CN110596228B - Differential ion mobility spectrometry-mass spectrometry combined epimer separation method - Google Patents
Differential ion mobility spectrometry-mass spectrometry combined epimer separation method Download PDFInfo
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Abstract
The invention discloses a method for separating and identifying O-glycosylated peptide segment epimers based on differential ion migration-mass spectrometry, and belongs to the field of detection and analysis of biological medicines and foods/health-care products. The method disclosed by the invention can be used for detecting the O-acetylglucosamine glycosylated epimeric protein or polypeptide, which lays a good foundation for researching and developing new drugs for treating nervous system diseases and the like, and has very important significance in the fields of biological medicines and food/health care product analysis. In addition, the separation and identification of O-glycosylated protein or peptide segment which is a very important substance in organisms are realized by the technology of differential ion mobility spectrometry-mass spectrometry for the first time, and the development of the identification technology of biomacromolecule O-glycosylated protein or peptide segment is promoted.
Description
Technical Field
The invention relates to a differential ion mobility spectrometry-mass spectrometry combined epimer separation method, and belongs to the field of detection and analysis of biological medicines and foods/health-care products.
Background
The information in this background section is only for enhancement of understanding of the general background of the disclosure and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
In organisms, protein glycosylation is a complex post-translational modification of proteins that is widely found in cells, with approximately 50% of human protein molecules being glycosylated. Glycosylation has important effects on the structure and function of proteins, for example, almost all key molecules in the immune system are glycoproteins, and immunoglobulin sugar chain abnormalities are the cause of autoimmune diseases; the zona pellucida glycoprotein ZP3 determines the combination of ovum and sperm, and the sugar chain is a person with sperm-egg combination; and so on.
The glycosylation pattern can be largely divided into two types: n-glycosylation and O-glycosylation, wherein the O-glycosylation contains a monosaccharide unit and plays an important role in the aspects of cell functions and disorder, for example, O-acetylglucosamine of protein is found to be involved in a plurality of signal networks and is closely related to diseases such as nervous system diseases (such as Alzheimer disease and the like), autoimmune diseases and the like. Therefore, the method has very important biological significance and clinical application value for the research of the glycoprotein, and particularly has important significance for the research of the complex glycoprotein/peptide as biological medicine and the like.
In the pharmaceutical and food/health product field, this epimeric glycopeptide often has only one form being active, or different epimeric glycopeptides have different functional activities, or only one form is capable of characterizing the efficacy of the drug, so in practice, it is necessary to accurately separate, identify the presence and determine the content of non-target epimeric glycopeptides.
Disclosure of Invention
In view of the above background technologies, the present disclosure establishes a method for separating and identifying O-glycosylated peptide segment epimers based on differential ion mobility-mass spectrometry. By injecting a trace amount of volatile organic regulator into the differential ion mobility spectrometry, the baseline separation of the glycopeptide epimers is realized, the foundation is laid for the complete separation of the glycopeptide epimers, and the method has very important significance in the fields of biological medicine and food/health care product analysis.
Specifically, the following technical scheme is adopted in the disclosure:
in a first aspect of the present disclosure, there is provided a method for separating epimers of O-glycosylated peptide segments, the method comprising:
taking a mixture containing at least two mutually O-glycosylation peptide segment epimers as a sample to be analyzed, and separating by adopting a differential ion mobility spectrometry technology; wherein, n-propanol (also known as 1-propanol), n-butanol (also known as 1-butanol), acetonitrile or 1-propanethiol are used as gas regulators.
In a second aspect of the present disclosure, there is provided a method for detecting and analyzing epimers of O-glycosylated peptide segments, the method comprising:
taking a mixture containing at least two mutual O-glycosylation peptide segment differential isomers as a sample to be analyzed, and separating by adopting differential ion mobility spectrometry-mass spectrometry; wherein, n-propanol, n-butanol, acetonitrile or 1-propanethiol is used as the gas regulator.
In a third aspect of the present disclosure, there is provided a method for determining whether O-glycosylated peptide segment epimers are contained, the method comprising:
taking a mixture suspected of containing O-glycosylated peptide segment epimers as a sample to be analyzed, and determining by adopting differential ion mobility spectrometry; wherein, n-propanol, n-butanol, acetonitrile or 1-propanethiol is used as the gas regulator.
