Method for synchronous and rapid analysis of characteristic polypeptides and small molecule metabolites in donkey-hide gelatin

Abstract

The application discloses a method for synchronous and rapid analysis of characteristic polypeptides and small molecule metabolites in donkey-hide gelatin. The method is characterized in that: on the basis of qualitative analysis, polypeptides and small molecule metabolites with good matching degree and high response value are selected as target analytes by means of LC-MRM technology for on-line optimization of mass spectrometry parameters of donkey-hide gelatin sample solution, and 14 MRM 3 ion pairs of 7 polypeptide sequences and 13 MRM 3 ion pairs of 13 small molecule metabolites 3 The detection method is more specific and has higher sensitivity.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry technology, specifically a method for the simultaneous and rapid analysis of characteristic polypeptides and small molecule metabolites in donkey-hide gelatin. Background Technology

[0002] Donkey-hide gelatin (Ejiao) is a solid gelatin made from the dried or fresh hide of the donkey (Equus asinus L.) through boiling and concentration. It is a commonly used tonic in traditional Chinese medicine, known for its blood-nourishing, yin-tonifying, dryness-moistening, and hemostatic effects, and is often called a "holy medicine for blood nourishment." Furthermore, donkey-hide gelatin is also a food and medicine product, commonly found in pharmaceuticals, various health products, and foods. The main components of donkey-hide gelatin are collagen, polypeptides, and amino acids, with a content of approximately 60-80%. Due to the scarcity of donkey hide resources and the complex production process, donkey-hide gelatin commands a high market price. The quality of donkey-hide gelatin on the market varies greatly, often adulterated with lower-priced hides from horses, cattle, sheep, and pigs, which may ultimately affect its efficacy. Therefore, there is an urgent need to establish rapid qualitative and quantitative analysis methods for donkey-hide gelatin. Existing research has established methods for identifying polypeptides from horses, cattle, sheep, pigs, camels, and deer in donkey-hide gelatin based on protease digestion and mass spectrometry, thereby achieving quality control of donkey-hide gelatin. However, existing methods are mostly based on liquid chromatography-mass spectrometry (LC-MS), with analysis times ranging from 25 to 40 minutes, resulting in long analysis times, low throughput, and high costs. Furthermore, LC-MS methods for the analysis of amino acid components in donkey-hide gelatin have been established. However, as the main components of donkey-hide gelatin, small molecule metabolites such as proteins, peptides, and amino acids are difficult to analyze simultaneously due to their different chemical properties. Currently, there are no reports on the simultaneous analysis of peptides and small molecule compounds in donkey-hide gelatin.

[0003] Direct injection-mass spectrometry (DI-MS) directly introduces the sample into the mass spectrometer, offering advantages such as short analysis time, high throughput, and the ability to simultaneously analyze compounds of varying polarity (high, medium, and low). DI combined with multiple reaction monitoring (MRM) modes of triple quadrupole mass spectrometry has been used in metabolomics analysis. However, under DI conditions, the analyte response is often suppressed due to matrix effects, resulting in a low signal-to-noise ratio, especially when applied to peptide analysis, where the suppression is more pronounced. 3 (or MRM) 3 The scanning mode is unique to the quadrupole-linear ion trap (QTrap-MS). Its working principle involves the parent ion colliding in the first collision cell (q2) to generate daughter ions, which are then transported to the third quadrupole to screen for designated daughter ions and switch to linear ion trap (LIT) mode for further fragmentation to generate grandchild ions for scanning. 3 Compared to MRM, scanning can significantly improve the signal-to-noise ratio, and after multiple screenings, the established characteristic ion pairs have stronger specificity and higher sensitivity.

[0004] Currently, there are many patents related to the component detection of donkey-hide gelatin. For example, existing technology (CN115015409A) describes a method for establishing LCMS characteristic spectra of donkey-hide gelatin peptides and its quality evaluation method, which uses LCMS technology to detect peptide components in donkey-hide gelatin. Existing technology (CN109293741A) describes a method for identifying horse-hide-derived peptides in donkey-hide gelatin and products containing donkey-hide gelatin, which uses mass spectrometry to identify peptide components in donkey-hide gelatin. It is evident that existing technologies do not include methods for simultaneously detecting peptides and small molecule metabolites, nor do they utilize direct injection-multiple-reaction monitoring mass spectrometry (DI-MRM). 3 The present invention analyzes donkey-hide gelatin using analytical methods such as direct injection-multiple-reaction monitoring mass spectrometry (DI-MRM). 3 This method allows for the rapid and simultaneous detection of peptides and small molecule metabolites in donkey-hide gelatin, enabling quick and effective identification of the gelatin. It is more specific and has higher sensitivity. Summary of the Invention

[0005] The purpose of this invention is to provide a direct injection-multi-stage multiple reaction monitoring mass spectrometry (DI-MRM)-based mass spectrometry. 3 This method enables rapid qualitative and quantitative analysis of proteins, peptides, and small molecule metabolites in donkey-hide gelatin, and has higher specificity and sensitivity.

