CN111208237A - Method for improving mass spectrum fragmentation efficiency and response based on peptide fragment C-terminal chemical derivatization - Google Patents
Method for improving mass spectrum fragmentation efficiency and response based on peptide fragment C-terminal chemical derivatization Download PDFInfo
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Abstract
The invention relates to a method for improving mass spectrum fragmentation efficiency and response based on peptide fragment C-terminal chemical derivatization. The method comprises three steps of reactions, namely, blocking carboxyl at the C tail end of a peptide segment, or carboxyl at the C tail end of the peptide segment and carboxyl of side chain glutamic acid or aspartic acid; then, specific hydrolysis is carried out by utilizing peptide fragment amidase, and carboxyl at the C terminal of the peptide fragment is exposed; finally, the exposed C-terminal carboxyl group is subjected to chemical derivatization of a basic molecule. The basic molecule marked on the C terminal of the peptide segment can increase the number of y ions in a secondary mass spectrum, and improve the fragmentation efficiency and response of the peptide segment in the mass spectrum, thereby being applied to improving the detection sensitivity of polypeptide drug peptide.
Description
Technical Field
The invention relates to a method for improving mass spectrum fragmentation efficiency and response based on peptide fragment C-terminal chemical derivatization and application thereof.
Background
The polypeptide medicine is polypeptide with specific therapeutic action, which is obtained by chemical synthesis, gene recombination or extraction from animal and plant. The polypeptide medicine can be widely applied to endocrine system, immune system, digestive system, cardiovascular system, musculoskeletal system, etc. The growth of the polypeptide drug market is extremely fast, and the growth is one of the trends of drug market research and development in recent years. More than 80 polypeptide drugs are approved to be on the market globally at present, such as insulin for treating diabetes, human growth hormone for treating senile diseases and dwarfism, natriuretic hormone for treating cardiovascular diseases, and the like. The polypeptides are highly active and are administered at low doses, with the concentration of drug circulating in the body generally being in the sub-nanogram per milliliter range. In vivo, a large amount of interfering substances, such as endogenous protein polypeptide substances with the same or similar structures, exist, so that the research of polypeptide drugs needs an ultrahigh-sensitivity detection method. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) technology integrates the high separation performance of high performance liquid chromatography and the high sensitivity of mass spectrometry, is widely applied to the research of drug metabolism and pharmacokinetics of polypeptide drugs, and will become a trend of future development. Many drug peptides lack basic amino acids at their termini, which results in poor fragmentation efficiency in mass spectrometry, and are not conducive to mass spectrometry identification. The ionization efficiency and the ion transmission rate of the polypeptide drug can be improved by marking basic molecules at the tail end of the peptide segment, so that the detection sensitivity of the polypeptide drug is improved. The derivative based on the C terminal of the peptide segment can shield the electronegativity of the carboxyl, thereby enhancing the mass spectrometry capability of the peptide segment. By applying a cationic derivatization reagent brominated 1-butyl, 3- (3-aminopropyl) imidazole to carboxyl derivatization of a peptide fragment by Qiaoqiang et al, the mass spectrometric detection sensitivity of a model peptide fragment can be improved by more than 42 times (Sci China Life Sci,2013,43, 96-102). Therefore, the chemical derivation of the basic molecule based on the C terminal of the peptide segment can improve the fragmentation efficiency and response of the polypeptide drug in mass spectrum, thereby improving the detection sensitivity of the polypeptide drug.
Disclosure of Invention
The invention aims to provide a method for improving the fragmentation efficiency and response of mass spectrometry based on C-terminal chemical derivatization of a peptide fragment. In order to realize the purpose, the method adopts the technical scheme that:
the method for improving the fragmentation efficiency and response of mass spectrum based on chemical derivatization of the C-terminal of peptide segment comprises the following steps of firstly carrying out amidation reaction or esterification reaction on carboxyl at the C-terminal of peptide segment or carboxyl at the C-terminal of peptide segment and carboxyl of side chain glutamic acid or aspartic acid in a solvent containing an organic phase; then, specific hydrolysis is carried out by utilizing peptide fragment amidase, and carboxyl at the C terminal of the peptide fragment is exposed; finally, the exposed C-terminal carboxyl group is subjected to chemical derivatization of a basic molecule in a solvent containing an organic phase, and mass spectrometry detection is carried out.
