibration enzymolysis 4 hours add the same enzyme amount, continue reaction more than 4 hours, finish the release of glycopeptide;
The following steps of also can interting before (2) purification procedures of embodiment 1 can quantitatively go out the content of hydrazine functionalized magnetic particles and diazanyl group, referring to Fig. 4, specifically comprise
1) quantitatively goes out the content of suspension hydrazine functionalized magnetic particles
After getting the oven dry of 2ml suspension, weigh, calculate the hydrazine functionalized magnetic particles content in the suspension;
2) measure the content that diazanyl is rolled into a ball on the hydrazine functionalized magnetic particles
With 0.1 milliliter of massfraction be 5% hydrazine hydrate respectively water do the different multiples dilution, as standard hydrazine sample;
With 0.01 milliliter of each concentration standard hydrazine sample, 0.05 milligram of hydrazine magnetic grain, 0.05 milligram of epoxidation magnetic grain, the latter two volumes are 0.01 milliliter, with 0.2 milliliter of dicinchonine acid work reagent mix, and 37 ℃ of oscillatory reactions 0.5 hour, magnetic resolution;
Make blank with dicinchonine acid work reagent, ultraviolet-visible pectrophotometer is surveyed supernatant 562 nanometers light absorption values in each pipe; It is for referencial use to draw the hydrazine typical curve, calculates diazanyl content on the magnetic grain with the difference of 562 nanometers light absorption values of supernatant behind hydrazine magnetic grain and epoxy magnetic grain and the dicinchonine acid reagent react.
The following steps of also can interting before (2) purification procedures of embodiment 2 can quantitatively go out the content of hydrazides functionalized magnetic particles and hydrazides group, referring to Fig. 4, specifically comprise
1) quantitatively goes out the content of suspension hydrazides functionalized magnetic particles
After getting the oven dry of 2ml suspension, weigh, calculate the hydrazides functionalized magnetic particles content in the suspension;
2) measure the content that diazanyl is rolled into a ball on the hydrazides functionalized magnetic particles
With adipic dihydrazide solution respectively water do different multiples dilution, as standard hydrazides sample;
With 0.01 milliliter of each concentration standard hydrazides sample and 0.05 milligram of hydrazides magnetic grain, 0.05 milligram of carboxylated magnetic grain, the latter two volumes are 0.01 milliliter, with 0.2 milliliter of dicinchonine acid work reagent mix, and 37 ℃ of oscillatory reactions 0.5 hour, magnetic resolution;
Make blank with dicinchonine acid work reagent, ultraviolet-visible pectrophotometer is surveyed supernatant 562 nanometers light absorption values in each pipe; It is for referencial use to draw the hydrazides typical curve, calculates hydrazide group mass contg on the magnetic grain with the difference of 562 nanometers light absorption values of supernatant behind hydrazides magnetic grain and carboxylated magnetic grain and the dicinchonine acid work reagent react.
Can identify glycoprotein and glycosylation site by the glycopeptide that aforesaid method obtains, concrete method can be with reference to following document:
1?Zhang?H，Li?XJ，Martin?DB，Aebersold?R(2003)Nat?Biotech?21：660-666；
2?Zhou?Y，Aebersold?R，Zhang?H(2007)Anal?Chem?79：5826-5837。
The effect of identifying glycoprotein and glycosylation site in this method can be referring to Fig. 7, Fig. 8 and Fig. 9.
