CN110286153A - A method of polypeptide drugs are detected based on tracer method - Google Patents
A method of polypeptide drugs are detected based on tracer method Download PDFInfo
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Abstract
The present invention provides a kind of method based on tracer method detection polypeptide drugs.Method includes the following steps: proteolytic cleavage: 20-50 mM/ls of dihydrogen sulfate sodium solution of pharmaceutical polypeptide is dissolved, the cosolvent Hapst SD that concentration is 0.2% and the FDAP that concentration is 5 mM/ls is added, 30-50 DEG C at a temperature of restore 1-3 hours, add the iodine Isopropamide that concentration is 20-50 mM/ls, dark place is placed in restore 1-3 hours, the tryptic digestion that pancreas enzyme-substrate (1:50, w/w) is added is stayed overnight；Step 2: polypeptide marker and trypsase inactivation: the peptide fragment solution after digestion being freeze-dried, then it re-dissolves, is shaken in ultrasonator after mixing, reacted 10 minutes, the iodine Isopropamide for finally adding 50-200 mM/ls is placed 1-3 hours in dark place.The method of the present invention has the advantages that efficient, labeling effciency is high.
Description
Technical field
The present invention relates to polypeptide drugs detection field, relate more specifically to that polypeptide can be detected with highly sensitive and accuracy
The tagging method of drug.
Background technique
Polypeptide is to be keyed a kind of compound formed by peptide by multiple amino acid, usually by 10-100 amino
Acid molecule composition, connection type is identical as protein, and relative molecular mass is lower than 10000.Polypeptide is prevalent in organism
Interior, the polypeptide found in vivo so far has reached tens of thousands of kinds, each system, organ, tissue in wide participation and adjusting body
With the functional activity of cell, play a significant role in vital movement.
In recent years, one of the hot spot that medicament research and development is had become using the polypeptide drugs that modern biotechnology synthesizes, because suitable
It should demonstrate,prove wide, highly-safe and significant in efficacy, be widely used to the pre- of the diseases such as tumour, hepatitis, diabetes, AIDS at present
Anti-, diagnosing and treating has wide development prospect.
Polypeptide drugs are a kind of specific drugs between traditional chemicals and macromolecular drug paper, have poison secondary
Act on the features such as low, dosage is few, bioactivity is strong, good effect.The synthesis class polypeptide drugs that the whole world has listed now have more than 60 kinds,
The synthesis polypeptide class drug included in domestic pharmacopeia has more than 30 kinds.The dosage form of polypeptide drugs is mostly injection-type and injection powder needle,
There are also novel dose, such as microsphere for injection, sustained release agent, nasal spray.
Protein and peptide drugs molecular structure, physicochemical property and in terms of with traditional small-molecule drug all
There is essential distinction, there is its own pharmacokinetic characteristics and rule in vivo.For example, compared with small-molecule drug,
The apparent volume of distribution (Vd) of protein and peptide drugs is usually smaller, and the ratio of Tissue and blood concentration is about 1%
~10%, lower about 0.1% in brain tissue.For new drug antineoplastic polypeptide mPEG-SC20k-HM-3, the Vd in SD rat body
It is 0.5%~5% in liver, kidney, the heart, spleen, lung and the ratio between muscle drug concentration and blood concentration for 0.11L/kg.
Radio isotope tracer technique has the characteristics that sensitivity is high, applied widely, easy to detect, is animal body
The prefered method of interior pharmacokinetic.Commonly labelled nuclide includes125I、3H etc., wherein125I because specific radioactivity activity compared with
High, half-life period is suitable for, it is simple and the most commonly used to mark.Yang Tingting etc. passes through chloramine-t method handle125I is tagged to scorpion active peptides
On tyrosine residue in ADWX-1 molecule, tracer has been obtained125I-ADWX-1.It is examined through radiochemical purity and bioactivity
It surveys, shows that its radiochemical purity is 96.77%, significant change does not occur for biological activity.In the detection of drug, radioactivity
Isotope tracer technique is often used in combination with acid precipitation method, SDS-PAGE, HPLC.Jiang Guohua etc. is to containing125The detection of I-NGF
After sample is separated by electrophoresis, ray signal detection is carried out, is obtained125I-NGF is in the intracorporal blood concentration of mouse, tissue point
The whole data such as cloth, excretion.
