CN112763636B - Peptide map analysis method - Google Patents
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- 238000004458 analytical method Methods 0.000 title claims abstract description 21
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- 235000019253 formic acid Nutrition 0.000 claims abstract description 20
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- 238000001514 detection method Methods 0.000 claims description 19
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- 108010033276 Peptide Fragments Proteins 0.000 claims description 10
- 101001018085 Lysobacter enzymogenes Lysyl endopeptidase Proteins 0.000 claims description 8
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- 239000003153 chemical reaction reagent Substances 0.000 claims description 8
- 238000010828 elution Methods 0.000 claims description 8
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- 238000012510 peptide mapping method Methods 0.000 claims description 7
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- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
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- 229960000789 guanidine hydrochloride Drugs 0.000 description 5
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- HQVFCQRVQFYGRJ-UHFFFAOYSA-N formic acid;hydrate Chemical compound O.OC=O HQVFCQRVQFYGRJ-UHFFFAOYSA-N 0.000 description 1
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Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
Abstract
The invention discloses a peptide map analysis method, which comprises the following steps: carrying out denaturation enzymolysis on the sample to obtain polypeptide fragments; detecting the polypeptide fragments by using UPLC-MS, and attributing the polypeptide fragments according to the collected peptide map data; the mobile phase of the UPLC-MS comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is a formic acid aqueous solution, and the mobile phase B is a formic acid acetonitrile solution; the UPLC-MS was gradient eluted using an ACQUITY UPLC HSS T3 column.
Description
Technical Field
The invention relates to the technical field of polypeptide analysis, in particular to a peptide map analysis method.
Background
Peptide map analysis can provide rich structural information on the confirmation of a primary structure, and a peptide map analysis method based on a liquid chromatography-mass spectrometry coupling technology is widely applied to the quality control of the existing biological medicines because the peptide map analysis method can be matched with a theoretical amino acid sequence. Reverse phase chromatography (RP-HPLC) is currently the main tool for the isolation and identification of polypeptides.
The C18 chromatographic column has wide application in peptide map analysis with its specific resolution, but some hydrophilic peptide fragments generated by restriction enzyme are difficult to be monitored on the C18 column. In addition, to obtain a good peak profile, a strong ion pairing reagent, trifluoroacetic acid (TFA), which can improve the peak profile, is usually added to the mobile phase of the peptide mapping analysis. In many cases, however, LC separation is often used in conjunction with ESI-MS in order to characterize a compound or CDR (complementary-determining region) peptide fragment in a peptide map, although strong ion pairing reagents (such as TFA) can cause a significant reduction in mass spectrum signal intensity.
Disclosure of Invention
Therefore, it is necessary to provide a peptide map analysis method for solving the problem of high difficulty in hydrophilic polypeptide detection in the conventional liquid chromatography-mass spectrometry separation.
A method of peptide map analysis comprising the steps of:
carrying out denaturation enzymolysis on the sample to obtain polypeptide fragments;
detecting the polypeptide fragments by using UPLC-MS, and attributing the polypeptide fragments according to the collected peptide map data;
the mobile phase of the UPLC-MS comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is a formic acid aqueous solution, and the mobile phase B is a formic acid acetonitrile solution;
the chromatographic column used by the UPLC-MS is an ACQUITY UPLC HSS T3 chromatographic column.
In one embodiment, the volume percentage of formic acid in the mobile phase a is 0.1% to 1%, and the volume percentage of formic acid in the mobile phase B is 0.1% to 1%.
In one embodiment, the UPLC-MS employs a gradient elution with parameters of:
in the first stage, the volume fraction of the mobile phase A is 100 percent, the volume fraction of the mobile phase B is 0, the time lasts for 4.5 min-5.5 min,
in the second stage, the volume fraction of the mobile phase A is 100 percent, the volume fraction of the mobile phase B is 0, the time lasts for 39.5 min-40.5 min,
in the third stage, the volume fraction of the mobile phase A is 35 to 45 percent, the volume fraction of the mobile phase B is 55 to 65 percent, the time lasts for 4.5 to 5.5min,
in the fourth stage, the volume fraction of the mobile phase A is 0, the volume fraction of the mobile phase B is 100 percent, the time lasts for 4.5min to 5.5min,
and in the fifth stage, the volume fraction of the mobile phase A is 0, the volume fraction of the mobile phase B is 100%, and the time lasts for 0.1-0.2 min.