Hydrazine activation magnetic grain is identified the ultimate principle of glycoprotein and glycosylation site
The adjacent hydroxyl of sugar is an aldehyde radical through periodate oxidation in the glycoprotein, and glycoprotein forms stable hydrazone key by the group of the diazanyl on aldehyde radical and the hydrazine magnetic grain and is fixed on the magnetic grain; Non-glycosylated protein is removed by cleaning the magnetic grain, and glycoprotein obtains separation and purification on the magnetic grain; Glycoprotein on the magnetic grain only stays glycopeptide on the magnetic grain after sex change, reduction, alkylation, trypsinase Trypsin enzyme are cut and cleaned; Glycopeptide can use PNGase F and β-null method to discharge N-respectively to be connected polypeptide and to be connected polypeptide with O-on the magnetic grain.Polypeptide is determined glycoprotein and is identified glycosylation site by the method for LC-MS/MS and data base querying comparison.This method is more effective to identifying the N-glycosylation site, because the N-glycosylation always occurs on the Asn residue, and have the conserved sequence (the wherein any amino acid of X representative except that proline(Pro)) of a N-X-S/T, can be used as the index that protein glycosylation site and glycoprotein are identified; And N-glycoprotein have can this cardohydrata-peptide linkage of specificity enzymolysis PNGase F, Asn becomes Asp behind the enzymolysis, cause relative molecular weight to increase by 0.98, play the effect of quality status stamp N-glycosylation site, thereby further increase the accuracy that identify in the protein glycosylation site.
Referring to Fig. 5 and Fig. 6, hydrazine functionalized magnetic particles that the present invention obtains or hydrazides functionalized magnetic particles record every gram magnetic grain hydrazine content and are about 180 μ mol.Under the room temperature condition, glycoprotein and hydrazine functionalized magnetic particles or hydrazides functionalized magnetic particles coupling time should be greater than 4 hours, but every milligram of hydrazine functionalized magnetic particles or hydrazides functionalized magnetic particles extraction separation purifying 10 μ mol glycoprotein, and extraction efficiency is greater than 80%.Verified its specificity that glycoprotein is extracted by standard protein, and detected target polypeptides with MALDI-TOF-MS.
Claims (8)
1. the method for a separation and purification glycopeptide is characterized in that, comprises the steps
(1) preparation hydrazine functionalized magnetic particles
A disappears and washes
Get 200 milligrams of epoxidation magnetic grains and place flask, water cleans, and the flask that will fill suspension after the cleaning is put into and carries out magnetic resolution on the magnetic separator, removes the clear liquid that the upper strata does not contain the magnetic grain;
B reacts modification
Add 50 milliliters of massfractions and be 5% hydrazine hydrate in flask, flask is placed water-bath, insert agitator in flask, turn on agitator stirs and makes magnetic grain and hydrazine hydrate thorough mixing, reaction again, and epoxidation magnetic grain is modified into the hydrazine functionalized magnetic particles;
(2) separation and purification of glycopeptide
A cleans
After reaction is finished, the hydrazine functionalized magnetic particles is cleaned respectively with dehydrated alcohol and water, carry out magnetic resolution, remove the clear liquid that the upper strata does not contain the magnetic grain; Use 40 ml waters resuspended again, suspension is preserved after resuspended standby;
The oxidation of b glycoprotein
With the dissolving of a certain amount of protein sample or be diluted in the coupling buffer, add sodium periodate to its ultimate density 10～15mM, reacting at normal temperature without light 0.5～1.5 hour;
C removes sodium periodate
Remove salt plug with G-25 and remove unreacted sodium periodate;
The coupling of d glycoprotein
Protein solution behind the desalination is joined in the pretreated hydrazine functionalized magnetic particles of coupling buffer, room temperature vibration coupling 4～24 hours, magnetic resolution after the coupling is removed supernatant; Wash the magnetic grain 3～6 times with scavenging solution, to remove not covalently bound albumen;
The e enzymolysis obtains glycopeptide
Glycoprotein on the magnetic grain is room temperature reductase 12 more than 0 minute in the solution of 7.8 the three propyloic phosphorus ammonium hydrogencarbonate that is dissolved in 0.1M with 5mM, pH; Adding iodo-acetamide to its ultimate density then is 8～15mM, and room temperature continued reaction more than 30 minutes, and magnetic resolution is removed supernatant; With pH is 7.8 the resuspended magnetic grain of ammonium bicarbonate solution, adds trypsinase to its ultimate density 20 mg/litre, and 37 ℃ of vibration enzymolysis 4 hours are added the trypsinase of same amount, continue enzymolysis more than 10 hours;
F cleans
The magnetic grain cleans respectively earlier with 2 mol sodium-chlor and dehydrated alcohol, is that 7.8 0.1 mol ammonium bicarbonate solution is washed with pH again, removes fully until non-glycosylated polypeptide;
G discharges glycopeptide
With pH is 7.8 the resuspended magnetic grain of 0.1 mol ammonium bicarbonate solution, adds the glycopeptidase F of 0.5 microlitre, and 37 ℃ of vibration enzymolysis 4 hours add the glycopeptidase F of same amount, continue reaction more than 4 hours, finish the release of glycopeptide.
