CN101846649A - Phosphorylated and/or glycosylated protein or peptide one-step enrichment modification determination method - Google Patents
Phosphorylated and/or glycosylated protein or peptide one-step enrichment modification determination method Download PDFInfo
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Abstract
The invention discloses a phosphorylated and/or glycosylated protein or peptide one-step enrichment modification determination method. The method includes the following steps: 1) modification on end alkynyl or triazon of protein or peptide; 2) resin capturing of protein or peptide with modified end alkynyl; 3) cutting of the peptide/protein with modified alkynyl on resin. The protein or peptide enrichment modification determination method provided by the invention not only can enrich and purify the modified protein or peptide after translation but also can add one molecule segment on the protein or peptide, and the molecule segment has the functions of mass spectrum label and mass spectrum signal gain. The method has wide application prospect in the science analysis field.
Description
Technical field
The method for separating and concentrating and the site that the present invention relates to phosphorylation and/or glycosylated protein and polypeptide are measured.
Background technology
Phosphorylation is modal protein covalent modification, all has this modification in 1/3rd eukaryotic gene expression product.It is regulating basic cell behavior: cell division, growth, differentiation.Owing to can't predict phosphorylation and other posttranslational modifications with gene order, researchers have just been developed some other method.
The method of traditional mensuration phosphorylation site generally comprises: in bacterial classification incubation growth base to the radioactive labeling of phosphorylation site, then with immuno-precipitation or SDS-PAGE method, through enzymic digestion, carry out Edman degraded separation and contain the isotope-labeled albumen of 32P.This method, except the potential hazard to operator's health, the low abundance of phosphopeptide has also reduced the invalid combination of susceptibility and radioactive labels, and the muting sensitivity of edman order-checking has limited the application of large number of biological sample automatically.
Mass spectrum is used to identification of protein always, is considered to characterize the best technique of posttranslational modification protein now.This method fast, sensitive and do not need radioactive labels.Two kinds of soft ionization technology---machine-processed auxiliary laser desorption ionization mass spectrum and electrospray ionization mass spectrum is by the detection that is applied to the protein phosphorylation site of success.But owing to following reason, still there is restriction in the widespread use of carrying out conventional phosphorylating protein sign with mass spectrum.
The first, because phosphate group self has negative charge and suppressed Ionization Efficiency, under mass spectral positive ion mode, the intensity of characteristic peak is very low, is difficult to be detected.
The second, during protein mixed, with respect to the non-phosphorylating polypeptide, phosphorylation chemiluminescent polypeptide content was very low, made that the phosphorylation polypeptide all characterizes the difficulty that becomes in the potpourri.
Three, perhaps the water-wet behavior of phosphoeptide can cause being washed off in RP-HPLC.
Four, be difficult to special big phosphorylated protein hydrolysis fragment is comprehensively checked order.
Therefore, design a kind of method, catch label by on the posttranslational modification site of protein or polypeptide, adding, and carry out enrichment by efficient chemical reaction, and in single-minded cutting process, produce mass spectrum quality tab and mass signal gain label, can reach rapidly and efficiently enrichment modification protein or polypeptide to improve the purpose of posttranslational modification site determination efficiency.
Summary of the invention
An one-step enrichment modification determination method that the purpose of this invention is to provide a kind of phosphorylation and/or glycosylated protein or polypeptide.
The enrichment modification determination method of phosphorylation provided by the invention and/or glycosylated protein or polypeptide comprises the steps:
1) the Terminal Acetylenes base of protein or polypeptide or azido are modified:
The ethanolic solution of target substrates with saturated baryta water, end alkynyl label compound mixed, obtain terminal alkynyl modified target substrates behind the incubation;
Wherein, target substrates is phosphorylation and glycosylated protein or polypeptide, specifically can be phosphorylation serine, oxygen connection glycosylation serine, phosphorylation threonine or oxygen and connects the glycosylation threonine residues;
2) the terminal alkynyl modified protein or the resin of polypeptide are caught:
In above-mentioned terminal alkynyl modified target substrates, add the polystyrene resin that azido is modified, and the potpourri or the cuprous iodide of adding ascorbic acid and copper sulphate, terminal alkynyl modified target substrates is captured on the polystyrene resin of this azido modification with the click chemistry reaction;
3) cutting of Terminal Acetylenes modified polypeptide/albumen on the resin:
The terminal alkynyl modified target substrates molecule that connects on the polystyrene resin with the azido modification cuts down, automatically form a mass signal gain label in terminal alkynyl modified target substrates molecular end simultaneously, utilize mass spectrum to finish the mensuration of phosphorylation and/or glycosylated protein or polypeptide afterwards.
In the step 1) of this method, the general structure of end alkynyl label compound is suc as formula shown in the I,
In the formula I general structure, R
0Be R-X-, tablet held before the breast by officials base protection aminoterminal halfcystine ester group or tablet held before the breast by officials base protection aminoterminal lysine ester group;
Wherein, among the R-X-, R be SH-or-NH
2, X is for the backbone c atoms number is 1～6, total carbon atom number is 1～10 alkyl chain or contain ester group or the backbone c atoms number of amide group is 2～8, total carbon atom number is 2～12 alkyl chain.
