CN107085032A - A kind of polypeptide derivatization method and its application in MALDI TOF MS detect drug metabolite - Google Patents
A kind of polypeptide derivatization method and its application in MALDI TOF MS detect drug metabolite Download PDFInfo
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- CN107085032A CN107085032A CN201710083984.8A CN201710083984A CN107085032A CN 107085032 A CN107085032 A CN 107085032A CN 201710083984 A CN201710083984 A CN 201710083984A CN 107085032 A CN107085032 A CN 107085032A
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- polypeptide
- aldehyde
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
- G01N33/6851—Methods of protein analysis involving laser desorption ionisation mass spectrometry
Abstract
The invention discloses a kind of chemical derivatization MALDI-TOF-MS technology (MALDI TOF MS) analysis method.The present invention is first by twain-aldehyde compound compound to being modified containing amino structure drug metabolite so that drug metabolism metabolin can derive the group with aldehyde radical, reuse polypeptide and aldehyde radical derivative is performed the derivatization, and is detected by MALDI TOF MS.Succinct single quasi-molecular ion peak will can be obtained after drug metabolite polypeptide, identification of these metabolites in mass spectrum is not only significantly improved, also greatly improves drug metabolite detection sensitivity in mass spectrum.This method sensitivity is high, have a wide range of application, simple operation, matrix interference are minimum, sample requirements are low, without preparing the matrix being attached to and derivatization reagent, you can quantitatively detect the extremely low amino metabolite of content in metabolin.There is very big application prospect in metabolism group research.
Description
Technical field
The present invention relates to Mass Spectrometer Method field, specific is to utilize many derived chemotactic peptide metabolites, with reference to MALDI-TOF-
MS method and its application in metabolism group field.
Background technology
Metabolism group is the another research field for characterizing life system situation after genomics and protein science, raw
The detection of metabolin in object to research gene expression, explore life process, diagnose the illness and prevention disease in terms of all have
There is profound meaning.Therefore, to metabolite analysis it is important directions in analytical chemistry field, or even life science.
The important means that mass spectrum is studied as small molecule metabolites, with sensitivity is high, selectivity is good, qualitative ability is strong etc.
Remarkable advantage.It is at present liquid chromatograph mass spectrography to the most common mass spectrum detection of small molecule metabolites.Although LC-
ESI-MS sensitivity is high, but LC-MS detection time is long, does not apply to and rapid high throughput assays.
Matrix Assisted Laser Desorption lonization-Time of Flight (MALDI-TOF-MS) is because detection is quick, easy, high pass
The features such as amount, it is widely used in the detection of the macromolecular samples such as DNA, protein, polypeptide, polysaccharide.In view of MALDI-TOF-
The advantages such as the quick, easy of MS detections, high sensitivity, high flux, we attempt to be extended to the inspection to small molecule metabolites
Survey.Although MALDI-DOF-MS has significant superiority on detection macromolecular substances, be not suitable for detection metabolin this
Small-molecule substance, mainly due to its conventionally used matrix CHCA, DHB, 3-HPA, 2,4,6-THP etc. small molecule (<
500Da) section can have serious matrix interference to target analytes.To overcome this shortcoming, the method master of existing literature report
It is divided into two classes：1) using the small matrix of matrix interference, such as porous silicon, nanoparticle, metal oxide, carbon material etc., but
Still no one kind is convenient, be widely used in matrix is developed；2) design macromolecular derivatization reagent derives small molecule and treated
Survey thing, the preparation generally troublesome and certain limitation of the derivatization reagent of existing literature report.Therefore, in small-molecule chemical thing
In the urgent need to developing a kind of easy, high sensitivity, pervasive deriving method in MALDI-TOF-MS analyses.
The present invention in view of the above-mentioned problems, develop using polypeptide as derivatization reagent to derive the new of small molecule metabolites first
Type MALDI-TOF-MS detection means, so as to overcome MALDI-TOF-MA in the advantage of small-molecule substance context of detection, developing
Applications of the MALDI-TOF-MS in metabolism group detection field.Have the advantages that easy to operate, quick, amount of samples is few, especially
Suitable for the analysis of biological sample.