2. the method for a separation and purification glycopeptide is characterized in that, comprises the steps:
(1) preparation hydrazides functionalized magnetic particles
A cleans
Get 200 milligrams of carboxylated magnetic grains and place flask, place flask, water cleans, and adds 0.1 mole of pH and be 4.75 2-(N-morpholine) ethyl sulfonic acid damping fluid pre-treatment, the flask that will fill suspension after the cleaning is put into and carries out magnetic resolution on the magnetic separator, removes the clear liquid that the upper strata does not contain the magnetic grain;
B reacts modification
Add 50 milliliters of 2-(N-morpholine) ethyl sulfonic acid damping fluids that contain 1 gram adipic dihydrazide in flask flask is put into water-bath, insert agitator, turn on agitator stirs, and adds 0.5 gram carbodiimide in the stirring, continues stirring reaction more than 3 hours;
(2) separation and purification of glycopeptide
A cleans
After reaction is finished, the hydrazides functionalized magnetic particles is cleaned respectively with dehydrated alcohol, sodium chloride solution and water, carry out magnetic resolution, remove the clear liquid that the upper strata does not contain the magnetic grain; Use 40 ml waters resuspended again, suspension is preserved after resuspended standbyly, suspension normal temperature is preserved or 4 ℃ of preservations;
The oxidation of b glycoprotein
With the dissolving of a certain amount of protein sample or be diluted in and add sodium periodate in the coupling buffer to its ultimate density 10～15mM, reacting at normal temperature without light 0.5～1.5 hour;
C removes sodium periodate
Remove salt plug with G-25 and remove unreacted sodium periodate;
The coupling of d glycoprotein
Protein solution behind the desalination is joined in the pretreated hydrazides functionalized magnetic particles of coupling buffer, room temperature vibration coupling 4～24 hours, magnetic resolution after the coupling is removed supernatant; Wash the magnetic grain 3～6 times with scavenging solution, to remove not covalently bound albumen;
The e enzymolysis obtains glycopeptide
Glycoprotein on the magnetic grain is room temperature reductase 12 more than 0 minute in the solution of 7.8 the three propyloic phosphorus ammonium hydrogencarbonate that is dissolved in 0.1M with 5mM, pH; Adding iodo-acetamide to its ultimate density then is 8～15mM, and room temperature continued reaction more than 30 minutes, and magnetic resolution is removed supernatant; With pH is 7.8 the resuspended magnetic grain of ammonium bicarbonate solution, adds trypsinase to its ultimate density 20 mg/litre, and 37 ℃ of vibration enzymolysis 4 hours are added the trypsinase of same amount, continue enzymolysis more than 10 hours;
F cleans
The magnetic grain cleans respectively earlier with 2 mol sodium-chlor and dehydrated alcohol, is that 7.8 0.1 mol ammonium bicarbonate solution is washed with pH again, removes fully until non-glycosylated polypeptide;
G discharges glycopeptide
With pH is 7.8 the resuspended magnetic grain of 0.1 mol ammonium bicarbonate solution, adds the glycopeptidase F of 0.5 microlitre, and 37 ℃ of vibration enzymolysis 4 hours add the glycopeptidase F of same amount, continue reaction more than 4 hours, finish the release of glycopeptide.