[0006] The technical solution of the present invention:

[0007] A method for simultaneous and rapid analysis of characteristic peptides and small molecule metabolites in donkey-hide gelatin (Ejiao) involves online optimization of mass spectrometry parameters of the Ejiao test solution using LC-MRM technology. Based on qualitative analysis, peptides and small molecule metabolites with good matching and high response values ​​are selected as target analytes. Furthermore, 14 MRM sequences of 7 peptide sequences are constructed. 3 13 MRMs of ion pairs and 13 small molecule metabolites 3 DI-MRM of ion pairs 3 Detection methods.

[0008] The aforementioned method for simultaneous and rapid analysis of characteristic polypeptides and small molecule metabolites in donkey-hide gelatin includes the following steps:

[0009] (1) Preparation of test solution: Extract the donkey-hide gelatin solution and enzymatically hydrolyze it to obtain the donkey-hide gelatin enzymatic hydrolysis solution as the test solution;

[0010] (2) Online parameter optimization of chromatographic conditions: The chromatographic column was a Waters Acquity UPL CHS ST3 column, 100×2.1mm, 1.8μm; the injection volume was 2μL, the flow rate was 0.25mL / min; gradient elution was performed using 0.1% formic acid water as mobile phase A and acetonitrile as mobile phase B; the column temperature was 40℃.

[0011] (3) Online parameter optimization ion source parameter setting conditions: positive ion mode acquisition, atomizer, heater, curtain gas or collision gas are all N2; atomizer gas GS1 is 55psi; heater gas GS2 is 55psi; curtain gas is 30psi; ion spray needle voltage is 5500V; collision activated dissociation CAD gas is High; ionization temperature TEM is 550℃; injection voltage EP is 10V, collision chamber emission voltage CXP is 13V; declustering voltage DP is 45V, using SRM-IDA-EPI scanning mode; for EPI scanning, IDA threshold is 2000cps, CE is 30eV, CES is 15eV;

[0012] (4) Chromatographic conditions for donkey-hide gelatin polypeptides: The chromatographic column was EASY-Spray. TM C 18 100×0.75mm, 3μm; injection volume 2μL, flow rate 300nL / min; gradient elution using 0.1% formic acid water as mobile phase A and 0.1% formic acid acetonitrile as mobile phase B; chromatogram acquisition time 85min;

[0013] (5) Ion source parameter settings for donkey-hide gelatin polypeptides: Mass spectrometry detection was performed using ESI positive ionization mode, with a spray voltage of 4.5 kV, a sheath flow rate of 30 arp, a normalized collision energy of 47%, and an activation time of 10 ms; MS 1 The spectra were acquired using FTMS mode, with a scan range of 350–2000 Da and a resolution of 60,000 FWHM; MS 2 The spectrum was acquired using ITMS mode at a resolution of 7500 FWHM.

[0014] (6) The test solution is injected according to the methods of steps (2) and (3) above to construct ion pairs with ion fragments of the parent ion and daughter ion of small molecule metabolites, and then derived into small molecule metabolite pseudo ion pairs. Each pseudo ion pair corresponds to a gradient change in CE in the range of 5 to 80 eV with a step size of 3 eV. Different collisions can produce different relative response values, and the relative response values ​​of small molecule metabolite pseudo ion pairs are obtained.

[0015] (7) Inject the test solution according to the methods in steps (4) and (5) above to obtain its high-resolution mass spectrometry information. Combine this with Swiss-Prot search to perform peptide qualitative analysis and obtain the peptide amino acid sequence and mass spectrometry (MS) data. 1 and MS 2Information; then inject the sample according to the above steps (2) and (3), take the double-charged quasi-molecular ion peak as the parent ion, construct ion pairs with the ion fragments of the parent ion and daughter ion, and then derive peptide pseudo-ion pairs. Each pseudo-ion pair corresponds to the gradient change of CE in the range of 5 to 80 eV, with a step size of 3 eV. Different collisions can produce different relative response values, and the relative response values ​​of peptide pseudo-ion pairs are obtained.

[0016] (8) Import the relative response values ​​of the small molecule metabolite pseudo-ion pairs and peptide pseudo-ion pairs from steps (6) and (7) into GraphPadPrism 7.0 software. Fit the fragmentation curve of the ion pairs using Gaussian curves. The collision energy corresponding to the vertex of the fitted curve is the optimal collision energy OCE. Select the pair of ion pairs with the best response for small molecule metabolites and the two pairs of ion pairs with the best response for peptides. Construct the MRM with the parent ion, daughter ion, and daughter ion. 3 Ion pairs were ultimately used to establish MRMs for 13 ion pairs of 13 small molecule metabolites and 14 ion pairs of 7 peptides. 3 method;

[0017] (9) Select 13 ion pairs of 13 small molecule metabolites and 14 ion pairs of 7 peptides from step (8) as target analytes. Use DI to directly introduce the test solution into the electrospray ionization (ESI) source of the mass spectrometer for separation. Use MRM 3 Qualitative and quantitative analysis was performed using scanning.

