CN111089969B - Method for rapid enzymolysis release and solid-phase enrichment of N-sugar chains and mass spectrometry - Google Patents
Method for rapid enzymolysis release and solid-phase enrichment of N-sugar chains and mass spectrometry Download PDFInfo
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Abstract
The application belongs to the field of glycoproteomics and glycogenomics analysis, and relates to a method for rapid enzymolysis release and solid phase enrichment and mass spectrometry of N-sugar chains, which comprises the following steps: absorbing a solution containing glycoprotein samples by using absorbent cotton materials, and performing high-temperature rapid PNGase F enzymolysis on cotton to release N-sugar chains; and after enzymolysis, regulating the solution environment, adsorbing free sugar chains on the absorbent cotton material by utilizing the actions of high-density hydroxyl groups on the surface of the absorbent cotton material, such as hydrogen bonds of sugar chains, and the like, then cleaning and removing non-sugar chain molecules which are not combined with the absorbent cotton material, such as proteins, polypeptides and the like, eluting the adsorbed sugar chains by using an aqueous solution, and sending the eluted sugar chains to mass spectrometry. The method has the advantages of low-cost and easily-obtained materials, easy preparation and simple and convenient operation, and can realize high-speed enzymolysis of the N-glycoprotein, high-selectivity enrichment of the N-sugar chain and high-sensitivity mass spectrometry.
Description
Technical Field
The application belongs to the field of glycoprotein and glycogenomics analysis, and relates to a method for rapid enzymolysis release, solid-phase enrichment and mass spectrometry of N-sugar chains in glycoprotein. Compared with the existing common N-sugar chain enrichment and mass spectrometry methods, the method has higher selectivity and higher analysis sensitivity, can obviously shorten the time required for acquiring sugar chains from samples such as serum and the like, and has the characteristics of simple steps, convenient and quick operation and the like.
Background
The prior art discloses that protein N-glycosylation is one of the most ubiquitous post-translational modifications in organisms, and it has been reported that more than 50% of proteins in mammals can undergo N-glycosylation modifications. Research shows that glycosylation has important biological significance, and N-sugar chains connected with protein play an important role in cell recognition and molecular recognition, protein folding and maintenance of correct conformation of the protein; the changes in the distribution, composition and structure of the N-sugar chains in turn affect these biological processes. Studies have shown that abnormalities in N-glycosylation play an important role in a variety of diseases such as cancer, cardiovascular disease, autoimmune disease, and the like. Therefore, highly selective, highly sensitive analysis of N-sugar chains in organisms would be beneficial for physiological and pathological studies, as well as providing potential biomarkers.
However, N-sugar chain studies have faced the following difficulties: first, although the N-glycosylated protein is abundant in species, the glycosylation modification ratio is relatively low, in other words, the overall abundance of N-sugar chains is at a low level; secondly, the N-sugar chains have microscopic heterogeneity, namely N-sugar chains with different compositions possibly exist on the same glycosylation site, so that the abundance of single N-sugar chains is also at a lower level, and the sensitivity of an analysis method is higher; in the mass spectrometry process, the N-sugar chain lacks a hydrophobic group and a group with stable charge, the ionization efficiency is low, and the signal is more easily inhibited by substances such as peptide fragments, proteins and the like which are easy to ionize in a sample; finally, although some tools such as microcolumns and microtanks for N-sugar chain enrichment exist at present, the tools often have the problems of low selectivity, high price and long operation time.
Based on the current state of the art, the inventors of the present application have devised a method for rapid enzymolysis, solid-phase enrichment and mass spectrometry of N-sugar chains based on cheaper and readily available materials, which would be advantageous for achieving rapid pretreatment, high-selectivity enrichment and high-sensitivity mass spectrometry identification of N-sugar chains in complex biological samples, thereby further facilitating the study of glycoproteomics and glycogenomics.
Disclosure of Invention
The application aims to overcome the defects of a sample processing method before N-sugar chain mass spectrometry in the prior art, provides a novel method for realizing rapid enzymolysis, selective enrichment and high-sensitivity mass spectrometry identification of N-sugar chains in an N-glycoprotein sample, and particularly relates to a method for rapid enzymolysis release, solid-phase enrichment and mass spectrometry analysis of N-sugar chains, which has the advantages of low cost, simple steps and convenient operation.