In one embodiment, the flow rate of the mobile phase is 0.15mL/min to 0.25 mL/min.
In one embodiment, the detection column temperature of the UPLC-MS is 58-62 ℃.
In one embodiment, the mass spectrometry detection mode in the UPLC-MS is a positive ion mode.
In one embodiment, the analysis mode is a sensitivity mode.
In one embodiment, the mass spectrum cone hole voltage in the UPLC-MS is 30V-50V.
In one embodiment, the capillary voltage is between 2.00kV and 3.00 kV.
In one embodiment, the detector voltage is 2800V-3000V.
In one embodiment, the ionization source temperature is 100-.
In one embodiment, the desolvation gas temperature is 400-600 ℃.
In one embodiment, the column is of the specification
，2.1×150mm，1.8μm。
In one embodiment, the step of subjecting the sample to denaturing enzymatic digestion comprises:
mixing the sample with a denaturing reagent and then denaturing;
mixing the denatured sample with endopeptidase for enzymolysis, and mixing with acid to terminate enzymolysis reaction;
in one embodiment, the endopeptidase is selected from any one of Trypsin/Lys-C mix, Trypsin, Lys-C.
In one embodiment, the sample is a protein or polypeptide that generates a hydrophilic peptide fragment after denaturing enzymatic digestion.
According to the invention, an ACQUITY UPLC HSS T3 chromatographic column is selected, and a formic acid and formic acid acetonitrile system is selected as a mobile phase, and the result shows that the hydrophilic target peptide segment is well retained, and a better separation effect can be obtained. In addition, the method uses a formic acid and formic acid acetonitrile system, realizes the compatibility of liquid chromatography and mass spectrum, and realizes the unification of qualitative detection and quantitative detection methods for the sample.
Drawings
FIG. 1 is a comparison graph of chromatographic conditions 1-blank control and mixed protein A enzymatic hydrolysis according to one embodiment of the present invention;
FIG. 2 is a comparison graph of chromatographic conditions 2-blank control versus mixed protein A enzymatic hydrolysis according to one embodiment of the present invention;
FIG. 3 is a peptide fingerprint of a CSH C18 column and a HSS T3 column in accordance with one embodiment of the present invention;
FIG. 4 is a diagram illustrating the result of mass spectrometric identification of a target peptide fragment of hydrophilicity according to an embodiment of the present invention.
Detailed Description
To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
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 invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The embodiment of the invention provides a peptide map analysis method, which comprises the following steps:
carrying out denaturation enzymolysis on the sample to obtain polypeptide fragments;
and detecting the polypeptide fragments by using UPLC-MS, and attributing the polypeptide fragments according to the collected peptide map data.
The mobile phase of the UPLC-MS comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is a formic acid aqueous solution, and the mobile phase B is a formic acid acetonitrile solution; the chromatographic column used by the UPLC-MS is an ACQUITY UPLC HSS T3 chromatographic column.
According to the invention, an ACQUITY UPLC HSS T3 chromatographic column is selected, and a formic acid and formic acid acetonitrile system is selected as a mobile phase, and the result shows that the hydrophilic target peptide segment is well retained, and a better separation effect can be obtained. In addition, the method uses a formic acid and formic acid acetonitrile system, realizes the compatibility of liquid chromatography and mass spectrum, and realizes the unification of qualitative detection and quantitative detection methods for the sample.
The sample can be protein or polypeptide which can generate hydrophilic peptide segment after denaturation and enzymolysis.
In some embodiments, the step of subjecting the sample to denaturing enzymatic digestion comprises:
mixing the sample with a denaturing reagent and then denaturing;
mixing the denatured sample with endopeptidase for enzymolysis, and mixing with acid to terminate the enzymolysis reaction.
In some embodiments, the denaturing agent may be selected from rapidest SF or guanidine hydrochloride, preferably guanidine hydrochloride.