The solvent containing the organic phase is a mixture of water and the organic phase, and the volume concentration of water in the solvent is 2-30%; the organic phase is one or more than two of acetonitrile, N-dimethylformamide, acetone and isopropanol.
The amidation reaction is to react the carboxyl at the C-terminal of the peptide segment, or the carboxyl of the C-terminal of the peptide segment and the carboxyl of the side chain glutamic acid or aspartic acid with one or more than two of methylamine, ethylamine, propylamine and ethanolamine to generate an amide substance; the esterification reaction refers to the reaction of carboxyl at the C terminal of the peptide segment, or the reaction of the carboxyl at the C terminal of the peptide segment and the carboxyl of side chain glutamic acid or aspartic acid with one or more than two of methanol, ethanol and isopropanol to generate ester substances.
The amidation reaction temperature is 4-50 ℃, and the reaction time is 0.5-12 h; the esterification reaction temperature is 4-50 ℃, and the reaction time is 0.5-12 h.
The peptide fragment amidase is extracted from an escherichia coli body, can specifically catalyze and hydrolyze an amido bond at the C terminal of the peptide fragment, and has no hydrolysis effect on the amido bond of the side chain of the peptide fragment and the peptide bond of the skeleton.
The pH value of the peptide fragment amidase catalytic hydrolysis reaction is 6.8-8.5, the temperature is 25-50 ℃, the time is 6-48h, and the mass ratio of amidase to peptide fragment is 1:50-20: 1.
The basic molecule is one or more than two of aminoguanidine, agmatine, N-dimethylethylenediamine, N-dimethyl-1, 3-propanediamine and (2-aminoethyl) trimethylammonium chloride.
The mass-volume ratio of the peptide segment exposing the C-terminal carboxyl group to the alkaline molecule is 0.005-1 g/ml, the reaction temperature is 4-50 ℃, and the reaction time is 0.5-12 h.
The basic molecule marked on the C terminal of the peptide segment can increase the number of y ions in the secondary mass spectrum, and improve the fragmentation efficiency and response of the peptide segment in the mass spectrum, thereby improving the detection sensitivity of the peptide segment.
The peptide segment is polypeptide drug peptide which is chemically synthesized, genetically recombined or extracted from animals and plants and has specific therapeutic action and consists of several to dozens of amino acids; the method is applied to improving the detection sensitivity of the polypeptide drug peptide.
(1) Dissolving 5-100 μ g peptide fragment (synthetic model peptide fragment or purchased medicinal peptide) powder in mixture of water and organic phase, wherein the volume concentration of water in the mixture is 2-30%, the organic phase is one or more of acetonitrile, N-dimethylformamide, acetone, and isopropanol, and the total volume of the solvent is 50-500 μ L. Adding one or more than two of methylamine, ethylamine, propylamine and ethanolamine with the final concentration of 25-1000mM into the peptide fragment solution after redissolution for amidation reaction, and adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide with the final concentration of 20-200mM and N-hydroxysuccinimide with the final concentration of 10-100mM for activating carboxyl, wherein the reaction temperature is 4-50 ℃, and the reaction time is 0.5-12 h. Or adding one or more of methanol, ethanol and isopropanol with final concentration of 25-1000mM to the peptide fragment solution after redissolution for esterification, and adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide with final concentration of 20-200mM and N-hydroxysuccinimide with final concentration of 10-100mM to activate carboxyl, wherein the reaction temperature is 4-50 ℃ and the reaction time is 0.5-12 h. After the reaction is finished, freeze-drying, and then performing liquid chromatography desalting and freeze-drying.
(2) Re-dissolving the product obtained in the step (1), and then carrying out hydrolysis reaction of peptide fragment amidase, wherein the volume of re-dissolution is 50-500 mu L, the pH of the hydrolysis reaction is 6.8-8.5, the temperature is 25-50 ℃, the time is 6-48h, and the mass ratio of amidase to peptide fragment is 1:50-20: 1. After the reaction, liquid chromatography desalting and freeze-drying are carried out.