Specifically, the application provides a method for carrying out rapid enzymolysis and release on N-sugar chains based on absorbent cotton material matrixes, enriching the N-sugar chains in situ and carrying out mass spectrometry; the application uses the strong adsorptivity of absorbent cotton to adsorb samples such as serum; the absorbent cotton has a loose and porous structure, and is beneficial to high-speed enzymolysis on cotton; the high-density hydroxyl on the surface of the material is favorable for enriching sugar chains based on hydrophilic interaction in situ, substances such as peptide fragments, proteins and salts can be removed from the material by washing with acetonitrile-water solution, and the sugar chains captured by the material can be eluted by using the water solution so as to be convenient for subsequent mass spectrometry analysis, thereby realizing the efficient pretreatment of the N-glycoprotein sample.
In order to achieve the above purpose, the technical scheme adopted by the application is as follows:
1. absorbing a solution containing glycoprotein samples by using absorbent cotton materials, and performing high-temperature rapid PNGase F enzymolysis on cotton to release N-sugar chains;
2. enriching free N-sugar chains in the solution by using absorbent cotton materials and using a centrifugal auxiliary method, and removing impurities such as peptide fragments, proteins and the like by proper cleaning;
3. the N-sugar chains were eluted from the absorbent cotton material and then mass-analyzed.
Specifically, the method for rapid enzymolysis release and solid-phase enrichment and mass spectrometry of N-sugar chains is characterized in that the method is based on absorbent cotton material matrix, performs rapid enzymolysis release on N-sugar chains, and enriches the N-sugar chains in situ and performs mass spectrometry, wherein absorbent cotton material is used as a suction carrier of an N-glycoprotein-containing sample, a platform for rapid enzymolysis release of the N-sugar chains, and an enrichment matrix of the N-sugar chains during solid-phase enrichment, and realizes rapid release and selective enrichment of the N-sugar chains, and comprises the following steps:
(1) Absorbing a glycoprotein sample-containing solution by using absorbent cotton materials, adding a proper buffer solution to infiltrate the materials, and placing the materials into a centrifuge tube for storage;
(2) Carrying out high-temperature rapid PNGase F deglycosylation treatment on the glycoprotein sample on absorbent cotton to release sugar chains on the glycoprotein;
(3) Adding a certain volume of organic solvent to adjust the sample solution, so that sugar chains in the solution are adsorbed by absorbent cotton materials;
(4) Filling absorbent cotton material into a gun head, and then placing the gun head on a centrifuge tube;
(5) Transferring the remaining sample solution to absorbent cotton material, and separating the solution from absorbent cotton by centrifugation;
(6) Washing the material with a suitable buffer, separating the solution from the absorbent cotton by centrifugation after each wash;
(7) Replacing a bottom centrifuge tube, adding an eluting solution, dissociating sugar chains from the material, and collecting a solution part by centrifugation;
(8) After the collected solution is freeze-dried, the solution can be directly re-dissolved and then analyzed by using matrix-assisted laser desorption/ionization mass spectrometry, or a methylamine reaction is carried out to protect sialic acid units;
(9) The sample after the amination is desalted by using the method of the steps (4) - (7) after being regulated by adding a certain volume of organic solvent, and can be analyzed by using matrix-assisted laser desorption/ionization mass spectrometry.
