Detection method of beta-enorphin

Abstract

The invention relates to a method for detecting beta-enorphin, and belongs to the technical field of protein analysis and detection. According to the detection method disclosed by the invention, the beta-enorphin is subjected to liquid mass spectrometry detection through the characteristic peptide fragment of the beta-enorphin. The mass spectrum parent ions and the mass spectrum daughter ions of the characteristic peptide fragment have better specificity, the accuracy of the detection method is improved, the mass spectrum parent ions and the mass spectrum daughter ions of the characteristic peptide fragment have stronger response signals, the signal-to-noise ratio (S / N) of the target peptide fragment is remarkably improved through concentration and purification, and the detection accuracy is improved. The accuracy, repeatability and sensitivity of the method for detecting the beta-enorphin in the sample are better.

Description

Technical Field

[0001] The present invention relates to the technical field of protein analysis and detection, and particularly relates to a method for detecting β-endorphin. Background Art

[0002] β-endorphin is widely distributed in the central nervous system and peripheral tissues, and participates in physiological processes such as pain regulation, stress response, and immune regulation. Abnormal expression levels are closely related to various diseases such as chronic pain, depression, and Parkinson's disease. Therefore, accurate detection and quantification of β-endorphin are of great significance for disease mechanism research, drug development, and clinical diagnosis. Endorphins include α-endorphin, β-endorphin, and γ-endorphin. These different subtypes have peptide segments with similar sequences. However, due to the extremely short half-lives (in the order of minutes) of α-endorphin and γ-endorphin, the actually detected stable concentration of endorphin is β-endorphin.

[0003] Currently, the detection of β-endorphin mainly relies on immunological methods, such as enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA). Although these techniques have the advantages of simple operation and low cost, their limitations are obvious: 1) Antibody dependence: Immunological methods rely on the specificity of antibodies. β-endorphin has a high sequence homology with other endogenous opioid peptides (such as enkephalin and dynorphin), which easily causes cross-reactions, resulting in false positives or quantitative deviations; 2) Limited detection range: The detection sensitivity of low-abundance β-endorphin (especially in plasma) is insufficient, and it is difficult to achieve linear quantification over a wide dynamic range; 3) Lack of polypeptide modification analysis: It is unable to distinguish post-translational modification forms of β-endorphin (such as phosphorylation and acetylation), affecting the accuracy of functional research.

[0004] Mass spectrometry (MS) has gradually become the core tool in the field of protein and polypeptide quantification due to its high resolution, high sensitivity, and multi-component parallel analysis capabilities. For example, liquid chromatography-tandem mass spectrometry (LC-MS / MS) can achieve precise analysis of target molecules through selective detection of characteristic peptide segments and isotope dilution quantification. However, existing mass spectrometry methods still face the following defects in the detection of target β-endorphin: 1) When detecting β-endorphin using existing liquid chromatography-mass spectrometry techniques, the ion suppression effect is often significant due to imperfect pretreatment methods or complex biological sample matrices, resulting in insufficient detection sensitivity and difficulty in accurately monitoring the content changes of β-endorphin in the physiological concentration range or the lower range (pg / mL level) caused by disease states; 2) Low ionization efficiency: The hydrophobicity and charge distribution characteristics of β-endorphin itself affect the electrospray ionization (ESI) efficiency. In addition, the complex components (such as high-concentration salt ions and metabolites) in clinical samples are prone to cause significant matrix effects, leading to unstable signal intensity and affecting the detection sensitivity and accuracy of mass spectrometry; 3) Lack of standardized procedures, resulting in the loss of target proteins during multiple operations and low detection amounts of target proteins, thus affecting subsequent research or result interpretation; 4) Conventional mass spectrometry acquisition modes (Full Scan / SIM) are difficult to effectively distinguish target characteristic ions from endogenous interfering substances in complex biological matrices, and the signal-to-noise ratio (S / N) decreases by 60%-80%, affecting the accurate quantification of low-abundance targets; 5) Existing technical processes are time-consuming (single-sample analysis time > 30 min) and cannot meet the requirements of high-throughput detection in clinical research (daily sample throughput < 50 cases); 6) Traditional sample pretreatment methods generally use acetonitrile to precipitate proteins. Macromolecular proteins (> 10 kDa) precipitate preferentially due to volume effects, while β-endorphin, as a small molecule protein (~3.5 kDa) with amphiphilicity, remains in the supernatant, resulting in incomplete protein precipitation. The remaining proteins in the supernatant lead to low detection results, and the residual solvent after using high-concentration acetonitrile affects the retention behavior of liquid chromatography.

[0005] Therefore, it is necessary to develop a mass spectrometry detection method that is efficient, stable, and suitable for clinical applications to achieve high-specificity quantification and analytical detection of β-endorphin, and to promote the monitoring, prediction, and diagnosis of related diseases. Summary of the Invention

[0006] The object of the present invention is to overcome the deficiencies of the prior art and provide a detection method for β-endorphin. The detection method of the present invention can detect β-endorphin in samples. The detection range of the detection method constructed by the present invention is 0.01 - 10 ng / mL, and the detection of low-concentration target sample can be achieved.