[0018] In step (1) above, the preparation of the test solution is as follows: accurately weigh about 0.1 g of donkey-hide gelatin sample, add 10 mL of aqueous solution containing 1% NH4HCO3, and extract by ultrasonication for 30 min. After centrifugation at 12000 rpm for 10 min, add 10 μL of 1 μg / μL trypsin solution to every 90 μL of supernatant. After enzymatic hydrolysis at 37℃ and 200 r / min on a shaker for 16 h, add 0.1% formic acid to terminate the reaction. After centrifugation at 12000 rpm for 15 min, filter through a 0.22 μm microporous membrane and take the filtrate to obtain the test solution.

[0019] In step (2) above, the elution program is 0–2 min, 0% B; 2–7 min, 0%–35% B; 7–8 min, 35%–90% B; 8–8.1 min, 90%–0% B; 8.1–11 min, 0% B.

[0020] In step (4) above, the elution program is 0–65 min, 5%–30% B; 65–75 min, 30%–50% B; 75–85 min, 50%–95% B.

[0021] In step (8) above, the 13 ion pairs of the 13 small molecule metabolites and the 14 ion pairs of the 7 polypeptides are as follows:

[0022]

[0023] In the aforementioned step (9), the parameters of the electrospray ESI ion source are set as follows: collection in positive ion mode, atomizer gas GS1 is 14psi, heater gas GS2 is 0psi, curtain gas is 20psi, ion spray needle voltage is 5500V, collision activation dissociation CAD gas is High, ionization temperature TEM is 100℃, and flow rate is set to 10μL / min.

[0024] In the aforementioned step (9), MRM 3 The scan parameters were set as follows: center, window size ±2 Da; AF2 energy set to 0.005 V; CE value set to the online optimized OCE value for each ion pair; fixed time set to 100 ms; excitation time set to 50 ms; Q0trap checked.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. This invention utilizes a method for simultaneous and rapid analysis of characteristic polypeptides and small molecule metabolites in donkey-hide gelatin, DI-MRM. 3 The average mass spectrometry response of peptides and small molecule metabolites was significantly improved, enhancing detection sensitivity. Furthermore, DI-MRM... 3 It enables simultaneous analysis of peptides and small molecule metabolites, and can be applied to targeted dual-omics analysis of small molecule metabolites and peptides simultaneously.

[0027] 2. DI-MRM was constructed using 14 MRM ion pairs of 7 polypeptide sequences and 13 MRM ion pairs of 13 small molecule metabolites. 3 The detection method is more specific and has higher sensitivity. Attached Figure Description

[0028] Figure 1 : Extraction ion chromatogram (A) of trypsin hydrolysate peptides from donkey-hide gelatin obtained by LC-MS / MS and MS of SGQPGTVGPAGVR 2 And structural analytical diagram (B);

[0029] Figure 2Fragmentation curves of six pseudo-ion pairs (SGQPGTVGPAGVR) with m / z 591.82→910.51, m / z 591.82→813.46, m / z 591.82→756.44, m / z 591.82→655.39, m / z 591.82→556.32 and m / z 591.82→499.30;

[0030] Figure 3 In this embodiment of the invention, LC-MRM was applied to 7 polypeptides and 13 small molecule metabolites in donkey-hide gelatin. 3 Select an ion detection chromatogram;

[0031] Figure 4 This refers to the DI-MRM targeting 7 polypeptides and 13 small molecule metabolites in donkey-hide gelatin in this embodiment of the invention. 3 Extraction ion chromatogram (A) and average response mass spectrum (B);

[0032] Figure 5 In this embodiment of the invention, the DI-MRM extraction ion chromatogram (A) and the average response mass spectrum (B) of 7 polypeptides and 13 small molecule metabolites in donkey-hide gelatin are shown. Detailed Implementation

[0033] Example 1:

[0034] Preparation method of test solution: Accurately weigh about 0.1g of donkey-hide gelatin sample, add 10mL of aqueous solution containing 1% NH4HCO3, and extract by sonication for 30min. After extraction, centrifuge the sample solution at 12000rpm for 10min, add 10μL of 1μg / μL trypsin solution to every 90μL of supernatant, and enzymatically hydrolyze at 37℃ and 200r / min on a shaker for 16h. Then add 0.1% formic acid to terminate the reaction, centrifuge at 12000rpm for 15min, filter through a 0.22μm microporous membrane, and collect the filtrate to obtain the test solution.

[0035] Example 2:

[0036] Online parameter optimization method for small molecule metabolites of donkey-hide gelatin

[0037] Online parameter optimization of chromatographic conditions: The chromatographic column was a Waters Acquity UPL CHS ST3 column, 100 × 2.1 mm, 1.8 μm; the injection volume was 2 μL, and the flow rate was 0.25 mL / min; gradient elution was performed using 0.1% formic acid water as mobile phase A and acetonitrile as mobile phase B; the elution program was 0–2 min, 0% B; 2–7 min, 0%–35% B; 7–8 min, 35%–90% B; 8–8.1 min, 90%–0% B; 8.1–11 min, 0% B; the column temperature was 40℃.