When X is when containing the label compound of alkyl chain of ester group or amide group, prepare the method for above-mentioned end alkynyl label compound, comprise the steps:
1) mercaptan carboxylic acid or halfcystine are carried out sulfhydryl protected reaction;
2) product that described step 1) is obtained carries out esterification or amidation process;
3) with described step 2) product that obtains carries out the deprotection reaction of sulfydryl, obtains described end alkynyl label compound.
In this method, in the described step 1), sulfhydryl protected reaction is to carry out according to any one method among the following a-d:
A, with mercaptan carboxylic acid or halfcystine and S, S-diphenyl-S-methoxyl sulphur nitrile reacts in organic solvent;
The mol ratio of described mercaptan carboxylic acid or halfcystine and p-methyl benzene sulfonic chloride is 1: 2; Reaction time is 30 ℃, and the reaction time is 4 hours;
B, mercaptan carboxylic acid or halfcystine are dissolved in the mixed liquor of ethanol and ammoniacal liquor, add benzyl chlorine and react;
The mol ratio of described mercaptan carboxylic acid or halfcystine and benzyl chlorine is 1: 1-2, preferred 1: 1.2; Reaction time is 30 minutes, and temperature of reaction is 25 ℃; In the mixed liquor of described ethanol and ammoniacal liquor, the volume ratio of ethanol and ammoniacal liquor is 3: 1-3;
C, mercaptan carboxylic acid or halfcystine are dissolved in the organic solvent, add benzyl chlorine and cesium carbonate and react;
The mol ratio of described mercaptan carboxylic acid or halfcystine and benzyl chlorine, cesium carbonate is 1 (1): preferred 1: 1.2: 0.5 of 1-2 (1.2): 0.2-1); Reaction time is 2 hours, and temperature of reaction is 20 ℃; Described organic solvent is N, dinethylformamide;
D, under alkali condition, mercaptan carboxylic acid or halfcystine and p-methyl benzene sulfonic chloride are reacted in organic solvent;
The pH value of described alkali condition is 9-12; The mol ratio of described mercaptan carboxylic acid or halfcystine and p-methyl benzene sulfonic chloride is 1: 1-2, preferred 1: 1.2; Reaction time is room temperature, and the reaction time is 1-6 hour;
Described step 2) in, esterification is carried out as follows: product that described step 1) is obtained and alkynyl alcohol, N, and N '-dicyclohexyl diimine reacts in organic solvent;
Product that described step 1) obtains and alkynyl alcohol, N, the mol ratio of N '-dicyclohexyl diimine is 1: 1-1.5: 1-2; Described temperature of reaction is 20-80 ℃, and the described reaction time is 8 hours;
Described step 2) in, amidation process carries out as follows: with the product of described step 1) and alkynylamine, benzotriazole-N, N, N ', N '-tetramethylurea hexafluorophosphate, 1-hydroxy benzo triazole and N, the N-diisopropylethylamine reacts;
Product that described step 1) obtains and alkynylamine, benzotriazole-N, N, N ', N '-tetramethylurea hexafluorophosphate, 1-hydroxy benzo triazole and N, the mol ratio of N-diisopropylethylamine is 1: 1.5-4: 0.9-1: 1-2: 1-3, preferred 1: 2: 0.9: 1: 2; Described temperature of reaction is 20-80 ℃, and the reaction time is 1.5-6 hour.
In the described step 3), the deprotection reaction of sulfydryl is to carry out according in the e-h method any one:
E, with described step 2) product be dissolved in ethanol, feed ammonia and also add sodium silk, back flow reaction;
F, with described step 2) product be dissolved in methyl phenyl ethers anisole, feed hydrogen fluoride and react;
G, under inert atmosphere protection, with described step 2) product with acetic acid ethyl dissolution after, add trifluoroacetic acid and react;
H, with step 2) product that obtains dissolves with strong aqua; The mass percent concentration of strong aqua is 25-28%.
Described organic solvent is benzene, N, dinethylformamide or acetonitrile.
After above-mentioned steps, also can carry out purifying to product; Concrete steps can be: after reaction finishes reaction dissolvent is removed, carried out purifying with silica gel column chromatography, moving phase is chosen the eluant, eluent that contains carboxylic acid, and carboxylic acid can be formic acid or acetate etc.
As X during, step 2 according to the method described above)-3) be prepared, only with step 2 for tablet held before the breast by officials base protection aminoterminal halfcystine ester group or tablet held before the breast by officials base protection aminoterminal lysine ester group) initial reactant replace with fluorenes methoxy carbonyl acyl group cysteine sulfydryl trityl.
When X is the label compound of alkyl chain, prepare the method for above-mentioned end alkynyl label compound, comprise the steps:
1) alkynyl alcohol is carried out the hydroxyl protection reaction;
2) prepare thiacetic cesium salt;
3) product that described step 1) is obtained carries out substitution reaction;
4) product that obtains of described step 3) carries out protecting group and removes reaction, obtains described end alkynyl label compound.