[0007] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides a method for detecting β-endorphin, and the detection method includes the following steps:

[0009] S1 Pretreat the sample:

[0010] S2 Detect the pretreated sample by a liquid chromatography-mass spectrometer to obtain the response signal of the β-endorphin characteristic peptide segment; wherein, the characteristic peptide segment of β-endorphin is YGGFMTSEK;

[0011] S3 Convert the response signal obtained by the characteristic peptide segment in the liquid chromatography-mass spectrometer in step S2 into the content of β-endorphin in the sample according to the standard curve.

[0012] The present invention obtains the characteristic peptide segment of β-endorphin by trypsin digestion, and its sequence is YGGFMTSEK. β-endorphin can be detected by liquid chromatography-mass spectrometry through this characteristic peptide segment. The mass spectrometry precursor ion and product ion of the characteristic peptide segment of the present invention have better specificity, which is beneficial to improving the accuracy of the detection method, and the mass spectrometry precursor ion and product ion of the characteristic peptide segment have stronger response signals, which can improve the accuracy, repeatability and sensitivity of the detection method for the target protein in the sample.

[0013] As a preferred embodiment of the first aspect of the present invention, the sample pretreatment method in step S1 includes the following steps:

[0014] I Ultrafilter and concentrate the sample, and collect the filtrate;

[0015] II Dialyze the filtrate to obtain the dialysate;

[0016] III Add a buffer solution to the dialysate and incubate with shaking;

[0017] IV Add a protein denaturant in step III and incubate;

[0018] V Add a protein stabilizer in step IV, incubate and then freeze-dry the sample;

[0019] VI Re-dissolve the freeze-dried sample in step V with a buffer solution, add trypsin for enzymatic digestion and then freeze-dry the sample.

[0020] The present invention uses trypsin to digest β-endorphin to produce a highly stable β-endorphin characteristic peptide segment, improving detection sensitivity and reproducibility. Centrifugal ultrafiltration and dialysis are used to remove most of the impurity proteins (albumin, immunoglobulin, fibrinogen, etc.) and salt ions in the biological matrix, greatly weakening the strong interference of the impurity proteins and salt ions on the target protein signal, and at the same time avoiding relying on high-cost immunoaffinity reagents and solid-phase extraction columns, which is easy to realize the screening and processing of a large number of samples.

[0021] The sample described in the present invention is a biological matrix sample, including blood, urine, saliva, milk, tears, cerebrospinal fluid, bile, gastric juice, feces, tissue homogenate, etc.

[0022] As a preferred embodiment of the first aspect of the present invention, the molecular weight cut-off for ultrafiltration in step I is 10 - 20 kDa; and / or, the molecular weight cut-off for dialysis in step II is 1 - 2 kDa.

[0023] As a preferred embodiment of the first aspect of the present invention, the molecular weight cut-off for ultrafiltration in step I is 10 kDa; and / or, the molecular weight cut-off for dialysis in step II is 2 kDa.

[0024] After optimization of the present invention, when the molecular weight cut-off for ultrafiltration is 10 kDa and the molecular weight cut-off for dialysis is 2 kDa, the treatment effect on the sample is better.

[0025] As a preferred embodiment of the first aspect of the present invention, the buffer solution is ammonium bicarbonate solution, the protein denaturant is dithiothreitol solution, and the protein stabilizer is iodoacetamide solution.

[0026] As a preferred embodiment of the first aspect of the present invention, the mass spectrometry conditions of the liquid chromatography - mass spectrometry in step S2 meet the following conditions:

[0027] (1) The parent ion of the characteristic peptide segment is 510.2, and the daughter ions are 136.1 - 221.1;

[0028] (2) The declustering voltage of the characteristic peptide segment is 80 - 90 V, and the collision energy is 29 - 34 eV.

[0029] As a preferred embodiment of the first aspect of the present invention, the mass spectrometry conditions of the liquid chromatography - mass spectrometry in step S2 meet the following conditions:

[0030] (1) The parent ion of the characteristic peptide segment is 510.2, and the daughter ion is 136.1;

[0031] (2) The declustering voltage of the characteristic peptide segment is 80 V, and the collision energy is 29 eV.

[0032] In the process of studying the sample detection method of the present invention, it is found that different mass spectrometry conditions will affect the subsequent detection results. Therefore, through optimization, it is found that when the parent ion of the characteristic peptide segment is 510.2, the daughter ion is 136.1, the declustering voltage of the characteristic peptide segment is 80 V, and the collision energy is 29 eV, the peak area is the largest and the effect is the best.