[0038] Online parameter optimization ion source parameter settings: positive ion mode acquisition; atomizer, heater, curtain gas, or collision gas are all N2; atomizer gas GS1 is 55 psi; heater gas GS2 is 55 psi; curtain gas is 30 psi; ion spray needle voltage is 5500V; collision-activated dissociation (CAD) gas is High; ionization temperature TEM is 550℃; injection voltage EP is 10V; collision chamber emission voltage CXP is 13V; declustering voltage DP is 45V; SRM-IDA-EPI scanning mode is used; for EPI scanning, IDA threshold is 2000cps, CE is 30eV, and CES is 15eV.

[0039] The test solution from Example 1 was injected according to the above-described methods for optimizing chromatographic conditions and ion source parameters using online parameters. Ion pairs were constructed from the parent ion and daughter ion fragments of the small molecule metabolites, which were then used to derive pseudo ion pairs of the small molecule metabolites. Each pseudo ion pair corresponded to a gradient change in CE within the range of 5–80 eV, with a step size of 3 eV. Different collisions could produce different relative response values, thus obtaining the relative response values ​​of the small molecule metabolite pseudo ion pairs.

[0040] Example 3:

[0041] Online parameter optimization method for donkey-hide gelatin polypeptides:

[0042] Chromatographic conditions for donkey-hide gelatin polypeptides: chromatographic column was EASY-Spray. TM C 18 The sample size was 100×0.75mm, 3μm; the injection volume was 2μL, and the flow rate was 300nL / min; gradient elution was performed using 0.1% formic acid water as mobile phase A and 0.1% formic acid acetonitrile as mobile phase B, with the elution program as follows: 0–65 min, 5%–30% B; 65–75 min, 30%–50% B; 75–85 min, 50%–95% B; the chromatogram acquisition time was 85 min.

[0043] The parameters for the donkey-hide gelatin polypeptide ion source were set as follows: mass spectrometry detection was performed using ESI positive ionization mode, with a spray voltage of 4.5 kV, a sheath flow rate of 30 arp, a normalized collision energy of 47%, and an activation time of 10 ms; MS 1 The spectra were acquired using FTMS mode, with a scan range of 350–2000 Da and a resolution of 60,000 FWHM; MS 2 The spectrum was acquired using ITMS mode at a resolution of 7500 FWHM.

[0044] The test solution was injected according to the chromatographic conditions and ion source parameter settings for donkey-hide gelatin peptides, respectively, to obtain its high-resolution mass spectrometry information. Qualitative peptide analysis was performed using Swiss-Prot search to obtain the peptide amino acid sequence and MS data. 1 and MS 2 Information; then, the sample was injected according to the method of optimizing the chromatographic conditions and the ion source parameter settings online in Example 2. The double-charged quasi-molecular ion peak was taken as the parent ion. Ion pairs were constructed with the ion fragments of the parent ion and the daughter ion, and then derived into peptide pseudo-ion pairs. Each pseudo-ion pair corresponds to a gradient change in CE in the range of 5 to 80 eV, with a step size of 3 eV. Different collisions can produce different relative response values, and the relative response values ​​of peptide pseudo-ion pairs are obtained.

[0045] Example 4:

[0046] MRMs were established for 13 ion pairs of 13 small molecule metabolites and 14 ion pairs of 7 peptides. 3 method:

[0047] The relative response values ​​of small molecule metabolite pseudo-ion pairs and peptide pseudo-ion pairs were imported into GraphPad Prism 7.0 software. The fragmentation curves of the ion pairs were fitted using Gaussian curves. The collision energy corresponding to the vertex of the fitted curve was the optimal collision energy (OCE). For small molecule metabolites, the pair of ion pairs with the best response was selected; for peptides, the two pairs of ion pairs with the best response were selected. MRM was performed using the parent ion, daughter ion, and daughter ion pairs. 3 The method was constructed, and the MRMs of 13 ion pairs for 13 small molecule metabolites and 14 ion pairs for 7 peptides were finally established. 3 method.

[0048] The 13 ion pairs of the 13 small molecule metabolites and the 14 ion pairs of the 7 polypeptides are as follows:

[0049]

[0050]

[0051] Example 5:

[0052] Quantitative analysis of small molecule metabolites and peptides in donkey-hide gelatin:

[0053] (1) The parameters of the electrospray ESI ion source are set as follows: in positive ion mode, the atomizer gas GS1 is 14psi, the heater gas GS2 is 0psi, the curtain gas is 20psi, the ion spray needle voltage is 5500V, the collision activation dissociation CAD gas is High, the ionization temperature TEM is 100℃, and the flow rate is set to 10μL / min.