Compared with traditional small-molecule drug, the pharmacokinetic analysis of protein and peptide class drug is faced with more challenges.It should
Class drug is in vivo in metabolic process, can be in conjunction with many kinds of substance, and how such as receptor, antigen, endogenous binding protein are distinguished
Free drug is metabolized segment and combines drug concentration, is at present still a big difficulty of detection and analysis.Enzyme linked immunosorbent assay (ELISA)
(ELISA) be current protein and peptide drugs pharmacokinetic main method.Pharmacokinetics is carried out using ELISA method to comment
When valence, what is primarily investigated is the specificity of method.Investigation object includes drug homologous protein, plasma protein, closed protein etc..One
As in the case of, using monoclonal antibody detect specificity will much higher than use polyclonal antibody.It is past that conventional method prepares antibody
Toward needing 6~8 months, preparation time can be foreshortened to 1 week using novel phage antibody library technique；Radioactive isotope shows
Track technology has high sensitivity, after electrophoresis or HPLC combination, can effectively distinguish tracer drug and radioactivity is metabolized
Segment.This method has unique advantage in the research that drug entities are distributed and are drained.Specific activity appropriate is for radioactivity
The detection of isotope tracer technique is most important, and specific activity is too low to will affect detection sensitivity and accuracy, excessively high, can improve
It marks cost and generates radiation effect；LC-MS/MS has preferable selectivity and repeatability, and can to drug metabolite into
Row monitoring.Currently, this method is mainly used for, some structures are simple, the stronger polypeptide drug of stability.LC-MS/MS is to sample
Product purity requirement is higher, should avoid target molecule that aggregation, denaturation or degradation etc. occurs in preprocessing process；Living imaging skill
Art can dynamic changing process carries out real time monitor in vivo in vivo to target sample, does not need at cumbersome sampling and sample
Reason, is as a result detected intuitive, easy to operate.But, these novel developing techniques development are not mature enough at this stage, method it is sensitive
Degree, stability, reproducibility etc. are all difficult to meet pharmacokinetics testing requirements.
In conclusion when evaluating protein and peptide drugs Non-clinical Pharmacokinetics, it should be according to this kind of drug
Molecular structure, physicochemical property and Pharmacokinetic Characteristics formulate reasonable experimental program, selection analysis method appropriate, most
That a variety of methods are used in combination well, complement one another verifying, absorbed, be distributed in vivo with obtaining drug, Metabolism Excretion it is comprehensive
Authentic communication.
For this purpose, in the art, it is still desirable to which one kind can be in the method for quickly, efficiently and accurately detecting polypeptide drugs.
Summary of the invention
1. technical problems to be solved
In existing detection method, when using conventional method detection polypeptide drugs, it is easy by intermolecular in polypeptide
Disulfide bond interference.In addition, conventional use of polypeptide detection methods program is complicated at present, accuracy rate is not high and inefficiency.
2. technical solution
To solve the above problems, the present invention adopts the following technical scheme that.
On the one hand, the present invention provides a kind of method based on tracer method detection polypeptide drugs, the method packet
Include following steps: step 1: proteolytic cleavage: 20-50 mM/ls of dihydrogen sulfate sodium solution dissolution of pharmaceutical polypeptide being added
The FDAP that the cosolvent Hapst SD and concentration that concentration is 0.2% are 5 mM/ls, 30-50 DEG C at a temperature of restore 1-3
Hour, the iodine Isopropamide that concentration is 20-50 mM/ls is added, dark place is placed in and restores 1-3 hours, pancreatin-is added
The tryptic digestion of substrate (1:50, w/w) is stayed overnight；Step 2: polypeptide marker and trypsase inactivation: by the peptide fragment after digestion
Solution freeze-drying, is then directly added into16NH3It re-dissolves, is shaken in ultrasonator after mixing, reaction 10 minutes, most
The iodine Isopropamide for adding 50-200 mM/ls afterwards is placed 1-3 hours in dark place；3. the peptide fragment after pair inactivation utilizes
Then capillary electrophoresis separation is analyzed by mass spectrometry.