In this method, described step 1) is carried out as follows: add the pure and mild pyridine of alkynyl and react in the mixed liquor of Methyl benzenesulfonyl chlorine and chloroform;
Described alkynyl alcohol is 3-butine-1-alcohol; Described temperature of reaction is 0-10 ℃, and the reaction time is 2-10 hour;
The mol ratio of alkynyl alcohol, Methyl benzenesulfonyl chlorine, chloroform and pyridine is 1: 1.2-2: 8-20: 8-20;
Described step 2) carries out as follows: thioacetic acid, methyl alcohol and cesium carbonate are mixed react, obtain thiacetic cesium salt;
The mol ratio of thioacetic acid, methyl alcohol and cesium carbonate is 1: 0.4-4: 0.2-1; Temperature of reaction is 40-85 ℃, and the reaction time is 0.5-6 hour;
Described step 3) is carried out as follows: the product and the described thiacetic cesium salt of described step 1) are reacted;
The product of described step 1) and the mol ratio of thiacetic cesium salt are 1: 1.2-4.
Described step 4) is carried out as follows: after product that described step 3) is obtained and the ammoniacal liquor reaction, acidifying obtains described end alkynyl label compound.
Above-mentioned end alkynyl label compound specifically can be as shown in the formula the compound shown in the II-formula VII structural formula:
In addition, the step 2 of the enrichment modification determination method of phosphorylation provided by the invention and/or glycosylated protein or polypeptide) in, the general structure of the polystyrene resin that used azido is modified is suc as formula shown in VIII or the formula IX.
Wherein, the polystyrene resin that azido is modified is prepared as follows: at 1-hydroxy benzo triazole, benzotriazole-N, N, N ', under the condition that N '-tetramethylurea hexafluorophosphate and 4-dimethylamino naphthyridine exist, with the terminal hydroxy group resin or hold amino photosensitive resin and carboxylic acid that azido is modified reacts, obtain the polystyrene resin that described azido is modified.
In this method, the terminal hydroxy group resin is Lin Ke-acid resin or 4-(4-methylol-3-benzyloxy) bytyry polystyrene resin;
The amino photosensitive resin of described end is Alpha-Methyl-6-nitro-veratrylamine polystyrene resin or Alpha-Methyl-6-nitro-veratryl alcohol polystyrene resin;
The carboxylic acid that described azido is modified is nitrine propionic acid or nitrine butyric acid;
The particle diameter of described terminal hydroxy group resin and the amino photosensitive resin of end is the 50-100 order;
Described terminal hydroxy group resin or hold amino photosensitive resin and the mol ratio of the carboxylic acid that azido is modified is 1: 2-1: 6; Described temperature of reaction is 20-60 ℃, and the reaction time is 0.5-8 hour; Described terminal hydroxy group resin and the amino photosensitive resin of end all are to use the ethylene dichloride swelling;
The medium that the carboxylic acid that described terminal hydroxy group resin and the amino photosensitive resin of end and azido are modified reacts is N, dinethylformamide.
In the step 3) of said method provided by the invention, cutting step specifically comprises the steps:
1) incubation 15 minutes-4 hours in 6-fluorine isopropyl alcohol, preferred 30 minutes; Temperature is 20～60 ℃, preferred 30 ℃;
2) volume ratio at acetonitrile and acetate is 1: incubation is 15 minutes-4 hours in the mixed liquor of 0.05-0.3, preferred 30 minutes; Temperature is 20～60 ℃, preferred 30 ℃; Preferred 1: 0.1 of the volume ratio of acetonitrile and acetate.
The invention provides a kind ofly based on mass spectrum, utilize β-elimination, the sulfur-bearing probe is modified phosphorylation site in conjunction with " click chemistry " again to the Micheal addition reaction of polypeptide, improve its in mass spectrum rate of ionization and measure the sequence of polypeptide.The present invention utilizes the sulfur-bearing probe in conjunction with the phosphorylation polypeptide phosphopeptide to be screened, then by solid-phase synthesis in conjunction with high selectivity, click chemistry obtains purer phosphopeptide efficiently, carries out the methodological study of MS/MS sequencing analysis then.This method has been used the click chemistry reaction very cleverly, easily synthetic, and click chemistry reacts resulting triazole ring and has very high mass spectrum inspection sensitivity, has replaced instability and difficult by the detected phosphate group of mass spectrum by it, has improved the susceptibility of analyzing.And in the first step that MS detects, the S-C key ratio that the sulphur probe links to each other with polypeptide is easier to the part fracture, also can well detect the mass spectra peak of phosphorylation polypeptide, is convenient to carry out next step analysis.
The enrichment modification determination method of protein provided by the invention or polypeptide, can not only carry out enriching and purifying to the protein or the polypeptide of posttranslational modification, and can on protein or polypeptide, increase a molecule fragment, this molecule fragment has the effect of mass spectrum label and mass signal gain.This method has broad application prospects at the life analysis field.
Embodiment
Method therefor is conventional method if no special instructions among the following embodiment.
First, synthetic end alkynyl label compound
End alkynyl label compound shown in embodiment 1, the synthesis type II structural formula
Add Fmoc-Cys (Trt)-OH (fluorenes methoxy carbonyl acyl group cysteine sulfydryl trityl) 585mg in flask, 3-butine-1-alcohol 151 μ L adds dry acetonitrile again, adds 50mg DMAP (4-dimethylamino naphthyridine).System is clarified the back fully and is added an amount of 2.2g DCC (1, the 3-dicyclohexylcarbodiimide), is warming up to room temperature naturally.React after 8 hours, remove by filter insolubles, silicagel column separates, and will filter gained solution and revolve steaming with oil pump, and the products therefrom oil pump is drained, and weighs.