[0054] (2) MRM 3 The scan parameters were set as follows: center, window size ±2 Da; AF2 energy set to 0.005 V; CE value set to the online optimized OCE value for each ion pair; fixed time set to 100 ms; excitation time set to 50 ms; Q0trap checked.

[0055] (3) Thirteen ion pairs of 13 small molecule metabolites and fourteen ion pairs of 7 peptides were used as target analytes. The test solution was directly introduced into the electrospray ionization (ESI) source of the mass spectrometer using the DI method for separation. MRM was used. 3 Qualitative and quantitative analysis was performed using scanning.

[0056] The inventor conducted numerous experiments; the following are some of the experimental studies.

[0057] The present invention will be further described below with reference to specific embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention.

[0058] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0059] Unless otherwise specified, the experimental methods used in the above embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0060] This invention has undergone extensive experimental research, and the following are the results of the experimental research process:

[0061] 1. Experimental Example:

[0062] 1.1 Experimental Materials

[0063] Donkey-hide gelatin was purchased from Shandong Fupai Donkey-hide Gelatin Co., Ltd. Tarasamine was purchased from Shanghai Biostandard Co., Ltd. Mass spectrometry grade methanol (MeOH), acetonitrile (ACN), and formic acid (FA) were all purchased from Thermo Fisher Scientific, USA. Deionized water was prepared in the laboratory using Milli-Q ultrapure water (18.2 MΩ·cm).

[0064] U-3000 dual ternary liquid chromatograph (including two ternary pumps, autosampler, column oven and UV detector, Thermo Fisher Scientific, USA); ACQUITY UPL CHS ST3 column (100mm × 2.1mm, 1.8μm, Waters Scientific, USA); Qtrap 5500 mass spectrometer (Sciex Scientific, USA); LTQ Orbitrap Velos ProMS (Thermo Fisher Scientific, USA); Milli-Q ultrapure water purification system (Millipore Scientific, USA); ME204 electronic analytical balance (Mettler Toledo, Switzerland, accuracy 0.1mg).

[0065] 1.2 Sample Preparation

[0066] Accurately weigh approximately 0.1 g of donkey-hide gelatin sample and add it to 10 mL of an aqueous solution containing 1% NH4HCO3. Extract by sonication for 30 min. After extraction, centrifuge the sample solution at 12000 rpm for 10 min. Add 10 μL of 1 μg / μL trypsin solution to every 90 μL of supernatant and incubate at a shaker at 200 rpm for 16 h (37℃). After hydrolysis, add 0.1% formic acid to terminate the reaction. Centrifuge at 12000 rpm for 15 min and filter through a 0.22 μm microporous membrane. Collect the filtrate to obtain the enzymatically hydrolyzed donkey-hide gelatin sample.

[0067] 1.3 Target compound DI-MRM 3 Method establishment

[0068] 1.3.1 Qualitative Analysis of Donkey-hide Gelatin Peptides

[0069] The enzymatically hydrolyzed donkey-hide gelatin sample was injected into a nanoLC-LTQ-Orbitrap-MS system, and high-resolution mass spectrometry data of the sample were acquired. The HPLC conditions were as follows: the column was EASY-Spray. TM C 18(100×0.75mm, 3μm, Thermo-Fisher, USA), injection volume 2μL, flow rate 300nL / min. Gradient elution was performed using 0.1% formic acid aqueous solution as mobile phase A and 0.1% formic acid acetonitrile as mobile phase B. The elution program was 0–65 min, 5%–30% B; 65–75 min, 30%–50% B; 75–85 min, 50%–95% B; chromatogram acquisition time was 85 min. Mass spectrometry was performed using ESI positive ionization mode, spray voltage 4.5 kV, sheath gas flow rate 30 arp, normalized collision energy 47%, activation time 10 ms; MS 1 The spectra were acquired using FTMS mode, with a scan range of 350–2000 Da and a resolution of 60,000 FWHM; MS 2 Mass spectra were acquired in ITMS mode at a resolution of 7500 FWHM. The mass spectrometry data were analyzed using ProteomeDiscoverer (Version 1.4) software and imported into the Swiss-Prot database for retrieval. The SEQUEST algorithm was used for peptide identification, with the following parameters: trypsin digestion method, trypsin cleavage failure rate of 2, parent ion deviation ≤10 ppm, and daughter ion deviation ≤0.8 Da.

[0070] 1.3.2 Qualitative Analysis of Small Molecule Metabolites

[0071] Literature reports that the small molecule metabolites in donkey-hide gelatin are mainly amino acids and nucleotides. An ion pair list was collected from previous reports, and the small molecule metabolites in donkey-hide gelatin were detected using the enhanced ion scanning (EPI) mode combined with predictive selected reaction (SRM) detection results. Ion pair, CE, and DP information were extensively collected from existing literature reports to construct a predictive SRM information collection list, resulting in 58 metabolite mass spectrometry data points. Correspondingly, 58 ion pair information was constructed, as detailed in Table 1.