In a fourth aspect of the present disclosure, there is provided a method for the isolation or identification of epimers of an O-glycosylated protein, the method comprising:
pretreating a sample to be analyzed by using a mixture containing at least two epimers of the O-glycosylated protein which are mutually O-glycosylated to generate a mixed solution of a plurality of peptide fragments containing at least two epimers of the O-glycosylated peptide fragment which are mutually O-glycosylated; then, the separation or detection analysis method of the O-glycosylated peptide segment epimer is adopted for operation, and the separation or identification of the O-glycosylated protein epimer can be realized.
In a fifth aspect of the present disclosure, there is provided a method for determining whether an epimer of an O-glycosylated protein is contained, the method comprising:
pretreating a sample to be analyzed by using a mixture suspected of containing O-glycosylated protein epimers as the sample to be analyzed to generate a multiple peptide fragment mixed solution containing at least two O-glycosylated peptide fragment epimers which are each other; measuring by adopting differential ion mobility spectrometry; wherein, n-propanol, n-butanol, acetonitrile or 1-propanethiol is used as the gas regulator.
Compared with the related technology known by the inventor, one technical scheme of the present disclosure has the following beneficial effects:
(1) the method realizes baseline separation of glycopeptide epimers, does not use an expensive separation chromatographic column, has the advantages of no consumption of an organic solvent (namely a volatile polar gas regulator) less than 20 microliter, environmental protection, shorter analysis time than an ultra-high performance liquid chromatography system, high sensitivity, capability of reaching the fM level to the maximum, no need of time-delay flushing of the system among samples, no cross contamination among samples and simple and convenient operation.
(2) The O-acetylglucosamine of the protein is found to be involved in a plurality of signal networks, and is closely related to diseases such as nervous system diseases (such as Alzheimer's disease and the like), autoimmune diseases and the like. The method disclosed by the invention can be used for detecting the O-acetylglucosamine glycosylated epimeric protein or polypeptide, so that a good foundation is laid for developing new medicines for treating nervous system diseases and the like.
(3) The separation and identification of O-glycosylated protein or peptide segment which is a very important substance in organisms are realized by using a differential ion mobility spectrometry-mass spectrometry combined technology for the first time, and the development of a biological detection technology of biomacromolecule O-glycosylated protein or peptide segment is promoted.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this disclosure, illustrate embodiments of the disclosure and, together with the description, serve to explain the disclosure and not to limit the disclosure.
FIG. 1 is a mass spectrum of three glycopeptides of the present disclosure;
FIG. 2 is a graph of the trend of compensation voltage of the present disclosure as a function of 1-propanol concentration;
FIG. 3 is a graph of the variation of compensation voltage versus regulator concentration for the present disclosure, (a) 1-butanol; (b) acetonitrile; (c) 1-propanethiol;
fig. 4 is a graph of the superimposed signal spectrum of a glycopeptide epimer of the present disclosure.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present disclosure. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of the stated features, steps, operations, and/or combinations thereof, unless the context clearly indicates otherwise.
Interpretation of terms:
epimer: in stereochemistry, a diastereomer having only one chiral center with a different configuration among stereoisomers having a plurality of chiral centers and the remaining configurations being the same is called an epimer; for example, epimers with glucose are: mannose (C2), allose (C3), galactose (C4).
O-sugar peptide bond: refers to an O-glycosidic bond formed by covalent bonding of the anomeric carbon of a monosaccharide and the hydroxyl O atom of a hydroxyl amino acid.
Carbon at the end of heterodromous: when monosaccharides are changed from linear to cyclic structures, the carbonyl carbon atom becomes the new chiral center, resulting in epimerization of C1 to yield two diastereomers, where the hemiacetal carbon atom is referred to as the anomeric carbon atom in the cyclic structure.
Baseline separation and quasi-baseline separation: the separation of the base line is generally considered to be that the separation degree of two adjacent peaks is more than 1.5, or the peak valley of two adjacent peaks is located on the base line, and the quasi-base line separation is understood to be a condition that the base lines of two peaks are basically separated.