Separate with silicagel column.
The product that obtains adds TFA (trifluoroacetic acid) with after the acetic acid ethyl dissolution, under the argon shield, and concussion 2h.Then dry up this thing, obtain final product with nitrogen.
End alkynyl label compound shown in embodiment 2, the synthesis type IV structural formula
Add 210 μ L mercaptoacetic acid in the solution of 10mL2N NaOH, slowly drip the 2.15mL p-methyl benzene sulfonic chloride while stirring in ice-water bath, drip the pH value of later stage mensuration system, hydro-oxidation sodium control pH value is 9～10.Behind the stirring reaction 1 hour, wash excessive p-methyl benzene sulfonic chloride off with ether, repeat twice of ether washing step after, with the dilution of 15mL ethyl acetate, be 1 with 6M hcl acidifying to pH value, isolate organic phase, the water ethyl acetate extraction.Merge organic phase, to neutral, anhydrous sodium sulfate drying revolves steaming and obtains thick liquid with the washing of 5% (wt) sodium chloride solution.
In the flask of previous step, add the dry acetonitrile of 10mL, add 50mg DMAP (4-dimethylamino naphthyridine).When system becomes clarification fully by the time, add 2.2g DCC, remove cooling, allow temperature be elevated to room temperature naturally.After reacting 8 hours, remove by filter insolubles.Filtrate is dissolved in the 1mL mixed solvent through the pressure reducing and steaming solvent again, this mixed solvent be petroleum ether-ethyl acetate (9: 1, V: V).If be necessary, can filter once more and remove residual urea. crude product in solution with petroleum ether-ethyl acetate (8: 2, V: V) for eluent through the silica gel THE SHORT COLUMN CHROMATOGRAPHY, must thick target product.
The product of previous step is dissolved with strong aqua, under the room temperature, after 1 hour deprotection reaction with petroleum ether-ethyl acetate (8: 2, V: V) for eluent through the silica gel THE SHORT COLUMN CHROMATOGRAPHY, must thick target product.
End alkynyl label compound SS-01 shown in embodiment 3, the synthesis type II structural formula
1, the Methyl benzenesulfonyl base of 3-butine-1-alcohol protection
The Methyl benzenesulfonyl chlorine and the 10mL chloroform that add 4.55g in there-necked flask stir and are cooled to 3 ℃ with ice-water bath; The pure and mild 2.06g pyridine of 0.701g 3-butine-1-that mixes is splashed in the flask, make temperature of reaction at 6-8 ℃, dropwise, keep 8-10 ℃ and continue stirring reaction 5h by ice-water bath and control rate of addition; With agitator treating in the reaction mixture impouring 40mL frozen water, use cold water washing twice again, with the abundant agitator treating twice of the aqueous sodium carbonate (temperature is below 3 ℃) of pH value 8-9, be washed with distilled water to the pH value stabilization at last at 6.57-6.70; Leave standstill branch water purification layer, organic layer with anhydrous sodium sulfate drying after, decompression distillation behind the first normal pressure steams all solvents, the colorless oil liquid product, use the absolute ethyl alcohol recrystallization, must the white plates crystallization.
2, prepare thiacetic cesium salt
Add 5mL methyl alcohol in the 379 μ L thioacetic acids, and add the 1.63g cesium carbonate, be stirred to cesium carbonate and dissolve fully, revolve and steam to doing.
3, the substitution reaction of the 3-butine-1-alcohol of Methyl benzenesulfonyl base protection
The product that the first step is obtained dissolves with the least possible DMF (N, dinethylformamide), adds in the product flask of previous step.Be heated to about 70 ℃ back flow reaction 5 hours.Add after an amount of sherwood oil, water is progressively with the DMF flush away.At last, sherwood oil being revolved steaming removes.Obtain product.
4, protecting group removes reaction
Get the product in the 3rd step, add the ammoniacal liquor of 30mL 1N, stirred 5 hours, after this use the hydrochloric acid of 4mol/L to carry out acidifying.Product extracts with ether, and after the drying, decompression distillation obtains product.Product is preserved under nitrogen environment.
End alkynyl label compound SS-11 shown in embodiment 4, the synthesis type XII structural formula
Add mercaptoacetic acid 210 μ L among the solution 10mL of 2N NaOH, in ice-water bath, under the stirring, slowly drip the p-methyl benzene sulfonic chloride of 2.15mL.Drip later stage measurement pH value, hydro-oxidation sodium makes the pH value be controlled at 9～10, after stirring reaction carried out 1 hour, at first use ether to wash excessive p-methyl benzene sulfonic chloride off, twice, with ethyl acetate 15mL dilution, be 1 to the pH value again with the 6M hcl acidifying, isolate organic phase, the water ethyl acetate extraction.Merge organic mutual-assistance and wash neutrality, use anhydrous sodium sulfate drying again, revolve steaming and obtain thick liquid with 5% sodium chloride.