(3) Dissolving the product obtained in the step (2) in a mixture of water and an organic phase, wherein the volume concentration of the water in the mixture is 2-30%, the volume concentration of the organic phase is one or more than two of acetonitrile, N-dimethylformamide, acetone and isopropanol, and the total volume of the solvent is 50-500 mu L. Adding one or more of aminoguanidine, agmatine, N-dimethylethylenediamine, N-dimethyl-1, 3-propanediamine and (2-aminoethyl) trimethylammonium chloride with a final concentration of 25-1000mM for labeling reaction, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide with a final concentration of 20-200mM and N-hydroxysuccinimide with a final concentration of 10-100mM for activation, wherein the reaction temperature is 4-50 ℃ and the reaction time is 0.5-12 h. After the reaction is finished, freeze-drying, and then performing liquid chromatography desalting and freeze-drying.
(4) And (3) analyzing the products obtained in the steps (1), (2) and (3) by LC-MS/MS, and comparing the number of fragment ions of the peptide fragment before and after labeling with the signal intensity.
The invention has the following advantages:
1. the reaction efficiency of chemically blocking C terminal, side chain glutamic acid and aspartic acid carboxyl by using small molecules at the level of peptide fragments is high.
2. The peptide fragment amidase specifically hydrolyzes amido bond at the C terminal of the peptide fragment, and has no hydrolysis effect on amido bond of a side chain and peptide bond of a skeleton.
3. The C terminal of the peptide fragment is marked with basic molecules, so that more y ions are generated in the secondary mass spectrum, and the fragmentation efficiency and response of the peptide fragment in the mass spectrum are improved.
Drawings
FIG. 1 is a flow chart of a method for improving mass spectrometry fragmentation efficiency and response by chemical derivatization of the C-terminal end of a peptide fragment;
FIG. 2 is a mass spectrum of matrix-assisted laser desorption ionization time-of-flight before and after marking methylamine with a model peptide fragment 1200;
FIG. 3 is a matrix-assisted laser desorption ionization time-of-flight mass spectrogram before and after marking methylamine with a model peptide fragment 9094.
Detailed Description
Example 1
(1) 1mg of synthesized model peptide fragment CT1200 (with the sequence of LVVSTQTALA) was weighed and added with 1000. mu.L of a mixed solution of N, N-dimethylformamide and water to prepare a peptide fragment mother liquor, the volume concentration of water was 5%. In another centrifuge tube, 20. mu.L of the peptide fragment mother liquor, 75. mu.L of acetonitrile, 2.5. mu.L of 1M N-hydroxysuccinimide, 2. mu.L of 2.5M 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide, 0.5. mu.L of 5M methylamine were added and reacted at 25 ℃ for 2 hours. After the reaction is finished, freeze-drying, and then performing liquid chromatography desalting and freeze-drying.
(2) The product is dissolved by adding 100 μ L of tris (hydroxymethyl) aminomethane buffer salt with pH7.5, 0.5 μ g peptide amidase is added, and hydrolysis is carried out for 12h at 25 ℃. And (3) after the reaction is finished, performing liquid chromatography desalting and freeze-drying.
(3) The product was redissolved by adding 93.5. mu.L of a mixed solution of N, N-dimethylformamide and water at a concentration of 2% by volume. Then, 2.5. mu.L of 1M N-hydroxysuccinimide, 2. mu.L of 2.5M 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide, 2. mu.L of 5M aminoguanidine were added and reacted at 25 ℃ for 2 hours. After the reaction is finished, freeze-drying, and then performing liquid chromatography desalting and freeze-drying.
(4) And (3) performing LC-MS/MS analysis on the products obtained in the steps (1), (2) and (3), and comparing the fragment ion number and the signal intensity of the peptide fragment before and after labeling to achieve the purpose of improving the detection sensitivity of the model peptide fragment.
Example 2
(1) Weighing 1mg of synthesized model peptide fragment 9094 (with the sequence of KEIFVGI), and adding 1000 mu L of mixed solution of N, N-dimethylformamide and water to prepare peptide fragment mother liquor, wherein the volume concentration of the water is 5%. In another centrifuge tube, 20. mu.L of the peptide fragment mother liquor, 74.5. mu.L of acetonitrile, 2.5. mu.L of 1M N-hydroxysuccinimide, 2. mu.L of 2.5M 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide, 1. mu.L of 5M methylamine were added and reacted at 25 ℃ for 2 hours. After the reaction is finished, freeze-drying, and then performing liquid chromatography desalting and freeze-drying.