The method according to aforementioned aspects, the pharmaceutical polypeptide are antineoplastic polypeptide, are selected from Derkartin, ND1012
With one of PIDDA or a variety of.
The method according to aforementioned aspects, the concentration of the dihydrogen sulfate sodium are 20-40 mM/ls.
The method according to aforementioned aspects, when carrying out ultrasonic vibration, the frequency of ultrasonic wave is 20kHz-38kHz.
The butyraldehyde of 0.1-0.8% is added into solution when carrying out ultrasonic vibration for the method according to aforementioned aspects.
3. beneficial effect
Compared with the prior art, the present invention has the advantages that
(1) unexpectedly, the inventors discovered that, it is possible to provide a kind of compound comprising with labelled with radioisotope,
For the selection of candidate compound, and then pass through and examine them, such as examines its (internal) binding site or detection and each text
The relevant internal metabolic characteristics of library member, to determine the final result of each compound.This, which to avoid, needs in the choice phase by ordering
Single requirement for carrying out drug candidate selected by radio-labeled, effectively makes entire option program without stopping because of time-consuming synthesis
?.In addition, the drug candidate of metabolic characteristics needed for can excluding to lack in very early stage, to significantly reduce the selection method
Cost.
(2) by using method of the present invention, the disulfide bond interference in polypeptide detection process is effectively eliminated, from
And improve the sensitivity and accuracy of polypeptide detection.It is not limited to any theory, the present inventor has found in an experiment, is carrying out
When ultrasonic vibration, by adding butyraldehyde into solution, pancreatin can be inactivated, prevents polypeptide marker from losing, thus when reducing label
Between, labeling effciency is improved, the durability of marked product is enhanced, meets actual needs.
(3) method of the invention is simple and practical, has very good cost-effectiveness.
Detailed description of the invention
Each exemplary implementation scheme according to the present invention described below with reference to the accompanying drawings.Those skilled in the art
It will be understood that the purpose that the present invention provides attached drawing be intended to help its sufficiently, thoroughly understand exemplary implementation scheme of the invention
And embodiment, and be tantamount to limit the invention in any way, in attached drawing:
Fig. 1 is alpha-casein digestion peptide fragment16N label front and back MALDI-TOF-TOF-MS mass spectrogram A；
Fig. 2 is16NH3Alpha-casein digestion peptide fragment after label is in -98% water of 2% acetonitrile-containing in 0.5% formic acid solution
The figure of stability.
Specific embodiment
Below in conjunction with attached drawing of the present invention, technical scheme in the embodiment of the invention is clearly and completely described；It is aobvious
So, it will be understood by those skilled in the art that embodiment described herein is used for the purpose of illustrating the present invention
Technical solution, and have no intention to limit the invention in any way.
For this purpose, presently, there are aiming at the problem that, the present inventor to the detection of polypeptide carried out deeply and widely study, and
And the method that highly efficient labeling polypeptide drugs have successfully been obtained.Specific technical solution is as follows: step 1: proteolytic cleavage: drug is more
Peptide is dissolved with 20-50 mM/ls of dihydrogen sulfate sodium solution, and the cosolvent Hapst SD and concentration that addition concentration is 0.2% are 5
MM/l FDAP, 30-50 DEG C at a temperature of restore 1-3 hour, it is different for 20-50 mM/ls of iodine to add concentration
Propionamide is placed in dark place and restores 1-3 hours, and the tryptic digestion that pancreas enzyme-substrate (1:50, w/w) is added is stayed overnight；Step 2:
Polypeptide marker and trypsase inactivation: the peptide fragment solution after digestion is freeze-dried, is then directly added into16NH3It re-dissolves, mixes
It is shaken in ultrasonator after even, reacts 10 minutes, 50-200 mM/ls of iodine Isopropamide is finally added, in dark
It places 1-3 hours at place；3. the peptide fragment after pair inactivation utilizes capillary electrophoresis separation, then it is analyzed by mass spectrometry.