In the previous step product, add 0.28g 3-butine-1-amine, DIEA (N, the N-diisopropylethylamine) 0.26g, HOBT (1-hydroxy benzo triazole) 0.14g, HBTU (benzotriazole-N, N, N ', N '-tetramethylurea hexafluorophosphate can commerce buys) 0.34g is dissolved among the 20mL DMF (N, dinethylformamide), under 60 degree, reflux and stirred 4 hours, reaction finishes and removes reaction dissolvent, carries out purifying with silica gel column chromatography, and moving phase is chosen ethyl acetate: sherwood oil: the mixed liquor of acetate=20: 80: 1 ratio.
The previous step product is dissolved with strong aqua, carries out 1 hour deprotection reaction in room temperature, with petroleum ether-ethyl acetate (8: 2, V: V) for eluent through the silica gel THE SHORT COLUMN CHROMATOGRAPHY, obtain thick target product.
The polystyrene resin that second portion, synthetic azido are modified
The polystyrene resin CR-01 that embodiment 5, synthetic azido are modified
1) get 3-bromo-propionic acid 70mg, sodium carbonate 106mg is put in the there-necked flask, behind injection DMF (N, the dinethylformamide) 10mL, adds NaN
3(sodium azide) 330mg refluxed 6 hours down in 60 degree, white opacity occurred.
2) after the filtration,, continue outstanding steaming with oil pump afterwards with remaining liq outstanding steaming in flask.
3) the outstanding steaming do not have liquid in the bottle, adds that 15mL is water-soluble to be separated, and transfers in the separating funnel, adds 15mL ethyl acetate, and extraction and aqueous phase discarded add entry extraction three times repeatedly, obtain 3-azido propionic acid.
4) with 32mg HOBT (1-hydroxy benzo triazole), 82mg HBTU (benzotriazole-N, N, N ', N '-tetramethylurea hexafluorophosphate), 49mg DMAP (4-dimethylamino naphthyridine) 3-azido propionic acid 28mg is dissolved in 5mL DMF (N, dinethylformamide) in, 100mg Lin Ke-acid resin (RinkAcid Resin) (the 50-100 order that adds the methylene chloride swelling, load 0.6mmol/g), concussion was alternately washed the resin of gained after 2 hours under the room temperature with DCM (methylene chloride) and DMF, obtain the polystyrene resin CR-01 that azido provided by the invention is modified, the structural formula of this resin is suc as formula shown in the VIII.
Wherein, Lin Ke-acid resin (Rink Acid Resin) available from Nankai with become company, production code member is HC RA01-1.
One one-step enrichment modification determination of third part, phosphorylation and glycosylated protein or polypeptide
Modification enrichment and the mass spectrophotometry of embodiment 6, phosphorylation polypeptide Kemptide
1) the phosphorylation polypeptide is terminal alkynyl modified
Phosphorylation polypeptide Kemptide (sequence is LRRApSLG) is mixed with the aqueous solution of 1mg/mL.Get the aqueous solution of this phosphorylation polypeptide of 10 microlitres, add the saturated baryta water of 100 microlitres, and the mass percent concentration of the 100 end alkynyl label compound SS-07 that prepare of microlitre the foregoing description 7 is 10% ethanolic solution, 45 ℃ of following incubations 4 hours, obtain terminal alkynyl modified phosphorylation polypeptide solution.
2) resin of terminal alkynyl modified phosphorylation polypeptide is caught
Utilize 1mL DMF (N, dinethylformamide), the saturated NaCl solution of 1mL, 1.3mgCuCl
2, 17.6mg VC (vitamin C) is mixed with resin capture reaction liquid.
Take by weighing the Rink Acid-N that 3mg embodiment 5 prepares
3After resin (the Rink-acid resin of end modified azido group) DCM (methylene chloride) swelling, water pump is taken out half-dried, in above-mentioned resin, add 1mL resin capture reaction liquid, the phosphorylation polypeptide solution that 20 microlitres are terminal alkynyl modified, concussion is spent the night, and has obtained catching the resin of the phosphorylation polypeptide of modifying.
3) cutting of terminal alkynyl modified polypeptide on the resin
Utilize 200 microlitre acetate, 1800 microlitre acetonitriles are mixed with resin cleavage liquid.
Clean step 2) resin of having caught the phosphorylation polypeptide of modifying that obtains, DMF (N, dinethylformamide) washing is three times, DCM washs once, and oil pump is drained, and cuts with resin cleavage liquid, the cutting rear pump or output pump is drained, carry out Mass Spectrometer Method behind the dissolve with methanol, observe under positive ion mode, what can observe phosphorylation Kemptide adds sodium peak 765.5 (m/z+23 Na
+) and modify back Kemptide add sodium peak 1062.8 (m/z+23Na
+) strong mass signal.
The modification enrichment and the mass spectrophotometry of embodiment 7, O-GlcNAc (oxygen connects the nitrogen acetylglucosamine) modified polypeptide
1) Terminal Acetylenes of posttranslational modification polypeptide is modified
O-GlcNAc modified polypeptide Kemptide (sequence is LRRAgSLG) is mixed with the aqueous solution of 1mg/mL.Get the aqueous solution of 10 microlitre polypeptide, add the saturated baryta water of 100 microlitres, and the mass percent concentration of the 100 end alkynyl label compound SS-07 that prepare of microlitre the foregoing description 1 is 10% ethanolic solution, 45 ℃ of following incubations 4 hours, and the polypeptide solution after obtaining modifying.