The content of the invention
The technical problem to be solved in the present invention is that providing one kind can apply in small molecule metabolites detection field, with reference to
The MALDI-TOF-MS analysis methods with high sensitivity and universality of chemical derivatization strategy；
The Derivative and analytical plan that the present invention is provided comprise the following steps：
(1) after molecular simulation being carried out to polypeptide and is calculated by PM3 methods, the polypeptide for derivatization is screened, this is more
Peptide contains the group such as the arginine of easily ionizable, phenylalanine in MALDI sources；
(2) polypeptide that step (1) is obtained derives small-molecule substance to be measured, makes the amino acid of easily ionizable on these small molecule bands
Residue chain.
The present invention is by the way that the compound containing amino, by dialdehyde derivatization, then is filtered out by molecular simulation highly sensitive
The polypeptide of degree is as derivatization reagent, and the polypeptide for modifying cysteine and maleimide with N-terminal derives aldehyde compound respectively
Class compound, then detect with MALDI-TOF-MS these small molecule metabolites for passing through polypeptide marker.
A kind of derivatization method：It is that KFRGLRGF (modifies horse on Lys for CFRGLRGF polypeptide P2 and sequence to use sequence
Carry out acid imide) polypeptide P7 respectively as the derivatization reagent of aldehyde material, water is used as reaction dissolvent, phosphate buffer solution conduct
Buffer；
Reaction condition is：Aldehyde compound/polypeptide P2 (20-100, c/c) is added in pH5-7 phosphate buffer solution,
Derivative 0.2-2h at 4-37 DEG C；
Described derivative reaction refers to the dehydration condensation that the polypeptide containing Cys occurs with aldehyde compound.
Preferably, aldehyde compound, polypeptide P2 are using mol ratio as 50:1 is added in the phosphate buffer solution that pH is 4, and 25
At DEG C, occur dehydration condensation, 5h.
Because described polypeptide derivatization reagent is with the amino acid easily ionized in MALDI-MS with reference to proton
Residue, including Arg (arginine), Phe (phenylalanine) etc., it is found that the polypeptide filtered out has higher proton affinity, from
And proton easily is combined in mass spectrum, with high sensitivity.
A kind of polypeptideization derives the quantitative analysis method for combining MALDI-TOF-MS：After small molecule determinand derivatization,
With corresponding stable isotope internal standard 1:1 is mixed, and 1 μ L matrix (CHCA) point plate is mixed according to 1 μ L prepare liquids；Room temperature drying is laggard
Enter the analysis of Matrix Assisted Laser Desorption lonization-Time of Flight, composed according to target analytes spectral strength and stable isotope
The ratio of peak intensity carries out quantitative analysis to it.
Described polypeptide derivatization method combination MALDI-TOF-MS analysis method answering in metabonomic analysis field
With：Contain the Arg (arginine) of the easily ionizable in MALDI sources, Phe (phenylalanine) base for the polypeptide of derivatization through screening
Group, can make these small molecules bring the amino acid residue chain of easily ionizable into, significantly increase them after marking small-molecule substance to be measured
Ionization efficiency in MALDI-TOF-MS, so as to cause the sensitivity of method to be greatly improved.Because polypeptide exists
Minimum detectability (LOD) in MALDI-TOF-MS can reach that small molecule determinand is in mass spectrum derived from fmol, polypeptide
LOD can also reach fmol ranks.Moreover, this polypeptideization, which derives strategy, can apply the inspection of many other small-molecule chemical things
Survey, in addition to the aldehyde compound described in invention, the small molecules such as ketone, carboxylic acids, alcohol phenols, esters can also be extended to
In the MALDI-TOF-MS analyses of compound, the detection of these micromolecular compounds is great to the Research Significance of metabolism group.
Beneficial effects of the present invention are as follows：
1. in the present invention, method is easy to operate, directly analyzed after substance markers to be measured with MALDI-TOF-MS.
2. the polypeptide deriving method that the present invention is designed can significantly improve small molecule determinand in MALDI-TOF-MS
Detection sensitivity (remolding sensitivity derive before improve 50000 times), detection limit can reach fmol ranks.
3. the polypeptide derivatization reagent that the present invention is used has made on small molecule band to be measured molecular weight very big label, it is to avoid
Low molecule section in MALDI-TOF-MS (<500Da) interference of the matrix effect to detection.
4. polypeptide of the present invention and isotope polypeptide can buy the product of commercialization, quantitative analysis is utilized.
5. the derivative reaction that the present invention is designed has higher universality, due to having many can on derivatization reagent polypeptide
For the group of modification, using the analysis of a variety of micromolecular compounds, with very high application prospect.