[0033] As a preferred embodiment of the first aspect of the present invention, the liquid chromatography conditions of the liquid chromatography - mass spectrometry in step S2 meet the following conditions:

[0034] (1) The aqueous phase of the mobile phase is a 0.1% formic acid aqueous solution, and the organic phase of the mobile phase is a 0.1% formic acid acetonitrile solution;

[0035] (2) The mobile phase gradient is as follows:

[0036] 0 - 0.50 min, the volume ratio of the aqueous phase to the organic phase is: 80%:20%;

[0037] 0.50 - 1.8 min, the volume ratio of the aqueous phase to the organic phase is: (50% - 80%):(20% - 50%);

[0038] 1.8 - 1.81 min, the volume ratio of the aqueous phase to the organic phase is: (15% - 50%):(50% - 85%);

[0039] 1.81 - 2.8 min, the volume ratio of the aqueous phase to the organic phase is: 15%:85%;

[0040] 2.8 - 2.81 min, the volume ratio of the aqueous phase to the organic phase is: (15% - 80%):(20% - 85%);

[0041] 2.81 - 3.8 min, the volume ratio of the aqueous phase to the organic phase is: 80%:20%.

[0042] In the process of studying the sample detection method of the present invention, it is found that different liquid phase conditions will affect the subsequent detection results. Therefore, through optimization, it is found that when the aqueous phase of the mobile phase is a 0.1% formic acid aqueous solution and the organic phase is a 0.1% formic acid acetonitrile solution, the peak area is the largest and the effect is the best.

[0043] As a preferred embodiment of the first aspect of the present invention, the liquid phase conditions of the liquid chromatograph - mass spectrometer in step S2 satisfy the following conditions:

[0044] (1) Chromatographic column: Waters ACQUITY UPLC BEH C8 1.7μm (2.1×100mm);

[0045] (2) Column temperature: 40°C; Autosampler temperature: 15°C;

[0046] (3) Flow rate: 0.3 mL / min;

[0047] (4) Injection volume: 10 μL.

[0048] As a preferred embodiment of the first aspect of the present invention, the mass spectrometry conditions of the liquid chromatograph - mass spectrometer in step S2 satisfy the following conditions:

[0049] (1) Ion source: Electrospray ionization source (ESI);

[0050] (2) Ion source temperature (°C): 550;

[0051] (3) Inlet voltage (KV): 10;

[0052] (4) Gas1 (L·h -1 ): 50;

[0053] (5) Gas2 (L·h -1 ): 50;

[0054] (6) Curtain Gas (L·h -1 ): 40;

[0055] (7) Collision Gas (L·h -1 ): Medium;

[0056] (8) Ionization mode: Positive ion;

[0057] (9) Signal acquisition mode: MRM.

[0058] As a preferred embodiment of the first aspect of the present invention, the sample concentration of the standard curve in step S2 is 0.01 - 10 ng / mL.

[0059] As a preferred embodiment of the first aspect of the present invention, it further includes the step of preparing a quality control sample, and the concentration of the quality control sample is 0.1 - 6 ng / mL.

[0060] In a second aspect, the present invention provides the application of the characteristic peptide segment YGGFMTSEK in the method for detecting β - endorphin by liquid chromatography - mass spectrometry.

[0061] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0062] 1. Achieve high - precision detection under complex biological sample systems: Screen characteristic ion pairs to avoid cross - interference of spectral signals, so that the quantitative repeatability (RSD) of β - endorphin all meets ≤ 15%.

[0063] 2. Ultra - sensitive detection of low - concentration proteins in samples: Combine ultrafiltration and dialysis operations before enzymatic digestion to achieve efficient extraction of β - endorphin and removal of impurities, so that the detection limit of proteins is as low as 10 pg / mL (~2.9 pM), which is at least 20 times higher than the β - endorphin detection limit of existing detection methods, and can accurately complete the quantification of proteins with extremely low abundance in complex clinical samples.

[0064] 3. Easy clinical translation: Optimize the standard operating procedures of the conventional LC-QqQ-MS platform. The detection time of target substances for each sample can be reduced to <3 min, the retention time of characteristic peptides is ~1.8 min, and the retention time of full-length peptides is ~2.5 min. Rapid detection of samples can be achieved, supporting a throughput of ~20 samples per hour. With pre-installed reagent kits and automated analysis modules, it significantly reduces labor and time costs compared to traditional mass spectrometry solutions.

[0065] 4. Wide range of applicable scenarios: This method is applicable to various precious and complex biological samples such as cerebrospinal fluid, plasma, and serum. The quantitative linear range is 0.01 ng / mL–10 ng / mL (r>0.99), which can meet the needs of clinical diagnosis and pathological research, and provide a general technology for scientific research and clinical diagnosis of various diseases such as chronic pain, depression, and Parkinson's disease.