2) resin of terminal alkynyl modified polypeptide is caught
Utilize 1mL DMF (N, dinethylformamide), the saturated NaCl solution of 1mL, 1.3mgCuCl
2, 17.6mg VC is mixed with resin capture reaction liquid.
The Rink Acid-N that 3mg embodiment 5 prepares
3After resin (the Rink-acid resin of end modified azido group) the DCM swelling, water pump is taken out half-dried, adds 1mL resin capture reaction liquid in above-mentioned resin, the phosphorylation polypeptide solution that 20 microlitres are terminal alkynyl modified, concussion is spent the night, and has obtained catching the resin of the glycosylated polypeptides of modifying.
3) cutting of terminal alkynyl modified polypeptide on the resin
Utilize 200 microlitre acetate, 1800 microlitre acetonitriles are mixed with resin cleavage liquid.
Clean step 2) resin of having caught the glycosylated polypeptides of modifying that obtains, DMF washing three times, the DCM washing once, oil pump is drained, cut with resin cleavage liquid, the cutting rear pump or output pump is drained, and carries out Mass Spectrometer Method behind the dissolve with methanol, observe under positive ion mode, what can observe phosphorylation Kemptide adds sodium peak 765.5 (m/z+23 Na
+) and modify back Kemptide add sodium peak 1014.5 (m/z+23Na
+) strong mass signal.
Claims (17)
1. the enrichment modification determination method of phosphorylation and/or glycosylated protein or polypeptide comprises the steps:
1) ethanolic solution of target substrates with saturated baryta water, end alkynyl label compound mixed, obtain terminal alkynyl modified target substrates behind the incubation;
Wherein, described target substrates is phosphorylating protein or polypeptide, or glycosylated protein or polypeptide;
2) in described terminal alkynyl modified target substrates, add the polystyrene resin that azido is modified, and add in cuprous iodide, ascorbic acid or the copper sulphate any one, with the click chemistry reaction described terminal alkynyl modified target substrates is captured on the polystyrene resin of described azido modification;
3) the described terminal alkynyl modified target substrates molecule that connects on the polystyrene resin that described azido is modified cuts down, automatically form a mass signal gain label in described terminal alkynyl modified target substrates molecular end simultaneously, utilize mass spectrum to finish the mensuration of described phosphorylation and/or glycosylated protein or polypeptide afterwards.
2. method according to claim 1 is characterized in that: in the described step 1), target substrates is that phosphorylation serine, oxygen connect glycosylation serine, phosphorylation threonine or oxygen connection glycosylation threonine residues; Heated culture temperature is 25-50 ℃, preferred 45 ℃; The incubation time is 2-24 hour, preferred 4 hours;
Described step 2) in, the general structure of the polystyrene resin that described azido is modified is suc as formula shown in VIII or the formula IX;
3. method according to claim 2 is characterized in that: described step 2), the polystyrene resin that described azido is modified is prepared as follows:
At 1-hydroxy benzo triazole, benzotriazole-N, N, N ' is under the condition that N '-tetramethylurea hexafluorophosphate and 4-dimethylamino naphthyridine exist, with the terminal hydroxy group resin or hold amino photosensitive resin and carboxylic acid that azido is modified reacts, obtain the polystyrene resin that described azido is modified.
4. method according to claim 3 is characterized in that: described terminal hydroxy group resin is Lin Ke-acid resin or 4-(4-methylol-3-benzyloxy) bytyry polystyrene resin;
The amino photosensitive resin of described end is Alpha-Methyl-6-nitro-veratrylamine polystyrene resin or Alpha-Methyl-6-nitro-veratryl alcohol polystyrene resin;
The carboxylic acid that described azido is modified is nitrine propionic acid or nitrine butyric acid;
The particle diameter of described terminal hydroxy group resin and the amino photosensitive resin of end is the 50-100 order;
Described terminal hydroxy group resin or hold amino photosensitive resin and the mol ratio of the carboxylic acid that azido is modified is 1: 2-1: 6;
Described temperature of reaction is 20-60 ℃, and the reaction time is 0.5-8 hour; Described terminal hydroxy group resin and the amino photosensitive resin of end all are to use the ethylene dichloride swelling;
The medium that the carboxylic acid that described terminal hydroxy group resin and the amino photosensitive resin of end and azido are modified reacts is N, dinethylformamide.
5. according to the arbitrary described method of claim 1-4, it is characterized in that: in the described step 3), cutting step comprises following concrete steps:
1) incubation 15 minutes-4 hours in 6-fluorine isopropyl alcohol, preferred 30 minutes; Temperature is 20～60 ℃, preferred 30 ℃;
2) volume ratio at acetonitrile and acetate is 1: incubation is 15 minutes-4 hours in the mixed liquor of 0.05-0.3, preferred 30 minutes; Temperature is 20～60 ℃, preferred 30 ℃; Preferred 1: 0.1 of the volume ratio of acetonitrile and acetate.