The principle illustrated the present invention below with reference to specific embodiment.Those skilled in the art will
Understand, provides the purpose of these embodiments just for the sake of illustrating the present invention principle, without constituting any limit to the present invention
System.
Material and reagent
Flight time tandem mass spectrometer is ionized using 4800 Matrix Assisted Laser Desorptions.Derkartin, ND1012 and
PIDDA, α Hydroxy-cinnamic acid (CHCA), thyroglobulin, alpha-casein (aldrich company, the U.S.)；Trypsase (the U.S.
Sigma) company, 97%NH3 (Qingdao pharmaceutical factory)；Three (2- carboxyethyl) phosphonium salt hydrochlorates (Aldrich)；16NH3Aqueous solution
Sigma Co., USA is purchased from Hapst SD；Other reagents are that domestic analysis is pure.
Embodiment 1:
Proteolytic cleavage is carried out first.By 20 mmol/L NH of Derkartin and alpha-casein albumen4HCO3After dissolution, add
Enter Hapst SD (final concentration 0.1%) and TCEP (final concentration 5mmol/L), in 56 DEG C of reduction 1h, adds IAA (final concentration 25
Mmol/L), open in dark place and restore 1h.The Trypsin digestion that pancreas enzyme-substrate (1: 50, w/w) is added is stayed overnight.
Then polypeptide drugs are marked, while inactivate pancreatin.It will be straight after the peptide fragment solution freeze-drying after digestion
Connect addition16NH3Weight is molten, shakes in ultrasonator after mixing, reacts 10 minutes.Finally add 50-200 mM/ls
The inactivation of iodine Isopropamide, after 37 DEG C of water-baths are incubated for 1h, microwave reaction 10min.IAA (final concentration 100mmol/L) is added,
1h is placed in dark place.
And then it is analyzed by mass spectrometry.Matrix uses CHCA.Instrument control software is 4800 Explorer TM
Software, data processing software are GPS Explorer TM software 2.0.The acquisition of first mass spectrometric data uses MS-
1kV reflective-mode, acceleration voltage 20kV, scanning range m/z 700~3500, laser energy 4500, every spectrogram cumulative 1500
It is secondary.Horse heart myoglobin pancreatin digestion peptide fragment carries out external standard correction to instrument as reference substance, calibrates to error≤0.1Da, phase
To standard deviation≤10 × 10- 6。
Embodiment 2:
Proteolytic cleavage is carried out first.By ND1012 and alpha-casein albumen 20mmol/L NH4HCO3After dissolution, it is added
Hapst SD (final concentration 0.1%) and TCEP (5 mmol/L of final concentration) adds IAA (final concentration in 55 DEG C of reduction 1h
30mmol/L), it opens in dark place and restores 1h.The Trypsin digestion that pancreas enzyme-substrate (1: 50, w/w) is added is stayed overnight.
Then polypeptide drugs are marked, while inactivate pancreatin.It will be straight after the peptide fragment solution freeze-drying after digestion
Connect addition16NH3Weight is molten, shakes in ultrasonator after mixing, reacts 10 minutes.Finally add 50-200 mM/ls
The inactivation of iodine Isopropamide, after 37 DEG C of water-baths are incubated for 1h, microwave reaction 10min.IAA (final concentration 100mmol/L) is added,
1h is placed in dark place.
And then it is analyzed by mass spectrometry.Matrix uses CHCA.Instrument control software is 4800 Explorer TM
Software, data processing software are GPS Explorer TM software 2.0.The acquisition of first mass spectrometric data uses MS-
1kV reflective-mode, acceleration voltage 20kV, scanning range m/z 700~3500, laser energy 4500, every spectrogram cumulative 1500
It is secondary.Horse heart myoglobin pancreatin digestion peptide fragment carries out external standard correction to instrument as reference substance, calibrates to error≤0.1Da, phase
To standard deviation≤10 × 10- 6.