[0066] 5. Achieve low-cost detection: Do not rely on antibodies, immunoaffinity reagents, and solid-phase extraction columns with high costs during sample pretreatment. Ultrafiltration and dialysis devices can be reused, the processing process is simple and convenient, and it greatly saves the costs of detection and application. It is suitable for large-scale sample screening and clinical promotion, shortens the time cost of sample processing, and provides significant economic benefits and environmental protection strategies. Description of the Drawings

[0067] Figure 1 It is the standard curve graph of the characteristic peptide segment YGGFMTSEK of β-endorphin (parent ion 510.2, daughter ion 136.1);

[0068] Figure 2 It is the standard curve graph of the characteristic peptide segment YGGFMTSEK of β-endorphin (parent ion 510.2, daughter ion 221.1);

[0069] Figure 3 It is the standard curve graph of the full-length peptide segment YGGFMTSEKSQTPLVTLFKNAIIKNAYKKGE of β-endorphin (parent ion 586.5, daughter ion 120.1);

[0070] Figure 4 It is the standard curve graph of the full-length peptide segment YGGFMTSEKSQTPLVTLFKNAIIKNAYKKGE of β-endorphin (parent ion 586.5, daughter ion 129.1);

[0071] Figure 5 It is the standard curve graph of the full-length peptide segment YGGFMTSEKSQTPLVTLFKNAIIKNAYKKGE of β-endorphin (parent ion 703.6, daughter ion 136.1);

[0072] Figure 6It is the standard curve graph of the full-length peptide of β-endorphin YGGFMTSEKSQTPLVTLFKNAIIKNAYKKGE (parent ion 703.6, daughter ion 120.1);

[0073] Figure 7 It is the standard curve graph of the full-length peptide of β-endorphin YGGFMTSEKSQTPLVTLFKNAIIKNAYKKGE (parent ion 703.6, daughter ion 129.1);

[0074] Figure 8 It is the detection result graph of different ion pairs of the characteristic peptide YGGFMTSEK of β-endorphin. Detailed implementation manners

[0075] To better illustrate the purpose, technical solution and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0076] Embodiment 1

[0077] This embodiment provides a synchronous detection method for β-endorphin, including the following steps:

[0078] S1 Pretreat the sample, including the following steps:

[0079] I Ultrafilter and concentrate the sample, and collect the filtrate;

[0080] II Dialyze the filtrate to obtain the dialysate;

[0081] III Add a buffer solution to the dialysate and incubate with shaking;

[0082] IV Add a protein denaturant in step III and incubate;

[0083] V Add a protein stabilizer in step IV, incubate and then lyophilize the sample;

[0084] VI Re-dissolve the lyophilized sample in step V with a buffer solution, add trypsin for enzymatic digestion and then lyophilize the sample.

[0085] S2 Detect the pretreated sample by a liquid chromatography-mass spectrometer to obtain the response signal of the characteristic peptide of β-endorphin; wherein, the characteristic peptide of β-endorphin is YGGFMTSEK;

[0086] The mass spectrometry conditions of the liquid chromatography-mass spectrometer in step S2 meet the following conditions:

[0087] (1) The parent ion of the characteristic peptide is 510.2, and the daughter ions are 136.1 - 221.1;

[0088] (2) The declustering voltage of the characteristic peptide segment is 80 - 90 V, and the collision energy is 29 - 34 eV;

[0089] (3) Ion source: Electrospray ionization source (ESI);

[0090] (4) Ion source temperature (°C): 550;

[0091] (5) Entrance voltage (KV): 10;

[0092] (6) Gas1 (L·h -1 ): 50;

[0093] (7) Gas2 (L·h -1 ): 50;

[0094] (8) Curtain Gas (L·h -1 ): 40;

[0095] (9) Collision Gas (L·h -1 ): Medium;

[0096] (10) Ionization mode: Positive ion;

[0097] (11) Signal acquisition mode: MRM.

[0098] The liquid phase conditions of the liquid mass spectrometer in step S2 satisfy the following conditions:

[0099] (1) Mobile phase: Aqueous phase is 0.1% formic acid aqueous solution, and organic phase is 0.1% formic acid acetonitrile solution;

[0100] (2) Mobile phase gradient is:

[0101] 0 - 0.50 min, the volume ratio of the aqueous phase to the organic phase is: 80%:20%;

[0102] 0.50 - 1.8 min, the volume ratio of the aqueous phase to the organic phase is: (50% - 80%):(20% - 50%);

[0103] 1.8 - 1.81 min, the volume ratio of the aqueous phase to the organic phase is: (15% - 50%):(50% - 85%);

[0104] 1.81 - 2.8 min, the volume ratio of the aqueous phase to the organic phase is: 15%:85%;

[0105] 2.8 - 2.81 min, the volume ratio of the aqueous phase to the organic phase is: (15% - 80%):(20% - 85%);

[0106] 2.81 - 3.8 min, the volume ratio of the aqueous phase to the organic phase is: 80%: 20%.

[0107] (3) Chromatographic column: Waters ACQUITY UPLC BEH C8 1.7 μm (2.1×100 mm);

[0108] (4) Column temperature: 40 °C; Autosampler temperature: 15 °C;

[0109] (5) Flow rate: 0.3 mL / min;

[0110] (6) Injection volume: 10 μL.