As described in the background, currently, diastereomer complex saccharide isomers containing a single epimeric monosaccharide are still not distinguishable by mass spectrometry, and in order to solve the above technical problems, in a first exemplary embodiment of the present disclosure, there is provided a method for separating epimers of O-glycosylated peptide segments, the method comprising: taking a mixture containing at least two mutually O-glycosylation peptide segment epimers as a sample to be analyzed, and separating by adopting a differential ion mobility spectrometry technology; wherein, n-propanol, n-butanol, acetonitrile or 1-propanethiol is used as gas regulator, and the purity is HPLC grade.
In one or more embodiments of the present disclosure, an O-glycosylated peptide segment is a diastereomer complex carbohydrate isomer comprising a single epimeric monosaccharide formed by an O-glycopeptide bond between a monosaccharide and a polypeptide (or peptide segment), wherein the monosaccharide includes, but is not limited to, glucose, mannose, galactose, allose, acetylglucosamine, acetylgalactosamine, or glucose, mannose, galactose, allose modified with other groups; and the like, wherein the length of the polypeptide is 2-100 amino acid molecules; further, the length of the polypeptide is 2-50 amino acid molecules; furthermore, the length of the polypeptide is 3-25 amino acid molecules, and researches show that the glycosylated peptide segment with the length in the range can better realize the separation of epimers. Peptide fragments of different lengths or of a specific sequence can be prepared mainly by biological methods (such as enzymatic hydrolysis), chemical methods (such as hydrolysis) and/or physical methods (such as ultrasound), which are conventional in the art and will not be described herein.
In one or more embodiments of the present disclosure, the sample can be a biological sample or a non-biological sample.
The biological sample may be from a mammalian subject or a non-mammalian subject. The mammalian subject can be, for example, a human or other animal species. Biological samples include biological fluids such as whole blood, serum, plasma, sputum, lymph, semen, vaginal mucus, fecal matter, urine, spinal fluid, saliva, stool, cerebrospinal fluid, tears, mucus, and the like; biological tissue, such as hair, skin, slices from organs or other body parts, or excised tissue; and so on. In many cases, the sample is whole blood, plasma, or serum.
Non-biological samples include, but are not limited to, for example, food products, health products, and the like, which can also be analyzed using O-glycosylated peptide fragments according to the principles described in the present disclosure.
In one or more embodiments of the present disclosure, in the differential ion mobility spectrometry technique, an electrospray ion source, a positive ion mode, is employed. Experiments prove that the electrospray voltage is 3.0-3.5 kV, so that the target signal intensity is high, and the subsequent separation between the targets is facilitated.
In one or more embodiments of the present disclosure, the parameters of the differential ion mobility spectrometry technique are: the flat plate electrode has a flat plate interval of 1.4mm, a length of 80mm, a width of 20mm, a scanning range of compensation voltage of-50V- +100V, and a dispersion field constant of 127 Td.
In one or more embodiments of the present disclosure, the parameters of the differential ion mobility spectrometry technique are: carrier gas: nitrogen gas of 99.999% purity was used; the flow rate of the carrier gas containing the gas regulator affects the effect of epimer separation to some extent, and the flow rate of the carrier gas containing the gas regulator is preferably 300 mL/min.
In one or more embodiments of the present disclosure, the parameters of the differential ion mobility spectrometry technique are: the sample introduction speed of a sample to be analyzed is 0.4-0.6 mu L/min; further, 0.5. mu.L/min was selected.
In one or more embodiments of the present disclosure, the concentration of the gas regulator is 0.2 to 0.5%; further selected to be 0.45%. The concentration in the present disclosure means a molar ratio of the gas conditioning agent to the carrier gas, and 0.45% means a molar ratio of the gas conditioning agent to the carrier gas of 0.45/100. Experiments prove that the gas regulator in the concentration range can ensure the quasi-baseline separation effect of different O-glycosylation peptide sections.
In one or more embodiments of the present disclosure, when the sample to be analyzed is an O-glycosyl protein or peptide fragment comprising glucose, galactose and/or mannose, the gas modifier is selected to be n-propanol, enabling efficient separation of epimers of the O-glycosyl protein or peptide fragment.