In the application, absorbent cotton material is adopted as an adsorbent, and N-sugar chains are adsorbed on absorbent cotton material by utilizing interaction of high-density hydroxyl groups on the surface of the material and hydrogen bonds and the like between the N-sugar chains with high hydrophilicity, and the absorbent cotton material is separated from solution by centrifugation, so that enrichment and mass spectrometry of the N-sugar chains are finally realized;
in the steps (1) and (2), protein samples are dissolved in 10-50mM ammonium bicarbonate aqueous solution, 0.5 mu L of PNGase F is added into each sample, and the mixture is mixed for 0.5-1 hour at 45-55 ℃ to enable N-sugar chains in the samples to be dissociated from the protein;
in the step (3), a certain volume of acetonitrile/trifluoroacetic acid/water solution is added into the solution after enzymolysis, so that the volume fraction of acetonitrile in the solution is 80-85%, the volume fraction of TFA is 0-0.1%, and the volume is 80-160 mu L;
in the step (5), a centrifugal machine is used for separating absorbent cotton materials from the solution, and the solution part is discarded; the interval between each liquid adding and the centrifugation is 0.5-1 min, and the centrifugation time is 10-30 seconds;
in the step (6), 50-100 mu L of acetonitrile/trifluoroacetic acid (TFA)/water solution (the volume fraction of acetonitrile is 75-85 percent and the volume fraction of TFA is 0-0.1 percent) is used for cleaning the material for 4-6 times, a centrifuge is used for separating absorbent cotton material from the solution after each cleaning, and the solution part is discarded; the interval between each liquid adding and the centrifugation is 0.5-1 min, the centrifugal force is 1500g, and the centrifugal time is 10-30 seconds;
in the step (7), eluting sugar chains 1-2 times with 50-100 mu L of 0-0.1% TFA aqueous solution, separating absorbent cotton material from the solution by using a centrifuge after each elution, and collecting a solution part; the interval between each liquid adding and the centrifugation is 0.5-1 min, the centrifugal force is 1500g, and the centrifugal time is 10-30 seconds;
in the step (8), the solution collected by elution is mixed with an organic matrix containing 2, 5-dihydroxybenzoic acid (DHB) without being subjected to methylamination, and is subjected to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis;
in the step (8), after the solution collected by elution is freeze-dried in the case of the methylamine, 10 to 15. Mu.L of 5M methylamine hydrochloride (dissolved in DMSO) and 10 to 15. Mu.L of 250mM (7-Azabenzotriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate (PyAOP) (dissolved in DMSO: N-methylmorpholine=7:3 system) are added and reacted at 37℃for 1 to 1.5 hours;
in the step (9), 400-500 mu L of acetonitrile/trifluoroacetic acid/water solution is added into the methylamine solution and mixed with the methylamine solution, the volume fraction of acetonitrile is 80-100%, and the volume fraction of TFA is 0-0.1%.
The application provides a method for carrying out rapid enzymolysis and release on N-sugar chains based on absorbent cotton material matrix, enriching the N-sugar chains in situ and carrying out mass spectrometry; the method utilizes the strong adsorptivity of absorbent cotton, and uses the absorbent cotton for the adsorption of samples such as serum and the like; the absorbent cotton material has loose and porous structure, so that the rapid enzymolysis of the glycoprotein sample on the surface of the absorbent cotton material is facilitated; the hydroxyl group with high density on the surface has strong affinity effect on the N-sugar chain, has high binding speed, can obviously improve the mass spectrum analysis selectivity of the N-sugar chain, and has the characteristics of simple steps, convenient operation, rapidness and the like.
Drawings
FIG. 1 is a flow chart of the present enrichment method.
FIG. 2 shows a MALDI-MS spectrum of a standard glycoprotein RNB, in which the spectrum is the relative Intensity (% Intensity) of a mass spectrum peak on the ordinate and the mass-to-charge ratio (m/z) on the abscissa, wherein the spectrum (a) is untreated RNB protein, the spectrum (b) is a sample obtained by adding 0.5. Mu.L PNGase F to 100. Mu.g RNB protein and then subjecting the sample to enzymolysis at 45-55℃for 30 minutes on cotton, and the spectrum (c) is a spectrum obtained by adding 0.5. Mu.L PNGase F to 100. Mu.g RNB protein and then subjecting the sample to enzymolysis at 37℃for 30 minutes on solution, and the spectrum (c) shows that the enzymolysis at 45-55℃for 30 minutes on absorbent cotton is sufficient to completely release N-sugar chains on 100. Mu.g RNB protein, and the conventional method (enzymolysis at 37℃in solution) cannot be performed completely during the same time.