[0111] S3 Convert the response signals obtained for the characteristic peptide segments in step S2 in the liquid chromatograph - mass spectrometer into the content of β - endorphin in the sample according to the standard curve.

[0112] Preferably, the molecular weight cut - off for ultrafiltration in step I is 10 - 20 kDa; and / or, the molecular weight cut - off for dialysis in step II is 1 - 2 kDa.

[0113] More preferably, the molecular weight cut - off for ultrafiltration in step I is 10 kDa; and / or, the molecular weight cut - off for dialysis in step II is 2 kDa.

[0114] Preferably, the buffer solution is ammonium bicarbonate solution, the protein denaturant is dithiothreitol solution, and the protein stabilizer is iodoacetamide solution.

[0115] Preferably, the mass spectrometry conditions of the liquid chromatograph - mass spectrometer in step S2 satisfy the following conditions:

[0116] (1) The precursor ion of the characteristic peptide segment is 510.2, and the product ion is 136.1;

[0117] (2) The declustering voltage of the characteristic peptide segment is 80 V, and the collision energy is 29 eV.

[0118] I. Specific experimental methods:

[0119] 1. Preparation of main reagents

[0120] 1.1 Preparation of artificial blank plasma: Dissolve 10.00 mg of bovine serum albumin (BSA) in 100.0 mL of physiological saline, and fully vortex and mix to obtain artificial blank plasma (blank matrix).

[0121] 1.2 β - endorphin stock solution: Take 20 μg of β - endorphin standard, dissolve it in 1 mL of blank matrix to 20 μg / mL as the stock solution, and store it at - 20 °C after aliquoting.

[0122] 1. 3β - Endorphin intermediate working solution: Take 10 μL of the β - endorphin stock solution, add 990 μL of blank matrix and dissolve it to 200 ng / mL as the intermediate working solution. After aliquoting, store it at -20 °C.

[0123] 1.4 Preparation of aqueous phase: Mix formic acid and water in a volume ratio of 1:1000, and prepare it according to this ratio when in use.

[0124] 1.5 Preparation of organic phase: Mix formic acid and acetonitrile in a volume ratio of 1:1000, and prepare it according to this ratio when in use.

[0125] 1.6 Preparation of standard curve: Dilute the β - endorphin intermediate working solution with blank matrix; the specific dilution method is shown in Table 1, and prepare it according to the ratio when in use.

[0126] 1.7 Preparation of quality control samples: Dilute the β - endorphin intermediate working solution with blank matrix, and the specific dilution method is shown in Table 2, and prepare it according to the ratio when in use.

[0127] Table 1 Preparation table of standard curve samples

[0128]

[0129]

[0130] Table 2 Preparation table of quality control samples

[0131]

[0132] 2. Determination of the pretreatment method for the samples to be tested (endorphin prepared with blank matrix, concentration from 0.01 to 10 ng / mL):

[0133] 2.1 Different pretreatment methods

[0134] Pretreatment method 1:

[0135] Sample pretreatment to obtain β - endorphin - specific peptide segments through denaturation and enzymatic digestion, including the following steps:

[0136] (1) Take 1000 μL of the sample in a 10 kDa cut - off ultrafiltration tube and centrifuge (3500 g / 15 min) to concentrate and remove most of the miscellaneous proteins, and collect the filtrate in the outer tube;

[0137] (2) Place the filtrate in the outer tube in a 2 kDa dialysis device and dialyze at 4 °C for 4 h to remove high - concentration salts;

[0138] (3) Add 50 μL of 100 mM ammonium bicarbonate solution and incubate with shaking at 90 °C for 25 min;

[0139] (4) Add 20 μL of 10 mM dithiothreitol (DTT) solution and incubate at 60 °C for 1 h;

[0140] (5) Add 40 μL of 100 mM iodoacetamide solution, incubate in the dark at room temperature for 30 min, and lyophilize the sample;

[0141] (6) Reconstitute with 200 μL of 100 mM ammonium bicarbonate solution, add 50 μL of 0.2 μg / μL trypsin and mix well, incubate at 37 °C overnight, and terminate the enzymatic digestion reaction with 0.1% formic acid in water, then lyophilize the sample.

[0142] In this example, the characteristic peptide segment sequence for detecting β - endorphin by trypsin hydrolysis is: YGGFMTSEK (SEQ ID NO: 1).

[0143] Pretreatment method 2:

[0144] Sample treatment of full - length β - endorphin: The sequence of the full - length β - endorphin peptide segment is: YGGFMTSEKSQTPLVTLFKNAIIKNAYKKGE (SEQ ID NO: 2).