In some embodiments, the endopeptidase can be Trypsin/Lys-C mix, Trypsin, Lys-C. Preferably, the enzymolysis time is 3-4 hours.
In some embodiments, the volume percentage of formic acid in mobile phase a is 0.1% to 1%, and the volume percentage of formic acid in mobile phase B is 0.1% to 1%. Preferably, the percentage by volume of formic acid in mobile phase a is 0.1%. Preferably, the percentage by volume of formic acid in mobile phase B is 0.1%.
In some embodiments, UPLC-MS employs gradient elution. In one embodiment, the parameters of the gradient elution may be: the mobile phase A volume fraction is 100%, the mobile phase B volume fraction is 0 and lasts for 4.5 min-5.5 min, the mobile phase A volume fraction is 100%, the mobile phase B volume fraction is 0 and lasts for 39.5 min-40.5 min, the mobile phase A volume fraction is 35% -45%, the mobile phase B volume fraction is 55% -65% and lasts for 4.5 min-5.5 min, the fourth stage is that the mobile phase A volume fraction is 0, the mobile phase B volume fraction is 100% and lasts for 4.5 min-5.5 min, and the fifth stage is that the mobile phase A volume fraction is 0 and the mobile phase B volume fraction is 100% and lasts for 0.1 min-0.2 min.
In some embodiments, the parameters of the gradient elution are as shown in table 1 below:
TABLE 1
In some embodiments, the flow rate of the mobile phase is from 0.15mL/min to 0.25 mL/min.
In some embodiments, the detection column temperature of the UPLC-MS is from 58 ℃ to 62 ℃. Specifically, the temperature can be 58 ℃, 59 ℃, 60 ℃, 61 ℃ and 62 ℃.
In some embodiments, the chromatography column has a column specification of
，2.1×150mm，1.8μm。。
In some embodiments, the loading amount of the UPLC-MS is 8 μ L to 12 μ L. Specifically, the concentration may be 8. mu.L, 9. mu.L, 10. mu.L, 11. mu.L or 12. mu.L.
In some embodiments, the mass spectrometry detection mode in the UPLC-MS is a positive ion mode.
In some embodiments, the analysis mode is a sensitivity mode.
In some embodiments, the voltage of the mass-spectral cone holes in the UPLC-MS is from 30V to 50V.
In some embodiments, the capillary voltage is between 2.00kV and 3.00 kV.
In some embodiments, the detector voltage is 2800V-3000V.
In some embodiments, the ionization source temperature is 100-.
In some embodiments, the desolvation gas temperature is 400-600 ℃.
The embodiment of the invention also provides an application of the peptide graph analysis method of any one embodiment in the detection of the hydrophilic peptide fragment.
The following are specific examples.
Example (b):
in the embodiment, the peptide segment containing the flexible linker (Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Ala-Glu-Ser-Lys) is synthesized and mixed with bovine serum albumin to be used as a test sample, and the effect of separating and identifying the hydrophilic target peptide segment in the protein sample by using the peptide map analysis method is verified.
(1) The experimental reagent consumables and instruments are shown in tables 2 and 3.
TABLE 2 Experimental reagent consumables
TABLE 3 Experimental instruments
| Name of instrument | Instrument type |
| UPLC | Waters-H-class plus |
| Constant temperature metal bath | DTK200-4 of Hangzhou Mi Europe Instrument Co., Ltd |
| Vortex oscillator | Hangzhou Miou Instrument Co Ltd MIX-25P |
| High-speed refrigerated centrifuge | Thermo-LEGEND MICRO 21R |
| Centrifugal machine | Thermo-ST16R |
(2) Reagent preparation
1 XPBS solution (pH7.4):
weighing 8g NaCl, 0.2g KCl and 1.44g Na2HPO4，0.24g KH2PO4Adding 800mL of ultrapure water for dissolving, adjusting the pH to 7.4, metering the volume of the ultrapure water to 1L, mixing uniformly, and carrying out suction filtration for later use.
1M guanidine hydrochloride solution:
weighing 95.53g of guanidine hydrochloride and 1 XPBS, dissolving, fixing the volume to 1L, mixing uniformly and filtering for later use.