Embodiment
1. the configuration of working solution：The polypeptide of derivatization is soluble in water, it is configured to 100 μM of solution for standby, adriamycin
The aldehyde derivative determinand of metabolin, which is dissolved in smuggled goods methanol, is configured to storing solution.
2. the preparation of aldehyde radical metabolite, is 1 according to mol ratio by metabolite and twain-aldehyde compound compound:1.2 ratio
Example is dissolved in alcohols solvent, is added 1 and is dripped stirring reaction at glacial acetic acid, 50 DEG C, is spin-dried for solvent standby.
3. polypeptide derivative method is：Polypeptide derivatization reagent box determinand is dissolved in aqueous or other are molten
Agent, is added in the phosphate buffer of certain pH according to appropriate proportioning, and it is 10 μM to make concentration of the determinand in reaction system,
Reaction is performed the derivatization after vortex mixed at a suitable temperature.Derivatising condition is aldehyde compound：Polypeptide P2 (20-100,
C/c) it is added in the phosphate buffer solution that pH is 4-6, derivatization 1-10 hours at 25-37 DEG C.
4. after derivative reaction terminates, reaction mixture is mixed with stable isotope inner mark solution, it is vortexed after mixing, according to
1 μ L samples mix 1 μ L matrix (CHCA) point plate；Room temperature enters Matrix Assisted Laser Desorption lonization-Time of Flight after air-drying,
Analyzed using cation reflective-mode.
5. the stable isotope internal standard preparation described in step 4 is, by stable isotope polypeptide P2 (polypeptide leucines
On N and 6 C flag), after being performed the derivatization with excessive aldehyde compound, vacuum is spin-dried for, to remove excessive small molecule, then
Multiple academic title's internal standard solution soluble in water.
6. the Mass Spectrometer Method condition described in step 4 is：Matrix-assisted desorption ionization-flight time matter of Waters companies
Spectrometer (MALDI-TOF-MS).MALDI-MS analyses use wavelength for 337nm nitrogen laser, and its pulse width is 3ns, is adopted
With 20kV accelerating potential, detected under cation reflective-mode.Every spectrogram is the average knot of 200 laser scannings
Really.
The adriamycin of embodiment 1 detection limit after aldehyde radical, many derived chemotactic peptides is determined
Take 1mmol adriamycins to be dissolved in 5mL methanol, add 1.1mmol glyoxal, add one and drip glacial acetic acid catalysis instead
Should, stirring reaction 3 hours at 50 DEG C, thin-layer chromatography monitoring reaction process, gained Schiff after removal of solvent under reduced pressure after reaction completely
Alkali is standby.By the dissolving of polypeptide derivatization reagent box determinand in aqueous, it is added in the phosphate buffer of certain pH, makes to treat
It is 10 μM to survey concentration of the thing in reaction system, performs the derivatization reaction after vortex mixed at a suitable temperature.Derivatization bar
Part is aldehyde compound：Polypeptide P2 (20-100, c/c) is added in the phosphate buffer solution that pH is 4-6, derivative at 25-37 DEG C
Change 1-10 hours.According to 1 μ L matrix point plates；Enter Matrix Assisted Laser Desorption lonization-Time of Flight point after room temperature drying
Analysis, obtains the detection limit of adriamycin.
The measure of adriamycin metabolite content in the MCF-7 cells of embodiment 2
After the MCF-7 clasmatosises of prior dosing, adriamycin metabolin is extracted, 1 μ L adriamycin metabolin extracts are taken
It is dissolved in 5mL methanol, adds 1.1 μ L MDA, adds one and drip stirring reaction 3 hours at glacial acetic acid catalytic reaction, 50 DEG C,
Thin-layer chromatography monitors reaction process, and gained schiff bases is standby after removal of solvent under reduced pressure after reaction completely.By polypeptide derivatization reagent
Box determinand dissolves in aqueous, is added in the phosphate buffer of certain pH, makes concentration of the determinand in reaction system
For 10 μM, reaction is performed the derivatization after vortex mixed at a suitable temperature.Derivatising condition is aldehyde compound：Polypeptide P2
(20-100, c/c) is added in the phosphate buffer solution that pH is 4-6, derivatization 1-10 hours at 25-37 DEG C.Addition is stablized same
The plain internal standard in position, it is 1 μM to make its concentration, takes 1 μ L matrix (CHCA) point plate, room temperature to enter ground substance assistant laser solution after air-drying after mixing
Ionization time of flight mass spectrometry is analysed, is analyzed using cation reflective-mode, according to the intensity of spectral line of target analytes and stably same position
The ratio of plain spectral line strong team carries out quantitative analysis, and the content for finally measuring adriamycinol in dosing 3h MCF-7 cells is 2.3 μ
M。
Claims (5)
1. a kind of polypeptide derives with the method for changing amino metabolism structure micromolecular compound combination MALDI-TOF-MS, its feature
Comprise the following steps：
(1) micromolecular compound：Using twain-aldehyde compound compound as linking arm, modified to amino-compound, made by condensation reaction
Drug metabolite structure derives aldehyde groups.