[0072] Table 1: Information on 58 pairs of qualitative ion pairs of small molecule metabolites

[0073]

[0074]

[0075]

[0076] The chromatographic conditions were as follows: a Waters Acquity UPL CHS ST3 column (100 × 2.1 mm, 1.8 μm, Waters Corporation, USA), an injection volume of 2 μL, and a flow rate of 0.25 mL / min. Gradient elution was performed using 0.1% formic acid-water as mobile phase A and acetonitrile as mobile phase B. The elution program was: 0–2 min, 0% B; 2–7 min, 0%–35% B; 7–8 min, 35%–90% B; 8–8.1 min, 90%–0% B; 8.1–11 min, 0% B. The column temperature was 40 °C.

[0077] The ion source parameters were set as follows: positive ion mode acquisition; nebulizer, heater, curtain gas, or collision gas were all N2; nebulizer gas (GS1): 55 psi; heater gas (GS2): 55 psi; curtain gas: 30 psi; ion spray needle voltage: 5500 V; collision activated dissociation (CAD) gas: high; ionization temperature (TEM): 550 °C; injection voltage (EP): 10 V; collision chamber ejection voltage (CXP): 13 V; declustering voltage (DP): 45 V; SRM-IDA-EPI scanning mode was used. For EPI scanning, the IDA threshold was 2000 cps, CE was 30 eV, and CES was 15 eV.

[0078] 1.3.3 Selection of target analytes and online optimization of mass spectrometry parameters

[0079] Based on qualitative analysis, seven peptides and thirteen small molecule metabolites with good matching and high response values ​​were selected as target analytes. All target peptides possessed quasi-molecular ions with double charges and molecular weights ranging from 393.2 to 765.9 Da.

[0080] Taking SGQPGTVGPAGVR as an example, the process of establishing the method is briefly described. It uses its double-charged quasi-molecular ion peak ([M+2H)). 2+ The parent ion is m / z 591.82, and the single-charged ion fragment is m / z 910.51 (y 10 + ), m / z813.46(y9 + ), m / z756.44(y8) + ), m / z655.39(y7 + ), m / z556.32(y6 + ) and m / z499.30(y5 +Six candidate ion pairs were constructed: m / z 591.82→910.51, m / z 591.82→813.46, m / z 591.82→756.44, m / z 591.82→655.39, m / z 591.82→556.32, and m / z 591.82→499.30. Six pseudo-ion pairs were further derived. Transitions (PITs) are m / z 591.820→910.510, m / z 591.820→910.511, m / z 591.820→910.512, etc.; m / z 591.820→813.460, m / z 591.820→813.461, m / z 591.820→813.462, etc.; m / z 591.820→756.440, m / z 591.820→756.441, m / z 591.820 →756.442, etc.; m / z 591.820→655.390, m / z 591.820→655.391, m / z 591.820→655.392, etc.; m / z 591.820→556.320, m / z 591.820→556.321, m / z 591.820→556.322, etc.; m / z 591.820→499.300, m / z 591.820→499.301, m / z 591.820→499.302, etc. For each pair of pseudo-ions, there is a corresponding CE (energy gradient) with a step size of 3 eV in the range of 5–80 eV. Different collision energies produce different relative response values. The relative response values ​​of each pseudo-ion pair were imported into GraphPad Prism 7.0 software (Graphpad, San Diego, CA). The fragmentation curves of the ion pairs were fitted using Gaussian curves, and the collision energy corresponding to the vertex of the fitted curve was the optimal collision energy (OCE). The two ion pairs with the best responses, m / z 591.82→910.51 and m / z 591.82→556.32, were selected as candidate ion pairs to construct MS... 3 Scanning method.

[0081] Set Q3 and Q LIT MRM was performed using the same ions and at a relatively low excitation energy (AF2 = 0.005 V). 3 Scanning. Taking SGQPGTVGPAGVR as an example, the ion pairs m / z 591.82→910.51→910.51 and m / z 591.82→556.32→556.32 were constructed as qualitative and quantitative ion pairs, respectively, for MRM. 3 Scanning. Ultimately, MRMs were established for 13 ion pairs of 13 small molecule metabolites and 14 ion pairs of 7 peptides. 3The method and its mass spectrometry parameters are shown in Table 2.

[0082] Table 2: Information on 13 ion pairs of 13 small molecule metabolites and 14 ion pairs of 7 polypeptides of donkey-hide gelatin

[0083]

[0084]

[0085] Note: P Proline representing hydroxylation modification

[0086] 1.4DI-MRM 3 Measurement with DI-MRM (control group)

[0087] The enzymatic hydrolysis sample of donkey-hide gelatin was not separated by a chromatographic column. It was directly introduced into the ESI ion source of the SCIEX Qtrap-MS mass spectrometer via an external syringe pump (KDS100, KDS Scientific, Holliston, MA) through a PEEK line. The ion source parameters were as follows: positive ion mode acquisition; nebulizer, heater, curtain gas, or collision gas were all N2; nebulizer gas (GS1): 14 psi; heater gas (GS2): 0 psi; curtain gas: 20 psi; ion spray needle voltage: 5500 V; collision-activated dissociation (CAD) gas: High; ionization temperature (TEM): 100℃. The flow rate was set to 10 μL / min. The acquisition mode was MS. 3 Scan (i.e., MRM) 3 MRM 3 The scan parameters were set as follows: center, window size ±2 Da, CE optimized for each ion, AF2 energy set to 0.005 V, fixed time set to 100 ms, excitation time set to 50 ms, and Q0trap checked.