1mg/mL Trypsin/Lys-C mix solution:
taking a bottle of Trypsin/Lys-C mix (20 mu g/bottle), adding 20 mu L of purified water for full dissolution, subpackaging at-80 ℃ for preservation, and keeping the validity period for one year.
10% FA (stop solution):
put 900ul of ultrapure water into an EP tube, add 100ul of formic acid, and mix well for later use.
Mobile phase a (0.1% formic acid-water solution):
taking a clean measuring cylinder, measuring 1000mL of ultrapure water, adding 1mL of formic acid, uniformly mixing, and ultrasonically degassing for later use.
Mobile phase B (0.1% formic acid-acetonitrile solution):
taking a clean measuring cylinder, measuring 1000mL of acetonitrile, adding 1mL of formic acid, uniformly mixing, and ultrasonically degassing for later use.
(3) Sample preparation
Synthesizing a peptide fragment: the synthesized peptide fragment was taken, dissolved in 1 XPBS and diluted to a final concentration of 1 mg/mL.
BSA: BSA was taken and dissolved in 1 XPBS and diluted to a final concentration of 5 mg/mL.
(4) Experimental procedure
(4-1) denaturing enzymolysis of a sample:
6ug of NJP40735 synthetic peptide fragment (1mg/mL, 6. mu.L) and 100. mu.g BSA (5mg/mL, 20. mu.L) were taken, 80. mu.L of 1M guanidine hydrochloride was added, and mixed well. Adding 1: 20 Trypsin/Lys-C mix5.3uL (i.e., 5.3ug enzyme); the enzyme is cut for 4 hours at 37 ℃, finally 5uL 10% FA aqueous solution is added to stop the reaction, the vortex is repeated for 30s twice, the PH is less than 2, 13000rpm is 5min, the supernatant is taken, the sample is 10uL, and the UPLC-MS analysis is carried out.
(4-2) UPLC-MS detection:
UPLC-MS equipment is turned on: after starting the UPLC, configuring an instrument G2XS-Qtof + TUV + QSM + FTN, after starting the ion source, sequentially carrying out instrument inspection, checking a sensitivity mode (sensitivity mode) during correction, and paying attention to whether the argon is started or not before starting.
The mass spectrometry conditions are shown in table 4.
TABLE 4 Mass Spectrometry conditions
(4-3) sample detection:
according to the chromatographic conditions 1 and 2, firstly, the chromatographic column is balanced (80 min-100 min), and then the sample injection detection is carried out.
Chromatographic conditions 1: Column-ACQUITY UPLC Peptide CSH C18 Column
(2.1X 150mm, 1.7 μm) column temperature: 60.0 +/-2 ℃; flow rate: 0.2 mL/min; the detection wavelength is 214 nm; the sample injection volume is 10 mu L; run time 60min, mobile phase elution gradient as shown in table 5 below:
TABLE 5 chromatographic conditions 1
Chromatographic conditions 2: column-ACQUITY UPLC HSS T3 (2.1X 150mm, 1.8 μm), the packing of HSS T3 column pair is high strength silica gel particles, column temperature: 60.0 +/-2 ℃;
flow rate of mobile phase: 0.2 mL/min; the detection wavelength is 214 nm; the sample injection volume is 10 mu L; run time 60min, mobile phase elution gradient as shown in table 6 below:
TABLE 6 chromatographic conditions 2
(5) And (3) analyzing an experimental result:
and (4) processing a result:
and performing mirror image overlapping comparison on the mixed protein A in the chromatogram condition 1 and the chromatogram condition 2, and storing the result.
And (4) judging a result:
as can be seen from FIGS. 1 and 2, the blank control has no obvious absorption peak between 6min and 16 min;
the hydrophilic peptide of interest (GGGGGGGSGGGGSGGGGSAESK) was visible at 11.28min of chromatogram retention time of the ACQUITY UPLC HSS T3 column (chromatographic Condition 2), and was not found in the chromatogram of the CSH C18 column (chromatographic Condition 1) (FIG. 3) (FIG. 4).