(2) many derived chemotactic peptide micromolecular compounds：By aldehyde compound, polypeptide with mol ratio 50-100:1 is added to pH for 4-6
Phosphate buffer in, condensation reaction occurs at 5-37 DEG C, the amino acid sequence N-terminal cysteine modified of polypeptide is used as aldehyde
Class compound derivatization reagent.
(3) MALDI-TOF-MS is analyzed：Analyte derivative is according to 1 μ L sample mixed-matrix alpha-cyano -4- hydroxycinnamic acids CHCA
Point plate, room temperature carries out Matrix Assisted Laser Desorption lonization-Time of Flight after air-drying, using cation reflective-mode.
2. aldehyde radical derivatization method according to claim 1 is characterized in that, twain-aldehyde compound chemical combination is selected from glyoxal, the third two
Aldehyde, butanedial, glutaraldehyde, hexandial, dialdehyde in heptan, suberic aldehyde.The reaction dissolvent of twain-aldehyde compound compound and metabolite is selected from first
Alcohol, ethanol, isopropanol, n-butanol, the tert-butyl alcohol, the reaction time are 0.5-5h, and reaction temperature is 20-100 DEG C.
3. polypeptide derivatization method according to claim 1, it is characterised in that aldehyde derivative in step (1) with it is many
The mol ratio of peptide is 50-100:1 is added in the phosphate buffer that pH is 4-6, occurs condensation reaction, the reaction time is 0.5-
5h。
4. a kind of polypeptide derives with the analysis method for changing amino metabolism structure micromolecular compound combination MALDI-TOF-MS, its
It is characterised by, after small molecule determinand is combined according to 1-3 method aldehyde radical derivative and polypeptide, according to 1 μ L sample mixed-matrixes
Alpha-cyano -4- hydroxycinnamic acids CHCA point plates, room temperature carries out Matrix Assisted Laser Desorption lonization-Time of Flight after air-drying and determined
Property analysis.Can directly can be with corresponding stable isotope 1：1 mixes, according to 1 μ L sample mixed-matrix alpha-cyano -4- hydroxyl meat
Cinnamic acid CHCA point plates, room temperature carries out Matrix Assisted Laser Desorption lonization-Time of Flight analysis after air-drying, according to target analysis
The ratio of material spectral strength and stable isotope spectral strength carries out quantitative analysis to it.
5. the application in method described in any one of claim 1-3 metabonomic analysis field again.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114047269A (en) * | 2022-01-13 | 2022-02-15 | 浙江湃肽生物有限公司南京分公司 | Detection method of acetyl hexapeptide-8 |
| CN114166976A (en) * | 2021-12-08 | 2022-03-11 | 桂林医学院 | Method for analyzing drug content in health product by tracing auxiliary agent |
| 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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2017
- 2017-02-16 CN CN201710083984.8A patent/CN107085032A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114166976A (en) * | 2021-12-08 | 2022-03-11 | 桂林医学院 | Method for analyzing drug content in health product by tracing auxiliary agent |
| CN114166976B (en) * | 2021-12-08 | 2024-02-27 | 桂林医学院 | Method for analyzing drug content in health care product by tracing auxiliary agent |
| CN114047269A (en) * | 2022-01-13 | 2022-02-15 | 浙江湃肽生物有限公司南京分公司 | Detection method of acetyl hexapeptide-8 |
| CN114047269B (en) * | 2022-01-13 | 2022-04-15 | 浙江湃肽生物有限公司南京分公司 | Detection method of acetyl hexapeptide-8 |
| 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 |
| CN115201385B (en) * | 2022-06-22 | 2024-01-09 | 河北医科大学 | Derivatization reagent for electrospray mass spectrometry detection for enabling amino small molecules to carry two charges, and preparation method and application thereof |
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Application publication date: 20170822 |