[0088] The same samples were introduced into the Qtrap-MS mass spectrometer in the same manner, with the mass spectrometry acquisition mode set to MRM scan, and all other parameters were the same as those for MRM. 3 The scan settings are the same.

[0089] 2 Experimental Results

[0090] 2.1 Qualitative Analysis Results of Donkey-hide Gelatin

[0091] After extraction, donkey-hide gelatin was hydrolyzed with trypsin to obtain enzymatically hydrolyzed samples. Shotgun proteomics analysis of the gelatin was then performed using nanoLC-LTQ-Orbitrap-MS. The collected peptide profiles are shown below. Figure 1As shown in Figure A, by combining analysis with ProteomeDiscoverer v.1.4 software and searching the DonkeySwiss-ProtFASTA database, 47 peptides derived from 2 proteins were initially identified.

[0092] Peptides were identified using fragment information from mass spectrometry, with peptide 5(t) as an example. R Taking (e.g., 20.21 min), its double-charged quasi-molecular ion peak ([M+2H]) 2+ ) is m / z591.8168 ( Figure 1 B), the information for single-charged ion fragments is m / z 1008.56, 910.52, 852.42, 813.48, 756.47, 684.31, 655.40, 627.32, 556.34, 528.30, 499.30, 331.21, 273.09, and 175.09, which are respectively assigned to b. 12 + y 10 + b 10 + y9 + y8 + b8 + y7 + b7 + y6 + b6 + y5 + y3 + b3 + and y1 + Based on database comparison and analysis, the polypeptide sequence was inferred to be SGQPGTVGPAGVR. Ultimately, seven polypeptide fragments were identified, detailed in Table 3, and used for further analysis.

[0093] Table 3: Retention time, amino acid sequence, and MS of 7 polypeptides from donkey-hide gelatin 1 Error value and MS 2 Fragmented information

[0094]

[0095]

[0096] Small molecule metabolites in donkey-hide gelatin samples were detected using LC-SRM. Thirteen compounds with good responses were selected as target analytes for donkey-hide gelatin, and their structures were annotated by combining mass spectrometry information and database comparison.

[0097] 2. MRM 3 Method establishment

[0098] To achieve better sensitivity, an online mass spectrometry parameter optimization strategy was employed to optimize ion pairs and CE values ​​for peptides and small molecule metabolites. The peptide components were used as parent ions, with a double-charged quasi-molecular ion peak, and single-charged ion fragments were used to construct ion pairs. Candidate ion pairs were then constructed, and their CE values ​​were optimized. Taking SGQPGTVGPAGVR as an example, a total of 6 candidate ion pairs were constructed, and the fragmentation curves of each ion pair are shown below. Figure 2 As shown, the two ion pairs with the best response, m / z 591.82→910.51 and m / z 591.82→556.32, were finally selected as the MRM ion pairs for SGQPGTVGPAGVR for further method construction. Ultimately, 14 MRM ion pairs from 7 polypeptide sequences and 13 MRM ion pairs from 13 small molecule metabolites were selected for method construction.

[0099] Tried using LC-MS 3 MS of peptides and small molecule metabolites 3 Fragments. However, MS of small molecule metabolites 2 The fragments are relatively stable and difficult to disintegrate, or their MS... 3 Fragment m / z value not in MS 3 Within the scanning range of the pattern; although peptides can produce MS 3 The fragments were detected, but their response was too low, and the signal-to-noise ratio could not meet the requirements of subsequent experiments. Therefore, in order to improve the detection sensitivity, an MRM was established. 3 In the methodology, for peptides and small molecule metabolites, Q3 and Q2 are set. LIT With the same value, a lower AF2 (0.005V) was set for scanning to reduce its fragmentation. The acquired LC-MRM... 3 Typical chromatograms are as follows Figure 3 As shown, the signal-to-noise ratios of all compounds meet the analytical requirements, which is sufficient for subsequent DI-MRM. 3 The specificity of the detection laid the foundation.