3. according to the method for claim 1 separation and purification glycopeptide, it is characterized in that: also comprise before described (2) purification procedures
1) quantitatively goes out the content of suspension hydrazine functionalized magnetic particles
After getting the oven dry of 2ml suspension, weigh, calculate the hydrazine functionalized magnetic particles content in the suspension;
2) measure the content that diazanyl is rolled into a ball on the hydrazine functionalized magnetic particles
With 0.1 milliliter of massfraction be 80% hydrazine hydrate respectively water do the different multiples dilution, as standard hydrazine sample;
With 0.01 milliliter of each concentration standard hydrazine sample, 0.05 milligram of hydrazine magnetic grain, 0.05 milligram of epoxidation magnetic grain, the latter two volumes are 0.01 milliliter, with 0.2 milliliter of dicinchonine acid work reagent mix, and 37 ℃ of oscillatory reactions 0.5 hour, magnetic resolution;
Make blank with dicinchonine acid work reagent, ultraviolet-visible pectrophotometer is surveyed supernatant 562 nanometers light absorption values in each pipe; It is for referencial use to draw the hydrazine typical curve, calculates diazanyl mass contg on the magnetic grain with the difference of 562 nanometers light absorption values of supernatant behind hydrazine magnetic grain and epoxy magnetic grain and the dicinchonine acid reagent react.
4. according to the method for claim 2 separation and purification glycopeptide, it is characterized in that: also comprise before described (2) purification procedures
1) quantitatively goes out the content of suspension hydrazides functionalized magnetic particles
After getting the oven dry of 2ml suspension, weigh, calculate the hydrazides functionalized magnetic particles content in the suspension;
2) content of hydrazides group on the mensuration hydrazides functionalized magnetic particles
With adipic dihydrazide respectively water do different multiples dilution, as standard hydrazides sample;
With 0.01 milliliter of each concentration standard hydrazides sample and 0.05 milligram of hydrazides magnetic grain, 0.05 milligram of carboxylated magnetic grain, the latter two volumes are 0.01 milliliter, with 0.2 milliliter of dicinchonine acid work reagent mix, and 37 ℃ of oscillatory reactions 0.5 hour, magnetic resolution;
Make blank with dicinchonine acid work reagent, ultraviolet-visible pectrophotometer is surveyed supernatant 562 nanometers light absorption values in each pipe; It is for referencial use to draw the hydrazides typical curve, calculates hydrazide group mass contg on the magnetic grain with the difference of 562 nanometers light absorption values of supernatant behind hydrazides magnetic grain and carboxylated magnetic grain and the dicinchonine acid work reagent react.
5. according to the method for the arbitrary described separation and purification glycopeptide of claim 1～4, it is characterized in that: described coupling buffer is meant that pH is 5.5 100 mmoles/rise sodium-acetate and 150 mmoles/the rise solution of sodium-chlor.
6. according to the method for claim 5 separation and purification glycopeptide, it is characterized in that: described scavenging solution is meant that pH is 8.3 the 8M urea and the solution of 0.4M ammonium hydrogencarbonate.
7. according to the method for claim 6 separation and purification glycopeptide, it is characterized in that: described water is ultrapure water or distilled water.
8. according to the method for claim 7 separation and purification glycopeptide, it is characterized in that: described magnetic resolution is all separated on magnetic separator.
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| CN1958600A (en) * | 2005-11-03 | 2007-05-09 | 陕西西大北美基因股份有限公司 | Method for enriching and purifying saccharide binding protein |
| JP2008111703A (en) * | 2006-10-30 | 2008-05-15 | Fujifilm Corp | Water dispersible magnetic particle |
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| CN1958600A (en) * | 2005-11-03 | 2007-05-09 | 陕西西大北美基因股份有限公司 | Method for enriching and purifying saccharide binding protein |
| JP2008111703A (en) * | 2006-10-30 | 2008-05-15 | Fujifilm Corp | Water dispersible magnetic particle |
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| Cellular Proteomics》.2006,第1957-1967页. * |
| Richard R. Drake et.al.Lectin Capture Strategies Combined with Mass.《Molecular & Cellular Proteomics》.2006,第1957-1967页. |
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