FIG. 3 shows that the mass ratio of standard sugar chain maltoheptaose (DP 7) to Bovine Serum Albumin (BSA) enzymatic peptide is 1:100 (a, b) or 1:1000 (c, d) MALDI-TOF-MS spectra after mixing without enrichment (a, c) and after enrichment of the absorbent cotton material (b, d), wherein the spectra are plotted on the ordinate as the relative Intensity (% Intensity) of the mass spectrum peak and on the abscissa as the mass-to-charge ratio (m/z); comparing the graph (a) with the graph (b), and the graph (c) and the graph (d) show that the sugar chain sample can be selectively enriched in the presence of a large amount of peptide fragment impurities, so that the sugar chain sample can be detected by mass spectrometry with high sensitivity, and the detected maltohexaose (DP 6) is another sugar chain mixed in the maltoheptaose standard.
FIG. 4 shows that the mass ratio of standard glycoprotein Asialofetuin (ASF) to Bovine Serum Albumin (BSA) enzymatic peptide fragment is 1:50, wherein the spectrum is the relative Intensity (% Intensity) of the mass spectrum peak on the ordinate and the mass to charge ratio (m/z) on the abscissa. (a) The ASF protein is subjected to enzymolysis overnight in advance by using a traditional method to release sugar chains, and then is mixed with BSA peptide fragments; (b) After the ASF protein is mixed with the BSA peptide fragment in advance, the method is used for high-speed enzymolysis-solid phase enrichment on cotton and then analysis is carried out, and comparison of the graph (a) and the graph (b) shows that excessive peptide fragment impurities contained in the sample can be removed in the whole flow of the method, so that the high-selectivity enrichment on N-sugar chains is realized.
FIG. 5 is a MALDI-TOF-MS spectrum of N-sugar chains in a standard glycoprotein, the ordinate of the spectrum is the relative Intensity (% Intensity) of mass spectrum peaks, and the abscissa is the mass-to-charge ratio (m/z), wherein (a) the fetuin is directly analyzed after releasing sugar chains by overnight enzymolysis using a conventional method; (b) The fetuin is treated by the steps (1) - (8) of the method and is not analyzed by methylamine; (c) The method is used for treating the fetuin, analysis is carried out after the methyl amination, and the result shows that the method can effectively enrich sugar chains containing sialic acid, and the integral signal intensity is improved after the methyl amination treatment; the sialylated sialic acid also no longer undergoes in-source degradation in mass spectrometry.
FIG. 6 is a graph showing that 46N-sugar chains were successfully enriched and mass-identified, including highly sialylated sugar chains, after high-speed enzymolysis and enrichment, from solid phase enrichment of 0.1. Mu.L complex human serum samples.
Detailed Description
The following examples are further illustrative of a method of high speed enzymatic hydrolysis, solid phase enrichment and mass spectrometry on N-glycoprotein cotton as set forth herein.
Example 1
Solid phase enrichment for free N-sugar chain containing samples
3-7mg of absorbent cotton material is weighed and filled into a gun head, and the gun head is placed on a centrifuge tube. Activating with 50-80 μl water for 1 time, centrifuging to remove solution, adding liquid each time at intervals of 0.5-1 min, centrifuging for 10-30 seconds at a centrifugal force of 1500g, and the same applies below; then, the solution was washed once with 50 to 80. Mu.L of an aqueous solution of 80 to 85% acetonitrile, and centrifuged to remove the solution. After freeze-drying the N-sugar chain sample to be desalted (DP 7: BSA peptide=1:1000), redissolving the N-sugar chain sample by using 80-160 mu L of acetonitrile/trifluoroacetic acid/water solution to ensure that the volume fraction of acetonitrile in the solution after the reaction is 80-85% and the volume fraction of TFA is 0-0.1%, loading the sample, and centrifuging to remove the solution. Washing with 50-100 μl of 75-85% acetonitrile/water solution for 4-6 times, centrifuging to remove the solution; the bottom centrifuge tube was replaced, the adsorbed sugar chains were eluted with 50-100. Mu.L of 0-0.1% TFA in water, and the eluate was collected and lyophilized, and then redissolved with 10. Mu.L of water. 1. Mu.L of the solution was spotted on a MALDI target plate, and after drying, an equal volume of DHB matrix solution was spotted, and after drying crystallization, MALDI-TOF-MS analysis was performed, and the results were shown in FIG. 3 (d).