In a second exemplary embodiment of the present disclosure, there is provided a method for the detection and analysis of O-glycosylated peptide segmental epimers with the purpose of identifying and quantifying each O-glycosylated peptide segmental epimer, the method comprising:
taking a mixture containing at least two mutual O-glycosylation peptide segment differential isomers as a sample to be analyzed, and separating and identifying by adopting differential ion mobility spectrometry-mass spectrometry; wherein, n-propanol, n-butanol, acetonitrile or 1-propanethiol is used as the gas regulator.
In one or more embodiments of the present disclosure, the parameters of the mass spectrum are: selection of ion mode selection of protonated parent ion [ M + H ] of glycopeptide]+: 1238.62, collecting mass spectrum signals, wherein the sample injection flow rate of the ion source is as follows: 0.5. mu.L/min, the temperature of heating nitrogen gas is: the cumulative time of the ions in the hexapole rod was 0.5s at 220 ℃.
When other samples are made, the parameters can be optimally adjusted according to actual conditions, and specific values are not limited.
In one or more embodiments of the present disclosure, during identification, a standard database needs to be established, and a compensation voltage value corresponding to a peak of a differential ion mobility spectrum signal peak of different O-glycosylated peptide segment epimers is obtained, so as to identify a specific type and content of the O-glycosylated peptide segment epimers (presence of an analysis target substance is determined by the compensation voltage, and semi-quantitative or quantitative determination may be performed by applying a peak area or a peak height). Further, the solvent system of the standard solution is a methanol-water system with a volume ratio of 50:50, and 0.1 v/v% of acetic acid is added. The solvent system is beneficial to the stability of the glycosylated peptide section, the purpose of adding acetic acid is to promote the ionization of the glycosylated peptide section, and the acetic acid can provide protons for the glycopeptide.
In a third exemplary embodiment of the present disclosure, there is provided a method for determining whether O-glycosylated peptide segment epimers are present, the method comprising:
taking an analyte suspected of containing the O-glycosylated peptide segment epimer as a sample to be analyzed, and determining by adopting differential ion mobility spectrometry; wherein, n-propanol, n-butanol, acetonitrile or 1-propanethiol is used as the gas regulator.
In a fourth exemplary embodiment of the present disclosure, there is provided a method for separating or identifying epimers of an O-glycosylated protein, the method comprising:
pretreating a sample to be analyzed by using a mixture containing at least two epimers of the O-glycosylated protein which are mutually O-glycosylated to generate a mixed solution of a plurality of peptide fragments containing at least two epimers of the O-glycosylated peptide fragment which are mutually O-glycosylated; then, the separation or detection analysis method of the O-glycosylated peptide segment epimer is adopted for operation, and the separation or identification of the O-glycosylated protein epimer can be realized.
In one or more embodiments of the present disclosure, the method of pre-processing is: the O-glycosylated protein is degraded into a plurality of peptide fragments or peptide fragments with specific length by biological methods (such as enzymolysis), chemical methods (such as hydrolysis) and/or physical methods (such as ultrasonic wave), which belong to the conventional prior art and are not described in detail herein.
In a fifth exemplary embodiment of the present disclosure, there is provided a method for determining whether an epimer of an O-glycosylated protein is present, the method comprising:
pretreating a sample to be analyzed by using a mixture suspected of containing O-glycosylated protein epimers as the sample to be analyzed to generate a multiple peptide fragment mixed solution containing at least two O-glycosylated peptide fragment epimers which are each other; measuring by adopting differential ion mobility spectrometry; wherein, n-propanol, n-butanol, acetonitrile or 1-propanethiol is used as the gas regulator.
In order to make the technical solutions of the present disclosure more clearly understood by those skilled in the art, the technical solutions of the present disclosure will be described in detail below with reference to specific embodiments.
Test equipment used in the examples: the used device is connected with a Bruker 9.4TFTMS mass spectrometer by adopting a differential ion mobility spectrometer in the prior art.
Materials used in the examples: the glycopeptides selected are EIDYIT (Gal) PAR, EIDYIT (Glc) PAR and EIDYIT (Man) PAR, wherein E-threonine, I-valine, D-phenylalanine, Y-tyrosine, T-threonine, P-proline, A-alanine, R-lysine, Gal-galactose, Glc-glucose, Man-mannose, T (Gal) -galactose linked to a threonine residue, T (Glc) -glucose linked to a threonine residue, and T (Man) -mannose linked to a threonine residue. The amino acid sequence of the peptide fragment is derived from the protein Arginine Deiminase (ADI). The epimeric monosaccharide is glucose, galactose and mannose, and the glycosylation site is threonine residue.