(2) The product is dissolved by adding 100 μ L of tris (hydroxymethyl) aminomethane buffer salt with pH7.5, 0.5 μ g peptide amidase is added, and hydrolysis is carried out for 12h at 25 ℃. And (3) after the reaction is finished, performing liquid chromatography desalting and freeze-drying.
(3) The product was redissolved by adding 90. mu.L of a mixed solution of N, N-dimethylformamide and water at a volume concentration of 2%. Then 5. mu.L of 1M N-hydroxysuccinimide, 4. mu.L of 2.5M 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and 1. mu.L of 5M agmatine were added and reacted at 25 ℃ for 2 hours. After the reaction is finished, freeze-drying, and then performing liquid chromatography desalting and freeze-drying.
(4) And (3) performing LC-MS/MS analysis on the products obtained in the steps (1), (2) and (3), and comparing the fragment ion number and the signal intensity of the peptide fragment before and after labeling to achieve the purpose of improving the detection sensitivity of the model peptide fragment.
Example 3
(1) 1mg of the purchased drug peptide oxytocin (with the sequence of CYIQNCPLG) is weighed and added with 1000. mu.L of a mixed solution of acetone and water to prepare a peptide fragment mother liquor, and the volume concentration of the water is 5 percent. In another centrifuge tube, 20. mu.L of the peptide fragment mother liquor, 65.5. mu.L of N, N-dimethylformamide, 2.5. mu.L of 1M N-hydroxysuccinimide, 2. mu.L of 2.5M 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide, 10. mu.L of 5M ethanolamine were added and reacted at 25 ℃ for 2 hours. After the reaction is finished, freeze-drying, and then performing liquid chromatography desalting and freeze-drying.
(2) The product is dissolved by adding 100 μ L of tris (hydroxymethyl) aminomethane buffer salt with pH7.5, 0.1 μ g peptide amidase is added, and hydrolysis is carried out for 24h at 30 ℃. And (3) after the reaction is finished, performing liquid chromatography desalting and freeze-drying.
(3) The product was redissolved by adding 93.5. mu.L of a mixed solution of N, N-dimethylformamide and water at a concentration of 2% by volume. Then, 2.5. mu.L of 1M N-hydroxysuccinimide, 2. mu.L of 2.5M 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide, 2. mu.L of 5M N, N-dimethylethylenediamine were added thereto and reacted at 25 ℃ for 4 hours. After the reaction is finished, freeze-drying, and then performing liquid chromatography desalting and freeze-drying.
(4) And (3) performing LC-MS/MS analysis on the products obtained in the steps (1), (2) and (3), and comparing the fragment ion number and the signal intensity of the peptide fragments before and after labeling to achieve the purpose of improving the detection sensitivity of the medicinal peptide.
Example 4
(1) 1mg of the purchased pharmaceutical peptide corticotropin (with the sequence of SYSMEHFRWGKPVGKKRRPVKVYP) was weighed and added with 1000. mu.L of a mixed solution of acetone and water to prepare a peptide fragment mother liquor, the volume concentration of water being 5%. In another centrifuge tube, 20. mu.L of the peptide fragment mother liquor, 65.5. mu.L of N, N-dimethylformamide, 2.5. mu.L of 1M N-hydroxysuccinimide, 2. mu.L of 2.5M 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide, 10. mu.L of 5M ethanolamine were added and reacted at 25 ℃ for 2 hours. After the reaction is finished, freeze-drying, and then performing liquid chromatography desalting and freeze-drying.
(2) The product is dissolved by adding 100 μ L of tris (hydroxymethyl) aminomethane buffer salt with pH7.5, 0.1 μ g peptide amidase is added, and hydrolysis is carried out for 24h at 30 ℃. And (3) after the reaction is finished, performing liquid chromatography desalting and freeze-drying.