Example 2
High-speed enzymolysis and solid-phase enrichment of cotton containing N-glycoprotein sample
Weighing 3-7mg of absorbent cotton material, and placing at the bottom of a centrifuge tube. After lyophilization of the N-glycoprotein sample to be treated (ASF: BSA enzymatic peptide=1:50), it was reconstituted with 20-40. Mu.L of 25mM ammonium bicarbonate buffer to a final concentration of 10 ng/. Mu.L to 1000 ng/. Mu.L and transferred to a centrifuge tube containing absorbent cotton material. Treating in water bath at 100deg.C for 5 min, cooling, adding 0.5 μl PNGase F enzyme, and performing enzymolysis at 45-55deg.C for 0.5-1 hr. And adding a certain volume of acetonitrile/trifluoroacetic acid/water solution into the solution after enzymolysis, so that the volume fraction of acetonitrile in the solution is 80-85%, the volume fraction of TFA is 0-0.1%, and the volume is 80-160 mu L. Filling absorbent cotton material into the gun head and placing the gun head on a centrifuge tube. Sampling the residual solution, centrifuging to remove the solution, wherein the interval between each liquid adding and centrifuging is 0.5-1 min, the centrifugal force is 1500g, the centrifuging time is 10-30 seconds, and the same applies below; washing with 50-100 μl of 75-85% acetonitrile/water solution for 4-6 times, centrifuging to remove the solution; the bottom centrifuge tube was replaced, the adsorbed sugar chains were eluted with 50-100. Mu.L of 0-0.1% TFA in water, and the eluate was collected and lyophilized, and then redissolved with 10. Mu.L of water. 1. Mu.L of the solution was spotted on a MALDI target plate, and after drying, an equal volume of DHB matrix solution was spotted, and after drying and crystallization, MALDI-TOF-MS analysis was performed, and the results are shown in FIG. 4 (b).
Example 3
High-speed enzymolysis and solid-phase enrichment of complex sample human serum on cotton
Weighing 3-7mg of absorbent cotton material, and placing at the bottom of a centrifuge tube. 1. Mu.L of serum to be treated was diluted with 20-40. Mu.L of 25mM ammonium bicarbonate buffer and transferred to a centrifuge tube containing absorbent cotton material. Treating in water bath at 100deg.C for 5 min, cooling, adding 0.5 μl PNGase F enzyme, and performing enzymolysis at 45-55deg.C for 0.5-1 hr. And adding a certain volume of acetonitrile/trifluoroacetic acid/water solution into the solution after enzymolysis, so that the volume fraction of acetonitrile in the solution is 80-85%, the volume fraction of TFA is 0-0.1%, and the volume is 80-160 mu L. Filling absorbent cotton material into the gun head and placing the gun head on a centrifuge tube. Sampling the residual solution, centrifuging to remove the solution, wherein the interval between each liquid adding and centrifuging is 0.5-1 min, the centrifugal force is 1500g, the centrifuging time is 10-30 seconds, and the same applies below; washing with 50-100 μl of 75-85% acetonitrile/water solution for 4-6 times, centrifuging to remove the solution; the bottom centrifuge tube was replaced, and the adsorbed sugar chains were eluted with 50-100. Mu.L of 0-0.1% TFA in water. After collecting the eluate and freeze-drying, 10-15. Mu.L of 5M methylamine hydrochloride (dissolved in dimethylsulfoxide DMSO) and 10-15. Mu.L of 250mM (7-Azabenzotriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate (PyAOP) (dissolved in dimethylsulfoxide: N-methylmorpholine=7:3 system) were added, and reacted at 37℃for 1-1.5 hours, 400-500. Mu.L of acetonitrile/trifluoroacetic acid/water solution was added to the methylamine-post-solution and mixed with the methylamine-post-solution, the volume fraction of acetonitrile was 80-100%, and the volume fraction of TFA was 0-0.1%. 3-7mg of absorbent cotton material is weighed and filled into a gun head, and the gun head is placed on a centrifuge tube. Loading the solution, and centrifuging to remove the solution; washing with 50-100 μl of 75-85% acetonitrile/water solution for 4-6 times, centrifuging to remove the solution; the bottom centrifuge tube is replaced, and 50-100 mu L of 0-0.1% TFA aqueous solution is used for eluting the adsorbed sugar chains; after the eluate was collected and lyophilized, 1. Mu.L of the eluate was spotted on a MALDI target plate, and after the eluate was dried, an equal volume of DHB matrix solution was spotted, and after the eluate was dried and crystallized, MALDI-TOF-MS analysis was performed, and the results were shown in FIG. 6.