Example 1
Electrospray is an ionization method that can be used as an ion source for mass spectrometers. The glycopeptides EIDYIT (Gal) PAR, EIDYIT (Glc) PAR and EIDYIT (Man) PAR were each measured by electrospray mass spectrometry (ESI-MS).
The test conditions and parameters were: the electrospray voltage is 3.0kV, and the selected ion mode selects the protonated parent ion [ M + H ] of glycopeptide]+: 1238.62, collecting mass spectrum signals, wherein the sample injection flow rate of the ion source is as follows: 0.5. mu.L/min, the temperature of heating nitrogen gas is: the cumulative time of the ions in the hexapole rod was 0.5s at 220 ℃.
The experimental results are shown in FIG. 1, which shows the electrospray mass spectrum of three glycopeptides in positive ion mode, and its protonated parent ion [ M + H ]]+The mass numbers of all the components are 1238.63. The secondary spectrogram fragment ions generated by collision cracking and electron-excited cracking are also consistent, and it can be seen that effective differentiation of three glycopeptide epimers cannot be realized through mass spectrometry.
Example 2
Preparation of a sample to be analyzed 1:
the solvent system is a methanol-water system with a volume ratio of 50:50, 0.1 v/v% of acetic acid is added (namely, the system contains 0.1% of acetic acid by volume fraction), and two glycopeptides are added, so that the concentration of EIDYIT (Gal) PAR and EIDYIT (glc) PAR in the system are both 1.0 mu mol/L.
Sample to be analyzed 2 was prepared:
the solvent system is a methanol-water system with a volume ratio of 50:50, 0.1 v/v% of acetic acid is added (namely: the system contains 0.1% of acetic acid by volume fraction), and two glycopeptides are added, so that the concentration of EIDYIT (Man) PAR and EIDYIT (glc) PAR in the system are both 1.0 mu mol/L.
The technical parameters of the differential ion mobility spectrometry are set as follows:
the electrospray voltage was 3.0kV, positive ion mode.
Plate electrodes of 20mm width and 80mm length with a plate spacing of 1.4 mm.
The compensation voltage is swept over a range of-50 to +100V and the dispersion field constant is set to 127 Td.
The sample injection rate was 0.5. mu.L/min.
Carrier gas: nitrogen gas of 99.999% purity was used.
The flow rate of the carrier gas containing the gas regulator was 300 mL/min.
And adding a selected gas regulator into the gas path of the differential ion mobility spectrometer.
Mass spectrum conditions: selection of ion mode selection of protonated parent ion [ M + H ] of glycopeptide]+: 1238.62, collecting mass spectrum signals, wherein the sample injection flow rate of the ion source is as follows: 0.5. mu.L/min, the temperature of heating nitrogen gas is: the cumulative time of the ions in the hexapole rod was 0.5s at 220 ℃.
The first set of tests: 1-propanol with the concentration of 0, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.40 and 0.45 percent is taken as a gas regulator, and the sample 1 to be analyzed and the sample 2 to be analyzed are detected by adopting a differential ion mobility spectrometry technology. The specific process is as follows: after a sample to be analyzed is ionized, the sample is separated through a differential ion mobility spectrum, then selective ion collection is carried out on a mass spectrum, and the relation between compensation voltage and the concentration of a regulator and a corresponding separation effect graph are obtained through scanning the compensation voltage.
The second set of tests: 1-butanol with the concentration of 0, 0.025, 0.05, 0.075, 0.10, 0.125, 0.15, 0.175, 0.20, 0.25, 0.30, 0.35 and 0.4 percent respectively is used as a gas regulator, and a sample 1 to be analyzed is detected by adopting a differential ion mobility spectrometry technology.
The third set of tests: acetonitrile with the concentration of 0, 0.05, 0.10, 0.125, 0.15, 0.20, 0.25, 0.30, 0.35, 0.4 and 0.5 percent is taken as a gas regulator, and the sample 1 to be analyzed is detected by adopting a differential ion mobility spectrometry technology.