6. according to the arbitrary described method of claim 1-5, it is characterized in that: in the described step 1), the general structure of end alkynyl label compound is suc as formula shown in the I,
In the described formula I general structure, R
0Be R-X-or tablet held before the breast by officials base protection aminoterminal halfcystine ester group or tablet held before the breast by officials base protection aminoterminal lysine ester group;
Wherein, among the described R-X-, R be SH-or-NH
2, X is for the backbone c atoms number that the backbone c atoms number is 1～6, total carbon atom number is 1～10 alkyl chain, contain ester group or amide group is 2～8, total carbon atom number is 2～12 alkyl chain.
7. method according to claim 6 is characterized in that: in the described formula I general structure, and R
0Be R-X-; Among the described R-X-, R be SH-or-NH
2, X is that the backbone c atoms number that contains ester group or amide group is 2～8, total carbon atom number is 2～12 alkyl chain.
8. according to claim 6 or 7 described methods, it is characterized in that: in the described formula I general structure, R
0Be R-X-; Among the described R-X-, R be SH-or-NH
2, X is for the backbone c atoms number is 1～6, always the carbon atom number is 1～10 alkyl chain.
9. method according to claim 6 is characterized in that: in the described formula I general structure, and R
0Be tablet held before the breast by officials base protection aminoterminal halfcystine ester group or tablet held before the breast by officials base protection aminoterminal lysine ester group.
10. according to the arbitrary described method of claim 6-9, it is characterized in that: described end alkynyl label compound is the compound shown in the formula II-formula VII structural formula:
11. according to the arbitrary described method of claim 6-9, it is characterized in that: the method for the described end alkynyl label compound of described claim 7 is prepared as follows:
1) mercaptan carboxylic acid or halfcystine are carried out sulfhydryl protected reaction;
2) product that described step 1) is obtained carries out esterification or amidation process;
3) with described step 2) product that obtains carries out the deprotection reaction of sulfydryl, obtains described end alkynyl label compound.
12. method according to claim 11 is characterized in that: in the described step 1), sulfhydryl protected reaction is to carry out according to any one method among the following a-d:
A, with mercaptan carboxylic acid or halfcystine and S, S-diphenyl-S-methoxyl sulphur nitrile reacts in organic solvent;
Wherein, the mol ratio of described mercaptan carboxylic acid or halfcystine and p-methyl benzene sulfonic chloride is 1: 2; Reaction time is 30 ℃, and the reaction time is 4 hours;
B, mercaptan carboxylic acid or halfcystine are dissolved in the mixed liquor of ethanol and ammoniacal liquor, add benzyl chlorine and react;
Wherein, the mol ratio of described mercaptan carboxylic acid or halfcystine and benzyl chlorine is 1: 1-2, preferred 1: 1.2; Reaction time is 30 minutes, and temperature of reaction is 25 ℃; In the mixed liquor of described ethanol and ammoniacal liquor, the volume ratio of ethanol and ammoniacal liquor is 3: 1-3;
C, mercaptan carboxylic acid or halfcystine are dissolved in the organic solvent, add benzyl chlorine and cesium carbonate and react;
Wherein, the mol ratio of described mercaptan carboxylic acid or halfcystine and benzyl chlorine, cesium carbonate is 1: 1-2: 0.2-1, preferred 1: 1.2: 0.5; Reaction time is 2 hours, and temperature of reaction is 20 ℃; Described organic solvent is N, dinethylformamide;
D, under alkali condition, mercaptan carboxylic acid or halfcystine and p-methyl benzene sulfonic chloride are reacted in organic solvent;
Wherein, the pH value of described alkali condition is 9-12; The mol ratio of described mercaptan carboxylic acid or halfcystine and p-methyl benzene sulfonic chloride is 1: 1-2, preferred 1: 1.2; Reaction time is room temperature, and the reaction time is 1-6 hour;
Described step 2) in, esterification is carried out as follows: product that described step 1) is obtained and alkynyl alcohol, N, and N '-dicyclohexyl diimine reacts in organic solvent;
Wherein, product that described step 1) obtains and alkynyl alcohol, N, the mol ratio of N '-dicyclohexyl diimine is 1: 1-1.5: 1-2; Described temperature of reaction is 20-80 ℃, and the described reaction time is 8 hours;
Described step 2) in, amidation process carries out as follows: with the product of described step 1) and alkynylamine, benzotriazole-N, N, N ', N '-tetramethylurea hexafluorophosphate, 1-hydroxy benzo triazole and N, the N-diisopropylethylamine reacts;
Wherein, in the amidation process, product that described step 1) obtains and alkynylamine, benzotriazole-N, N, N ', N '-tetramethylurea hexafluorophosphate, 1-hydroxy benzo triazole and N, the mol ratio of N-diisopropylethylamine is 1: 1.5-4: 0.9-1: 1-2: 1-3, preferred 1: 2: 0.9: 1: 2; Described temperature of reaction is 20-80 ℃, and the reaction time is 1.5-6 hour.