Embodiment 3:
Proteolytic cleavage is carried out first.By PIDDA and alpha-casein albumen 20mmol/L NH4HCO3After dissolution, it is added
Hapst SD (final concentration 0.1%) and TCEP (5 mmol/L of final concentration) adds IAA (final concentration in 55 DEG C of reduction 1h
30mmol/L), it opens in dark place and restores 1h.The Trypsin digestion that pancreas enzyme-substrate (1: 50, w/w) is added is stayed overnight.
Then polypeptide drugs are marked, while inactivate pancreatin.It will be straight after the peptide fragment solution freeze-drying after digestion
Connect addition16NH3Weight is molten, shakes in ultrasonator after mixing, reacts 10 minutes.Finally add 50-200 mM/ls
The inactivation of iodine Isopropamide, after 37 DEG C of water-baths are incubated for 1h, microwave reaction 10min.IAA (final concentration 100mmol/L) is added,
1h is placed in dark place.
And then it is analyzed by mass spectrometry.Matrix uses CHCA.Instrument control software is 4800 Explorer TM
Software, data processing software are GPS Explorer TM software 2.0.The acquisition of first mass spectrometric data uses MS-
1kV reflective-mode, acceleration voltage 20kV, scanning range m/z 700~3500, laser energy 4500, every spectrogram cumulative 1500
It is secondary.Horse heart myoglobin pancreatin digestion peptide fragment carries out external standard correction to instrument as reference substance, calibrates to error≤0.1Da, phase
To standard deviation≤10 × 10- 6.
Embodiment 4
The labeling effciency of measurement enhancing protein digestion peptide fragment
Protein digestion peptide fragment16NH3The principle of labeling method is that enzyme-peptide fragment twice based on trypsase catalysis generation is multiple
Hydrate hydrolysis reaction.The reaction is quick dynamic reversible reaction.Therefore, labeling effciency is incomplete frequent occurrence and marks peptide fragment
The case where loss, efficiently control label and backcrossing inhibit the two links be the key that Success in Experiment.
It is effective to increase reaction efficiency by improving the degree of scatter and auxiliary heating of peptide fragment in the reaction system for this experiment
Improve labeling effciency in ground.After degree of scatter of the peptide fragment in system increases, be conducive to being completely exposed for peptide fragment C-terminal, increase and pancreas
Enzyme and16NH3Touch opportunity.Therefore, improve peptide fragment degree of scatter, label can be promoted to carry out to positive reaction direction.In an experiment,
Hapst SD cosolvent is added, the dissolubility of protein and polypeptide can be enhanced, improves degree of scatter, is conducive to16NH3Carry out the end C
The exchange reaction of carboxyl end group.In alpha-casein digestion peptide fragment16In N marked product mass spectrometry results, because being added
RapigestTM SF cosolvent, the labeling effciency of the peptide fragment of m/z 1660,1267 and 2316 significantly improve.And hydrotropy is not added
3 peptide fragments of agent group then mark not exclusively, however it remains a large amount of unlabelled16N peptide fragment.
Fig. 1 is alpha-casein digestion peptide fragment16N label front and back MALDI-TOF-TOF-MS mass spectrogram A.In conventional method,16NH3Label needs to be incubated in 37 DEG C of water-baths for 24 hours, to achieve the purpose that sufficiently to react.But nonetheless, mild reaction item
Part and longer reaction time are also difficult to ensure that label is complete.Protein digestion is more than 36 h plus the label spent time,
The process of experiment is lagged.And due to being placed in water bath for a long time, be easy because it is poorly sealed situations such as introduce16NH3, from
And cause label incomplete.This experiment has not only completely cut off moist environment by the way of ultrasonic vibration, but also only needs
10min can be marked completely.Compared with traditional heating water bath method, since microwave makes the heated more quick and equal of peptide fragment
It is even, therefore ideal label effect can be reached within a very short time.