[0145] Denaturation treatment of full - length β - endorphin sample

[0146] (1) Take 1000 μL of the sample in a 10 kDa cut - off ultrafiltration tube and centrifuge (3500 g / 15 min) to concentrate and remove most of the miscellaneous proteins, and collect the filtrate in the outer tube;

[0147] (2) Place the filtrate in the outer tube in a 2 kDa dialysis device and dialyze at 4 °C for 4 h to remove high - concentration salts;

[0148] (2) Add 50 μL of 100 mM ammonium bicarbonate solution and incubate with shaking at 90 °C for 25 min;

[0149] (3) Add 20 μL of 10 mM dithiothreitol (DTT) solution and incubate at 60 °C for 1 h;

[0150] (4) Add 40 μL of 100 mM iodoacetamide solution, incubate in the dark at room temperature for 30 min, and lyophilize the sample.

[0151] Pretreatment method 3:

[0152] Sample treatment of full - length β - endorphin: The sequence of the full - length β - endorphin peptide segment is: YGGFMTSEKSQTPLVTLFKNAIIKNAYKKGE.

[0153] b) No need to denature the full - length β - endorphin sample

[0154] (1) Take 1000 uL of the sample and centrifuge it (3500 g / 15 min) in a 10 kDa cut-off ultrafiltration tube to concentrate and remove most of the miscellaneous proteins, and collect the filtrate in the outer tube;

[0155] (2) Place the filtrate in the outer tube in a 2 kDa dialysis device and dialyze it at 4 °C for 4 h to remove the high-concentration salts, and lyophilize the sample.

[0156] 2.2 Influence of different pretreatment methods on the detection results

[0157] Detect the samples with the above different pretreatment methods by liquid chromatography-mass spectrometry, record the chromatographic peak areas of the target peptides, perform linear regression with the peak area y corresponding to its concentration x, obtain the regression equation of the standard curve, and the weighting coefficient is 1 / x 2 , and the inspection results are shown in the following table. The linear range of the detection method constructed in the present invention is 0.01 - 10 ng / mL, and its standard curve is as Figure 1-7 shown, and the detection results of the linear range are as follows in the table:

[0158] Table 3 Investigation results of the linear range of pretreatment method 1

[0159]

[0160]

[0161] Table 4 Investigation results of the linear range of pretreatment method 2

[0162]

[0163]

[0164] From the above table and Figure 1-7 it can be seen that the linearity of pretreatment methods 2 and 3 is poor (r < 0.99) in the detection range of 0.01 - 10 ng / mL, which is inferior to pretreatment method 1. Finally, pretreatment method 1 is selected as the sample pretreatment method of the present invention.

[0165] 2.3 Investigation on the stability of the pretreated samples

[0166] 2.3.1 Short-term stability of the samples placed at room temperature

[0167] Take 3 concentration quality control samples of LQC, MQC, and HQC, pretreat them using the above pretreatment method 1, store them at room temperature for 12 hours, and re-dissolve the samples with 100 uL of 0.1% formic acid aqueous solution during injection. Record the peak areas of the target peptides, calculate the protein concentration from the standard curve of the same day, and compare it with the labeled concentration. The average deviation of the accuracy is within ±15% of the labeled concentration. The investigation results are shown in the following table.

[0168] Table 6 Results of short-term stability

[0169]

[0170] 2.3.2 Long-term storage stability of samples

[0171] Take the quality control samples at 3 concentrations of LQC, MQC, and HQC prepared. After pretreatment using the pretreatment method, store them frozen at -80°C for 1 month. When injecting samples, reconstitute the samples with 100 μL of 0.1% formic acid aqueous solution. Record the peak area of the target peptide segment, calculate the protein concentration from the standard curve of the same day, and compare it with the labeled concentration. The average deviation of accuracy is within ±15% of the labeled concentration. The inspection results are shown in the following table.

[0172] Table 7 Long-term stability results

[0173]

[0174] It can be seen from Tables 6 and 7 above that the pretreatment method 1 of the present invention also has the effect of improving the stability of samples. After being stored frozen at -80°C for 1 month and stored at room temperature for 12 hours, the average deviation of accuracy of the samples is within ±15% of the labeled concentration.

[0175] 3. Determination of mass spectrometry chromatographic conditions

[0176] 3.1 Influence of different peptide segments and ion pairs on detection results

[0177] In LC-MS / MS detection, the selection of peptide segments and ion pairs has an important influence on the method sensitivity. During the optimization of mass spectrometry parameters, the characteristic peptide segments and ion pairs were selected and optimized. The candidate conditions are as follows in the table:

[0178] Table 8 Different ion information of peptide segments is as follows in the table:

[0179]

[0180] Results, as Figure 8 shown, when the ion pair of the characteristic peptide segment is the parent ion 510.2 and the daughter ion 136.1, the response and sensitivity are higher. Finally, the parent ion 510.2 and the daughter ion 136.1 of the characteristic peptide segment are selected as the ion pair of the present invention.