And (4) analyzing results:
the obtained acid UPLC HSS T3 chromatographic column has better retention of hydrophilic target peptide (GGGGGGGSGGGGSGGGGSAESK) than the general C18 chromatographic column, and can obtain better separation effect. The method uses a formic acid system to realize the compatibility of liquid phase and mass spectrum. The method realizes the unification of qualitative detection and quantitative detection of the sample.
After the sample collection is finished, the ion source is standby, the ultraviolet lamp is turned off, the chromatographic column is washed for 20min by using 80% acetonitrile (or 80% methanol), and the flow rate is turned off.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (9)
1. A method of peptide map analysis comprising the steps of:
performing denaturing enzymolysis on a sample to obtain a polypeptide fragment, wherein the used enzyme is selected from Trypsin/Lys-C mix;
detecting the polypeptide fragments by using UPLC-MS, and attributing the polypeptide fragments according to the collected peptide map data;
the mobile phase of the UPLC-MS comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is a formic acid aqueous solution, and the mobile phase B is a formic acid acetonitrile solution;
the chromatographic column used by the UPLC-MS is an ACQUITY UPLC HSS T3 chromatographic column;
the sample is protein or polypeptide which can generate hydrophilic peptide fragment after denaturation and enzymolysis, and the hydrophilic peptide fragment is GGGGGGGSGGGGSGGGGSAESK;
the volume percentage of formic acid in the mobile phase A is 0.1-1%, and the volume percentage of formic acid in the mobile phase B is 0.1-1%;
the UPLC-MS adopts gradient elution, and the parameters of the gradient elution are as follows:
in the first stage, the volume fraction of the mobile phase A is 100 percent, the volume fraction of the mobile phase B is 0, the time lasts for 4.5 min-5.5 min,
in the second stage, the volume fraction of the mobile phase A is 100 percent, the volume fraction of the mobile phase B is 0, the time lasts for 39.5 min-40.5 min,
in the third stage, the volume fraction of the mobile phase A is 35 to 45 percent, the volume fraction of the mobile phase B is 55 to 65 percent, the time lasts for 4.5 to 5.5min,
in the fourth stage, the volume fraction of the mobile phase A is 0, the volume fraction of the mobile phase B is 100 percent, the time lasts for 4.5min to 5.5min,
in the fifth stage, the volume fraction of the mobile phase A is 0, the volume fraction of the mobile phase B is 100%, and the time lasts for 0.1-0.2 min;
the flow rate of the mobile phase is 0.15 mL/min-0.25 mL/min;
the detection column temperature of the UPLC-MS is 58-62 ℃;
the specification of the chromatographic column is
2.1×150mm，1.8μm。
2. The method for peptide mapping analysis of claim 1, wherein the mass spectrometric detection mode in UPLC-MS is positive ion mode.
3. The peptide graph analysis method of claim 2, wherein the analysis mode in UPLC-MS is a sensitivity mode.
4. The peptide graph analysis method of claim 2, wherein the mass-spectral cone voltage in UPLC-MS is 30V-50V.
5. The peptide mapping analysis method of claim 2, wherein the capillary voltage in UPLC-MS is 2.00kV-3.00 kV.
6. The peptide mapping analysis method of claim 2, wherein the UPLC-MS detector voltage is 2800V-3000V.
7. The peptide mapping analysis method of claim 2, wherein the temperature of the ionization source in the UPLC-MS is 100-130 ℃.
8. The peptide mapping analysis method of claim 2, wherein the temperature of desolvation gas in the UPLC-MS is 400-600 ℃.
9. The peptide mapping analysis method of claim 1, wherein the step of denaturing the sample comprises:
mixing the sample with a denaturing reagent and then denaturing;
mixing the denatured sample with endopeptidase for enzymolysis, and mixing with acid to terminate the enzymolysis reaction.
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|---|---|---|---|
| CN202011419204.0A CN112763636B (en) | 2020-12-07 | 2020-12-07 | Peptide map analysis method |
| PCT/CN2020/141486 WO2022121040A1 (en) | 2020-12-07 | 2020-12-30 | Peptide mapping method |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011419204.0A CN112763636B (en) | 2020-12-07 | 2020-12-07 | Peptide map analysis method |
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