(3) The product was redissolved by adding 81. mu.L of a mixed solution of N, N-dimethylformamide and water at a concentration of 2% by volume. Then 5. mu.L of 1M N-hydroxysuccinimide, 4. mu.L of 2.5M 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and 10. mu.L of 5M N, N-dimethyl-1, 3-propanediamine were added and reacted at 25 ℃ for 4 hours. After the reaction is finished, freeze-drying, and then performing liquid chromatography desalting and freeze-drying.
(4) And (3) performing LC-MS/MS analysis on the products obtained in the steps (1), (2) and (3), and comparing the fragment ion number and the signal intensity of the peptide fragments before and after labeling to achieve the purpose of improving the detection sensitivity of the medicinal peptide.
Claims (10)
1. The method for improving the fragmentation efficiency and response of the mass spectrum based on the chemical derivatization of the C terminal of the peptide fragment is characterized by comprising the following steps: firstly, in a solvent containing an organic phase, carrying out amidation reaction or esterification reaction on carboxyl at the C tail end of a peptide segment or the carboxyl at the C tail end of the peptide segment and carboxyl of side chain glutamic acid or aspartic acid; then, specific hydrolysis is carried out by utilizing peptide fragment amidase, and carboxyl at the C terminal of the peptide fragment is exposed; finally, the exposed C-terminal carboxyl group is subjected to chemical derivatization of a basic molecule in a solvent containing an organic phase, and mass spectrometry detection is carried out.
2. The method of claim 1, wherein: the solvent containing the organic phase is a mixture of water and the organic phase, and the volume concentration of water in the solvent is 2-30%; the organic phase is one or more than two of acetonitrile, N-dimethylformamide, acetone and isopropanol.
3. The method of claim 1, wherein: the amidation reaction is to react the carboxyl at the C-terminal of the peptide segment, or the carboxyl of the C-terminal of the peptide segment and the carboxyl of the side chain glutamic acid or aspartic acid with one or more than two of methylamine, ethylamine, propylamine and ethanolamine to generate an amide substance; the esterification reaction refers to the reaction of carboxyl at the C terminal of the peptide segment, or the reaction of the carboxyl at the C terminal of the peptide segment and the carboxyl of side chain glutamic acid or aspartic acid with one or more than two of methanol, ethanol and isopropanol to generate ester substances.
4. A method according to claim 1 or 3, characterized by: the amidation reaction temperature is 4-50 ℃, and the reaction time is 0.5-12 h; the esterification reaction temperature is 4-50 ℃, and the reaction time is 0.5-12 h.
5. The method of claim 1, wherein: the peptide fragment amidase is extracted from an escherichia coli body, can specifically catalyze and hydrolyze an amido bond at the C terminal of the peptide fragment, and has no hydrolysis effect on the amido bond of the side chain of the peptide fragment and the peptide bond of the skeleton.
6. The method of claim 1 or 5, wherein: the pH value of the peptide fragment amidase catalytic hydrolysis reaction is 6.8-8.5, the temperature is 25-50 ℃, the time is 6-48h, and the mass ratio of amidase to peptide fragment is 1:50-20: 1.
7. The method of claim 1, wherein: the basic molecule is one or more than two of aminoguanidine, agmatine, N-dimethylethylenediamine, N-dimethyl-1, 3-propanediamine and (2-aminoethyl) trimethylammonium chloride.
8. The method of claim 1, wherein: the mass-volume ratio of the peptide segment exposing the C-terminal carboxyl group to the alkaline molecule is 0.005-1 g/ml, the reaction temperature is 4-50 ℃, and the reaction time is 0.5-12 h.
9. The method of claim 1, wherein: the basic molecule marked on the C terminal of the peptide segment can increase the number of y ions in the secondary mass spectrum, and improve the fragmentation efficiency and response of the peptide segment in the mass spectrum, thereby improving the detection sensitivity of the peptide segment.
10. The method of any of claims 1-9, wherein: the peptide segment is polypeptide drug peptide which is chemically synthesized, genetically recombined or extracted from animals and plants and has specific therapeutic action and consists of several to dozens of amino acids; the method is applied to improving the detection sensitivity of the polypeptide drug peptide.
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| CN115201385A (en) * | 2022-06-22 | 2022-10-18 | 河北医科大学 | Derivatization reagent for electrospray mass spectrometry detection and capable of enabling amino micromolecules to carry two charges, and preparation method and application thereof |
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