16NH3Another of reaction is marked common problem encountered is that the case where label peptide fragment is easy to happen label loss.Due to the mark
Note reaction is a kind of reversible reaction, so if will16NH3Complete peptide fragment is marked to be dissolved in16NH3In, it remains to make16N marks peptide fragment
It is returned to16NH3Flag state.Pancreatin is the principal element for causing label loss, this research uses chemical ablation method, by highly concentrated
The go back original reagent and alkylating reagent of degree thoroughly inactivate pancreatin remaining in solution.As shown in Fig. 2, after pancreatin inactivation,16N
Label peptide fragment is obvious still without observing at the middle preservation of liquid chromatogram mobile phase (- 0.5% formic acid of -98% water of 2% acetonitrile) 12 days
Backcrossing product, stable label peptide fragment can carry out later separation analysis in liquid chromatogram.
Fig. 2 is16NH3Alpha-casein digestion peptide fragment after label is in -98% water of 2% acetonitrile-containing in 0.5% formic acid solution
Study on the stability.Complicated peptide fragment mixing substance markers result: Derkartin molecular weight is 660kDa.MALDI-TOF- after digestion
TOF mass spectrum can detect wherein 24 peptide fragments.Method after optimization is used for the label of the protein digestion peptide fragment, with the side of investigation
The validity and tolerance that method is applied in complicated peptide fragment mixture.It can be seen that this method for complicated peptide fragment mixture still
Satisfactory labeling effciency can be generated.
16NH3Label reaction is one of the isotope labelling method being most widely used in quantitative proteomics.But due to
The reaction is a kind of dynamic reversible reaction,16NH3Exchange is influenced by factors, and labeling effciency and inhibition backcrossing are always real
Thorny problem during testing still cannot steadily be widely applied in multiple laboratories at present.This research is marking and is inhibiting back
It is right in two key points to hand over16NH3Labeling method is optimized, and changes auxiliary heating method, thoroughly inactivates in conjunction with chemical method
Pancreatin blocks the generation of backcrossing reaction, reduces the label time, and labeling effciency significantly improves, and enhances the durable of marked product
Property, meet the needs of differential protein group analysis.
The above is only the preferable specific embodiments of the present invention；But scope of protection of the present invention is not limited thereto.It is any
Those familiar with the art in the technical scope disclosed by the present invention, according to the technique and scheme of the present invention and its improves
Design is subject to equivalent substitution or change, should be covered by the scope of protection of the present invention.
Claims (5)
1. a kind of method based on tracer method detection polypeptide drugs, which is characterized in that the described method comprises the following steps:
Step 1: proteolytic cleavage: 20-50 mM/ls of dihydrogen sulfate sodium solution of pharmaceutical polypeptide is dissolved, and it is 0.2% that concentration, which is added,
The FDAP that cosolvent Hapst SD and concentration are 5 mM/ls, 30-50 DEG C at a temperature of restore 1-3 hours, add dense
The iodine Isopropamide that degree is 20-50 mM/ls is placed in dark place and restores 1-3 hours, is added pancreas enzyme-substrate (1:50, w/w)
Tryptic digestion stay overnight；Step 2: polypeptide marker and trypsase inactivation: the peptide fragment solution after digestion being freeze-dried, so
After be directly added into16NH3Aqueous solution re-dissolves, and shakes in ultrasonator after mixing, reacts 10 minutes, finally adds
50-200 mM/ls of iodine Isopropamide is placed 1-3 hours in dark place；3. the peptide fragment after pair inactivation utilizes capillary electricity
Swimming separation, is then analyzed by mass spectrometry.
2. being selected from the method according to claim 1, wherein the pharmaceutical polypeptide is antineoplastic polypeptide
One of Derkartin, ND1012 and PIDDA or a variety of.
3. method according to claim 1 or 2, which is characterized in that the concentration of the dihydrogen sulfate sodium is 20-40 mmoles
You/liter.
4. method according to claim 1 or 2, which is characterized in that when carrying out ultrasonic vibration, the frequency of ultrasonic wave is
20kHz-38kHz。
5. method according to claim 1 or 2, which is characterized in that when carrying out ultrasonic vibration, 0.1- is added into solution
0.8% butyraldehyde.
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