Claims (6)
1. A method for rapid enzymolysis release and solid-phase enrichment and mass spectrometry of N-sugar chains is characterized in that,
adopting a material based on absorbent cotton as a substrate for absorbing glycoprotein sample solution, carrying out rapid enzymolysis and enriching N-sugar chain solid phase; the method comprises the following steps:
(1) Absorbing a solution containing glycoprotein samples by using absorbent cotton materials, adding buffer solution to infiltrate the materials, and placing the materials into a centrifuge tube for preservation; the glycoprotein sample concentration is 10 ng/mu L-1000 ng/mu L, and the glycoprotein sample is dissolved in 10-50mM ammonium bicarbonate buffer solution; wherein the mass of the added absorbent cotton material is 3-7 mg;
(2) Performing high-temperature rapid PNGase F deglycosylation treatment on the glycoprotein sample on the absorbent cotton, and performing enzymolysis reaction with an absorbent cotton material co-system at 45-55 ℃ to release sugar chains on the glycoprotein;
(3) Adding a certain volume of acetonitrile/trifluoroacetic acid/water solution to ensure that the volume fraction of acetonitrile in the solution is 80-85%, the volume fraction of TFA is 0-0.1%, and the volume is 80-160 mu L, so that sugar chains in the solution are adsorbed by absorbent cotton materials;
(4) Filling absorbent cotton material into a gun head, and placing the gun head on a centrifuge tube;
(5) Transferring the remaining sample solution to absorbent cotton material, and centrifuging to separate the solution from absorbent cotton;
(6) Washing the material with a buffer solution, and centrifuging to separate the solution from absorbent cotton after washing;
(7) Replacing a bottom centrifuge tube, adding an eluting solution, dissociating sugar chains from the material, and centrifugally collecting a solution part;
(8) After freeze-drying the collected solution, directly redissolving the solution, analyzing the solution by using matrix-assisted laser desorption/ionization mass spectrometry,
or methylamine reaction to protect sialic acid units;
(9) After the sample after the methylamination is added into an organic solvent for adjustment, the salt is removed by the method of the steps (4) - (7), and the matrix-assisted laser desorption/ionization mass spectrometry is used for analysis.
2. The method of claim 1, wherein 0.5 μl of PNGase F enzyme is added to each glycoprotein sample of step (1).
3. The method of claim 1, wherein the absorbent cotton material in step (5) is subjected to sample enrichment for 0.5-2 hours, the sample is dissolved in acetonitrile/trifluoroacetic acid/water solution, the volume fraction of acetonitrile is 75-85%, and the volume fraction of TFA is 0-0.1%; the interval time from loading to centrifugation is 0.5-1 min, the centrifugal force is 1500g, and the centrifugal time is 10-30 seconds.
4. The method of claim 1, wherein the material is washed 4-6 times with 50-100 μl of 75-85% acetonitrile/water containing 0-0.1% TFA in step (6).
5. The method according to claim 1, wherein the step (7) is carried out by eluting the sugar chain 1 to 2 times with 50 to 100. Mu.L of the aqueous solution containing 0 to 0.1% TFA.
6. The process according to claim 1, wherein in step (9) 400 to 500. Mu.L of acetonitrile/trifluoroacetic acid/water is mixed with the post-methylamine solution, the volume fraction of acetonitrile is 80 to 100% and the volume fraction of TFA is 0 to 0.1%.
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