[0181] 3.2 Influence of different declustering voltages and collision energies on detection results

[0182] In LC-MS / MS detection, the declustering voltage and collision energy directly affect the loss and fragmentation degree of ions during the process of entering the mass spectrometer, and have an important influence on the method sensitivity. During the optimization of mass spectrometry parameters, for the above-determined optimal ion pair, optimize its declustering voltage and collision energy. The candidate conditions are as follows in the table:

[0183] Optimization of Mass Spectrometry Parameters for Characteristic Peptide Fragments

[0184]

[0185] As can be seen from the above table, when the declustering voltage of the characteristic peptide fragment is 80 V and the collision energy is 29 eV, the peak area is the largest and the effect is the best. Finally, this condition is determined as the declustering voltage and collision energy of the present invention.

[0186] 3.3 Other Mass Spectrometry Conditions:

[0187] Mass Spectrometer: SCIEX Triple Quad 6500+;

[0188] Ion Source: Electrospray Ionization Source (ESI);

[0189] Ion Source Temperature (°C): 550;

[0190] Entrance Voltage (KV): 10;

[0191] Gas1 (L·h -1 ): 50;

[0192] Gas2 (L·h -1 ): 50;

[0193] Curtain Gas (L·h -1 ): 40;

[0194] Collision Gas (L·h -1 ): Medium;

[0195] Ionization Mode: Positive Ion;

[0196] Signal Acquisition Mode: MRM.

[0197] 4. Determination of Liquid Chromatography Conditions

[0198] 4.1 Selection of Chromatographic Column: Waters ACQUITY UPLC BEH C8 1.7μm (2.1×100mm)

[0199] 4.2 Selection of Mobile Phase: The candidate mobile phase components are as follows in the table:

[0200] Table 10 Candidate Mobile Phase Components

[0201] Candidate conditions Aqueous phase Organic phase Condition 1 0.1% formic acid aqueous solution 0.1% formic acid acetonitrile solution Condition 2 0.1% formic acid aqueous solution Acetonitrile Condition 3 0.1% ammonia water Acetonitrile

[0202] Table 11 Comparison of Detection Areas of Characteristic Peptide Fragments under Different Mobile Phase Conditions

[0203] Candidate conditions Characteristic peptide peak area Condition 1 12299406 Condition 2 9854651 Condition 3 2145805

[0204] As can be seen from the above peak areas, when Condition 1 was selected, the peak area of the characteristic peptide segment was significantly higher than that of other conditions. Therefore, Condition 1 was finally selected as the mobile phase of the present invention.

[0205] 4.3 Other liquid phase conditions

[0206] Chromatographic column: Waters ACQUITY UPLC BEH C8 1.7μm (2.1×100mm);

[0207] Column temperature: 40°C; Autosampler temperature: 15°C;

[0208] Flow rate: 0.3 mL / min;

[0209] Injection volume: 10 μL;

[0210] Aqueous phase: 0.1% formic acid aqueous solution; Organic phase: 0.1% formic acid acetonitrile solution;

[0211] Gradient is shown in Table 12.

[0212] Table 12 Mobile phase gradient 1

[0213] Time (min) Flow rate (mL·min-1) Aqueous phase %(V) Organic phase %(V) 0.00 0.3 80 20 0.50 0.3 80 20 1.8 0.3 50 50 1.81 0.3 15.0 85.0 2.8 0.3 15.0 85.0 2.81 0.3 80 20 3.8 0.3 80 20

[0214] 5. Precision and accuracy evaluation

[0215] Samples at three levels of LQC, MQC, and HQC for quality control were pretreated and then injected; Detection was carried out for at least two days, with 3 replicates for each concentration in each batch. Requirements: The mean accuracy deviation within and between batches is within ±15% of the labeled value; The precision %CV within and between batches is within 15%. The inspection results are shown in the following table.

[0216] Table 13 Inspection results of precision and accuracy

[0217]

[0218]

[0219]

[0220] II. Application of the detection method

[0221] Extract the plasma samples of the subjects for detection, and the detection steps are as follows:

[0222] 1. Pretreat the plasma samples, and use Treatment Method 1 for pretreatment. 2. Reconstitute the pretreated plasma samples with 100 μL of 0.1% formic acid aqueous solution;

[0223] 3. Perform LC-MS / MS detection on the samples in Step 2:

[0224] The liquid chromatography conditions are as follows:

[0225] Column: Waters ACQUITY UPLC BEH C8 1.7μm (2.1×100mm) Mobile phase: Aqueous phase: 0.1% formic acid aqueous solution; Organic phase: 0.1% formic acid acetonitrile solution; Elution program: As shown in Table 12.

[0226] Column temperature: 40°C; Autosampler temperature: 15°C;

[0227] Flow rate: 0.3 mL / min;

[0228] Injection volume: 10 μL.