Fourth group of tests: 1-propanethiol with the concentration of 0, 0.05, 0.10, 0.20, 0.30 and 0.4 percent is taken as a gas regulator, and a sample 1 to be analyzed is detected by adopting a differential ion mobility spectrometry technology.
The experimental results are shown in fig. 2, fig. 3 and fig. 4, and fig. 2 and fig. 3 are graphs showing the variation of the compensation voltage of the differential ion mobility spectrometry with the concentration of the gas modifier in the carrier gas.
FIG. 2(a) shows the relationship between the compensation voltage of epimeric glycopeptides EIDYIT (Gal) PAR and EIDYIT (glc) PAR and the concentration of gas regulator 1-propanol, wherein the compensation voltage of the two glycopeptides gradually increases with the increasing concentration of the gas regulator 1-propanol, and the compensation voltage gradually separates with the increasing concentration.
FIG. 2(b) is a graph of the dependence of the compensation voltage of the epimeric glycopeptides EIDYIT (Man) PAR and EIDYIT (glc) PAR on the concentration of 1-propanol regulator, similar to FIG. 2 (a).
The principle of using 1-butanol as a gas regulator in FIG. 3(a) is similar to that in FIG. 2 (a). For eidyit (gal) PAR, two peaks appear when 1-propanol and 1-butanol are used as gas modifiers, presumably due to the presence of two conformation of protonated parent ions. It can be seen that alcohols (1-propanol and 1-butanol) are used as gas regulators, and with the increase of the concentration of the gas regulators, the influence of the compensation voltage change required by the glycopeptide epimer through differential ion mobility spectrometry is very obvious, and the change amount is 14-16V. When 1-butanol was used, the difference in compensation voltage between the two glycopeptides was not significant, and separation was difficult to achieve.
FIG. 3(b) is a graph showing the compensation voltage variation law using acetonitrile as a gas regulator. The variation in the compensation voltage of the glycopeptide epimer is about 5V, but the variation values of the compensation voltage of the glycopeptide epimer are the same, and separation is difficult.
FIG. 3(c) the compensation voltage increase of glycopeptide was less than 1V using 1-propanethiol as gas regulator. Since the epimer glycopeptides have the same variation values, it is difficult to achieve separation. Whereas with acetonitrile and propanethiol, only one peak was present in the EIDYIT (Gal) PAR.
As can be seen from FIGS. 2 and 3, the compensation voltages for the three glycopeptides were almost identical for the gas regulator 1-butanol, acetonitrile and 1-propanethiol, and it was difficult to achieve epimer separation. The epimer separation can be achieved using only 1-propanol as gas modifier.
FIG. 4 is a superimposed ionization diagram of glycopeptide epimers at 0.45% concentration of 1-propanol added as a gas regulator. As shown in fig. 4, when no gas regulator was added (0%), the glycopeptide epimer was not able to achieve separation; when the concentration of 1-propanol is 0.45 v/v%, the epimer quasi-baseline separation can be achieved separately for the glycopeptide mixture due to the large variation in the compensation voltage.
Example 3
The separation and identification of the epimeric peptide fragments of O-acetylglucosamine are also verified by the disclosure, and the experimental results show that the O-glycosylated peptide fragments (O-acetylglucosamine peptide fragments, O-acetylgalactosamine peptide fragments, O-acetylaminomannosylate peptide fragments and the like) which are epimeric can be effectively separated by taking n-propanol, n-butanol, acetonitrile, 1-propanethiol and the like as gas conditioning agents, particularly n-propanol and the like, and the separation effect is remarkable.
The above embodiments are preferred embodiments of the present disclosure, but the embodiments of the present disclosure are not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present disclosure should be regarded as equivalent replacements within the scope of the present disclosure.
Claims (16)
1. A method for separating O-glycosylated peptide segmental epimers is characterized by comprising the following steps:
taking a mixture containing at least two mutually O-glycosylation peptide segment epimers as a sample to be analyzed, and separating by adopting a differential ion mobility spectrometry technology;
the parameters of the differential ion mobility spectrometry technology are as follows: adopting a flat electrode, wherein the concentration of the gas regulator is 0.2-0.5%;
when the sample to be analyzed is an O-glycosyl protein or peptide fragment comprising glucose, galactose and/or mannose, the gas conditioning agent is selected to be n-propanol.