In the described step 3), the deprotection reaction of sulfydryl is to carry out according in the e-h method any one:
E, with described step 2) product be dissolved in ethanol, feed ammonia and also add sodium silk, back flow reaction;
F, with described step 2) product be dissolved in methyl phenyl ethers anisole, feed hydrogen fluoride and react;
G, under inert atmosphere protection, with described step 2) product with acetic acid ethyl dissolution after, add trifluoroacetic acid and react;
H, with step 2) the products therefrom mass percent concentration is the ammonia solvent of 25-28%;
Described organic solvent is benzene, N, dinethylformamide or acetonitrile.
13. according to the arbitrary described method of claim 6-9, it is characterized in that: the method for the described end alkynyl label compound of described claim 8 is prepared as follows:
1) alkynyl alcohol is carried out the hydroxyl protection reaction;
2) prepare thiacetic cesium salt;
3) product that described step 1) is obtained carries out substitution reaction;
4) product that obtains of described step 3) carries out protecting group and removes reaction, obtains described end alkynyl label compound.
14. method according to claim 13 is characterized in that: described step 1) is carried out as follows: add the pure and mild pyridine of alkynyl and react in the mixed liquor of Methyl benzenesulfonyl chlorine and chloroform;
Described step 2) carries out as follows: thioacetic acid, methyl alcohol and cesium carbonate are mixed react, obtain thiacetic cesium salt;
Described step 3) is carried out as follows: the product and the described thiacetic cesium salt of described step 1) are reacted;
Described step 4) is carried out as follows: after product that described step 3) is obtained and the ammoniacal liquor reaction, acidifying obtains described end alkynyl label compound.
15. method according to claim 14 is characterized in that: in the described step 1), described alkynyl alcohol is 3-butine-1-alcohol; Described temperature of reaction is 0-10 ℃, and the reaction time is 2-10 hour;
The mol ratio of alkynyl alcohol, Methyl benzenesulfonyl chlorine, chloroform and pyridine is 1: 1.2-2: 8-20: 8-20
Described step 2) in, the mol ratio of thioacetic acid, methyl alcohol and cesium carbonate is 1: 0.4-4: 0.2-1; Temperature of reaction is 40-85 ℃, and the reaction time is 0.5-6 hour;
In the described step 3), the product of described step 1) and the mol ratio of thiacetic cesium salt are 1: 1.2-4.
16. according to the arbitrary described method of claim 6-9, it is characterized in that: the method for the described end alkynyl label compound of described claim 9 is prepared as follows:
1) fluorenes methoxy carbonyl acyl group cysteine sulfydryl trityl is carried out esterification or amidation process;
2) product that described step 1) is obtained carries out the deprotection reaction of sulfydryl, obtains described end alkynyl label compound.
17. method according to claim 16, it is characterized in that: in the described step 1), esterification is carried out as follows: with fluorenes methoxy carbonyl acyl group cysteine sulfydryl trityl and alkynyl alcohol, N, N '-dicyclohexyl diimine reacts in organic solvent;
Wherein, fluorenes methoxy carbonyl acyl group cysteine sulfydryl trityl and alkynyl alcohol, N, the mol ratio of N '-dicyclohexyl diimine is 1: 1-1.5: 1-2; Described temperature of reaction is 20-80 ℃, and the described reaction time is 8 hours;
In the described step 1), amidation process carries out as follows: with fluorenes methoxy carbonyl acyl group cysteine sulfydryl trityl and alkynylamine, benzotriazole-N, N, N ', N '-tetramethylurea hexafluorophosphate, 1-hydroxy benzo triazole and N, the N-diisopropylethylamine reacts;
Wherein, fluorenes methoxy carbonyl acyl group cysteine sulfydryl trityl and alkynylamine, benzotriazole-N, N, N ', N '-tetramethylurea hexafluorophosphate, 1-hydroxy benzo triazole and N, the mol ratio of N-diisopropylethylamine is 1: 1.5-4: 0.9-1: 1-2: 1-3, preferred 1: 2: 0.9: 1: 2; Described temperature of reaction is 20-80 ℃, and the reaction time is 1.5-6 hour.
Described step 2) in, the deprotection reaction of sulfydryl is to carry out according in the e-h method any one:
E, the product of described step 1) is dissolved in ethanol, feeds ammonia and also add sodium silk, back flow reaction;
F, the product of described step 1) is dissolved in methyl phenyl ethers anisole, feeds hydrogen fluoride and react;
G, under inert atmosphere protection, with the product of described step 1) with acetic acid ethyl dissolution after, add trifluoroacetic acid and react;
H, the product that described step 1) is obtained dissolve with strong aqua;
Described organic solvent is benzene, N, dinethylformamide or acetonitrile.
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| CN109855929A (en) * | 2018-12-13 | 2019-06-07 | 复旦大学 | A kind of multi-mode capture, the continuously strong method for eluting glycosylated peptide and Phosphorylated Peptide |
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| CN111777696B (en) * | 2020-06-22 | 2022-04-01 | 复旦大学 | Method for specific reversible enrichment of nascent protein |
| CN116333759A (en) * | 2023-05-29 | 2023-06-27 | 上海柯珑清洁技术有限公司 | Preparation method of temperature-resistant surfactant |
| CN116333759B (en) * | 2023-05-29 | 2023-08-25 | 上海柯珑清洁技术有限公司 | Preparation method of temperature-resistant surfactant |
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