[0229] The mass spectrometry conditions are as follows:

[0230] Mass spectrometer: SCIEX Triple Quad 6500+

[0231] Ion source: Electrospray ionization source (ESI)

[0232] Ion source temperature (°C): 550

[0233] Entrance voltage (KV): 10

[0234] Gas1 (L·h -1 ): 50

[0235] Gas2 (L·h -1 ): 50

[0236] Curtain Gas (L·h -1 ): 40

[0237] Collision Gas (L·h -1 ): Medium

[0238] Ionization mode: Positive ion

[0239] Signal acquisition mode: MRM

[0240] The ion pair of characteristic peptide 1 is parent ion 510.2 and daughter ion 136.1;

[0241] The declustering voltage is 80 V and the collision energy is 29 eV.

[0242] The detection results are shown in the following table:

[0243] Table 14 Detection results of β-endorphin in the sample

[0244]

[0245] As can be seen from Table 14, the inspection method constructed by the present invention can separate and quantitatively detect β-endorphin in plasma samples, and the detection method of the present invention can complete the detection within a short time (3 - 4 minutes), greatly shortening the detection time and improving the detection efficiency.

[0246] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A detection method for β-endorphin, characterized in that, The detection method includes the following steps: S1 Pre-treat the sample: S2 Detect the pre-treated sample with a liquid chromatography-mass spectrometer to obtain the response signal of the β-endorphin characteristic peptide fragment; wherein, the characteristic peptide fragment of β-endorphin is YGGFMTSEK; S3 Convert the response signal of the characteristic peptide fragment obtained in step S2 in the liquid chromatography-mass spectrometer into the content of β-endorphin in the sample according to the standard curve.

2. The detection method according to claim 1, characterized in that The sample pre-treatment method in step S1 includes the following steps: I Ultrafilter and concentrate the sample, and collect the filtrate; II Dialyze the filtrate to obtain the dialysate; III Add a buffer solution to the dialysate and incubate with shaking; IV Add a protein denaturant in step III and incubate; V Add a protein stabilizer in step IV, incubate, and then lyophilize the sample; VI Re-dissolve the lyophilized sample in step V with a buffer solution, add trypsin for enzymatic digestion, terminate the reaction with formic acid, and then lyophilize the sample.

3. The detection method according to claim 2, wherein In step I, the ultrafiltration cut-off molecular weight is 10-20 kDa; and / or, in step II, the dialysis cut-off molecular weight is 1-2 kDa.

4. The detection method according to claim 1, wherein The mass spectrometry conditions of the liquid chromatography-mass spectrometer in step S2 meet the following conditions: (1) The parent ion of the characteristic peptide fragment is 510.2, and the daughter ions are 136.1-221.1; (2) The declustering voltage of the characteristic peptide fragment is 80-90 V, and the collision energy is 29-34 eV.

5. The detection method according to claim 1, characterized in that The liquid chromatography conditions of the liquid chromatography-mass spectrometer in step S2 meet the following conditions: (1) The aqueous phase of the mobile phase is a 0.1% formic acid aqueous solution, and the organic phase of the mobile phase is a 0.1% formic acid acetonitrile solution; (2) The mobile phase gradient is: 0-0.50 min, the volume ratio of the aqueous phase to the organic phase is: 80%:20%; 0.50-1.8 min, the volume ratio of the aqueous phase to the organic phase is: (50%-80%):(20%-50%); 1.8-1.81 min, the volume ratio of the aqueous phase to the organic phase is: (15%-50%):(50%-85%); 1.81-2.8 min, the volume ratio of the aqueous phase to the organic phase is: 15%:85%; 2.8-2.81 min, the volume ratio of the aqueous phase to the organic phase is: (15%-80%):(20%-85%); 2.81-3.8 min, the volume ratio of the aqueous phase to the organic phase is: 80%:20%.

6. The detection method according to claim 1, wherein The liquid chromatography conditions of the liquid chromatography-mass spectrometer in step S2 meet the following conditions: (1) Chromatographic column: Waters ACQUITY UPLC BEH C8 1.7 μm (2.1×100 mm); (2) Column temperature: 40 °C; Autosampler temperature: 15 °C; (3) Flow rate: 0.3 mL / min; (4) Injection volume: 10 μL.

7. The detection method according to claim 5, characterized in that The mass spectrometry conditions of the liquid chromatography-mass spectrometer in step S2 meet the following conditions: (1) Ion source: Electrospray ionization source (ESI); (2) Ion source temperature (°C): 550; (3) Entrance voltage (KV): 10; (4) Gas1 (L·h -1 ): 50; (5) Gas2 (L·h -1 ):50; (6)Curtain Gas(L·h -1 ):40; (7)Collision Gas(L·h -1 ):Medium; (8) Ionization mode: Positive ion; (9) Signal acquisition mode: MRM.

8. The detection method according to claim 1, characterized in that The sample concentration of the standard curve in step S2 is 0.01-10 ng / mL.

9. The detection method according to claim 1, wherein, It further includes the step of preparing a quality control sample, and the concentration of the quality control sample is 0.1 - 6 ng / mL.

10. Application of the characteristic peptide segment YGGFMTSEK in the method for detecting β - endorphin by liquid chromatography - mass spectrometry.

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