2. The method of claim 1, wherein the O-glycosylated peptide segment is a diastereomeric complex carbohydrate isomer comprising a single epimeric monosaccharide formed by O-glycopeptide bonding of a monosaccharide and a polypeptide.
3. The method of claim 2, wherein the monosaccharide is glucose, mannose, galactose, allose, acetylglucosamine mannose, acetylgalactosamine, or acetylgalactosamine.
4. The method of claim 2, wherein the polypeptide is 2 to 100 amino acid molecules in length.
5. The method of claim 4, wherein the polypeptide is 2 to 50 amino acid molecules in length.
6. The method of claim 1, wherein an electrospray ion source, positive ion mode, is used in the differential ion mobility spectrometry technique.
7. The method of claim 6, wherein the electrospray voltage is 3.0 to 3.5 kV.
8. The method of claim 1, wherein the parameters of the differential ion mobility spectrometry technique are: the plate interval is 1.4mm, the length is 80mm, the width is 20mm, the scanning range of the compensation voltage is-50V- +100V, and the dispersion field constant is set to 127 Td.
9. The method according to claim 8, wherein the sample size is 20 μ L.
10. The method according to claim 8, characterized in that the sample introduction speed of the sample to be analyzed is 0.4-0.6 μ L/min.
11. The method of claim 1, wherein the concentration of the gas conditioning agent is 0.45%.
12. A method for detecting and analyzing O-glycosylated peptide segmental epimer is characterized by comprising the following steps:
taking a mixture containing at least two mutual O-glycosylation peptide segment differential isomers as a sample to be analyzed, and separating and identifying by adopting differential ion mobility spectrometry-mass spectrometry;
the parameters of the differential ion mobility spectrometry technique are as follows: adopting a flat electrode, wherein the concentration of the gas regulator is 0.2-0.5%;
when the sample to be analyzed is an O-glycosyl protein or peptide fragment comprising glucose, galactose and/or mannose, the gas conditioning agent is selected to be n-propanol.
13. The detection and analysis method of claim 12, wherein the parameters of the mass spectrum are: the electrospray voltage is 3.0-3.5 kV, the protonation parent ion [ M + H ] of glycopeptide is selected in an ion-selective mode]+: 1238.62, collecting mass spectrum signals, wherein the sample injection flow rate of the ion source is as follows: 0.5 muL/min, the temperature of heating nitrogen gas is 220 ℃, and the accumulation time of ions in the hexapole rod is 0.5 s.
14. A method for determining whether an O-glycosylated peptide segment epimer is contained, the method comprising:
taking an analyte suspected of containing the O-glycosylated peptide segment epimer as a sample to be analyzed, and judging by adopting a differential ion mobility spectrometry;
the parameters of the differential ion mobility spectrometry technique are as follows: adopting a flat electrode, wherein the concentration of the gas regulator is 0.2-0.5%;
when the sample to be analyzed is an O-glycosyl peptide fragment containing glucose, galactose and/or mannose, the gas conditioning agent is selected to be n-propanol.
15. A method for separating or identifying epimers of an O-glycosylated protein, the method comprising: the method comprises the following steps:
pretreating a sample to be analyzed by using a mixture containing at least two epimers of the O-glycosylated protein which are mutually O-glycosylated to generate a mixed solution of a plurality of peptide fragments containing at least two epimers of the O-glycosylated peptide fragment which are mutually O-glycosylated; and then the separation or identification of the epimers of the O-glycosylated protein can be realized by adopting the separation method of the epimers of the O-glycosylated peptide segments as described in any one of claims 1 to 11 or the detection analysis method as described in claim 12 or 13.
16. A method for determining the presence of epimers of an O-glycosylated protein, the method comprising:
pretreating a sample to be analyzed by using a mixture suspected of containing O-glycosylated protein epimers as the sample to be analyzed to generate a multiple peptide fragment mixed solution containing at least two O-glycosylated peptide fragment epimers which are each other; judging by adopting a differential ion mobility spectrum;
the parameters of the differential ion mobility spectrometry technique are as follows: adopting a flat electrode, wherein the concentration of the gas regulator is 0.2-0.5%;
when the sample to be analyzed is an O-glycosyl protein comprising glucose, galactose and/or mannose, the gas conditioning agent is selected to be n-propanol.
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