[0100] 3. DI-MRM 3 Comparative analysis results of the technology and DI-MRM technology

[0101] DI-MRM 3 Typical total ion chromatograms are as follows: Figure 4 As shown in Figure A, the TIC plot can be divided into three stages, namely stages I-III, for the entire analysis process. The solvent in the tubing first enters the mass spectrometer to generate the matrix range (stage I), the subsequent ramp-up stage corresponds to the mixing of the carrier solvent and sample (stage II), and finally, complete equilibrium is achieved in the tubing to form a stable stage (stage III). The average intensity curve of stage III is subtracted from the average intensity curve of stage I to generate the dataset, whose MRM... 3 Average mass spectrum response Figure 4As shown in B. Typical TIC spectra and average response mass spectra of DI-MRM are shown in [reference]. Figure 5 A and B. Compared to DI-MRM, DI-MRM 3 The average mass spectrometry response of peptides and small molecule metabolites was significantly improved, enhancing detection sensitivity. Furthermore, DI-MRM... 3 It enables simultaneous analysis of peptides and small molecule metabolites, and can be applied to targeted dual-omics analysis of small molecule metabolites and peptides simultaneously.

[0102] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for simultaneous and rapid analysis of characteristic polypeptides and small molecule metabolites in donkey-hide gelatin, characterized by: The method described above involves online optimization of mass spectrometry parameters of the donkey-hide gelatin test solution using LC-MRM technology. Based on qualitative analysis, peptides and small molecule metabolites with good matching and high response values ​​are selected as target analytes. Furthermore, 14 MRMs for 7 peptide sequences are constructed. 3 13 MRMs of ion pairs and 13 small molecule metabolites 3 DI-MRM of ion pairs 3 Detection methods; The method includes the following steps: (1) Preparation of test solution: Extract the donkey-hide gelatin solution and enzymatically hydrolyze it to obtain the donkey-hide gelatin hydrolysate as the test solution; (2) Online parameter optimization of chromatographic conditions: The chromatographic column was a Waters Acquity UPLC HSS T3 column, 100×2.1mm, 1.8µm; the injection volume was 2µL, the flow rate was 0.25mL / min; gradient elution was performed using 0.1% formic acid water as mobile phase A and acetonitrile as mobile phase B; the column temperature was 40℃. (3) Online parameter optimization ion source parameter setting conditions: positive ion mode acquisition, atomizer, heater, curtain gas or collision gas are all N2; atomizer gas GS1 is 55psi; heater gas GS2 is 55psi; curtain gas is 30psi; ion spray needle voltage is 5500V; collision activation dissociation CAD gas is High; ionization temperature TEM is 550℃; injection voltage EP is 10V, collision chamber emission voltage CXP is 13V; declustering voltage DP is 45V, SRM-IDA-EPI scanning mode is used; for EPI scanning, IDA threshold is 2000cps, CE is 30eV, CES is 15eV; (4) Chromatographic conditions for donkey-hide gelatin polypeptides: The chromatographic column was EASY-Spray. ™ C 18 100×0.75mm, 3µm; injection volume 2µL, flow rate 300 nL / min; gradient elution using 0.1% formic acid water as mobile phase A and 0.1% formic acid acetonitrile as mobile phase B; chromatogram acquisition time 85min; (5) Ion source parameter settings for donkey-hide gelatin polypeptides: Mass spectrometry detection was performed using ESI positive ionization mode, with a spray voltage of 4.5 kV, a sheath flow rate of 30 arp, a normalized collision energy of 47%, and an activation time of 10 ms; MS 1 The spectra were acquired using FTMS mode, with a scan range of 350–2000 Da and a resolution of 60,000 FWHM; MS 2 The spectra were acquired using ITMS mode at a resolution of 7,500 FWHM. (6) The test solution is injected according to the methods of steps (2) and (3) above to construct ion pairs with ion fragments of the parent ion and daughter ion of small molecule metabolites, and then derived into small molecule metabolite pseudo ion pairs. Each pseudo ion pair corresponds to a gradient change in CE in the range of 5~80eV with a step size of 3eV. Different collisions can produce different relative response values, and the relative response values ​​of small molecule metabolite pseudo ion pairs are obtained. (7) Inject the test solution according to the methods in steps (4) and (5) above to obtain its high-resolution mass spectrometry information. Combine with Swiss-Prot search for peptide qualitative analysis to obtain the peptide amino acid sequence and mass spectrometry (MS) data. 1 and MS 2 Information; then inject the sample according to the above steps (2) and (3), take the double-charged quasi-molecular ion peak as the parent ion, construct ion pairs with the ion fragments of the parent ion and daughter ion, and then derive peptide pseudo-ion pairs. Each pseudo-ion pair corresponds to the gradient change of CE in the range of 5~80 eV, with a step size of 3 eV. Different collisions can produce different relative response values, and the relative response values ​​of peptide pseudo-ion pairs are obtained. (8) Import the relative response values ​​of the small molecule metabolite pseudo-ion pairs and peptide pseudo-ion pairs from steps (6) and (7) into GraphPad Prism 7.0 software. Fit the fragmentation curve of the ion pairs using Gaussian curves. The collision energy corresponding to the vertex of the fitted curve is the optimal collision energy OCE. Select the pair of ion pairs with the best response for small molecule metabolites and the two pairs of ion pairs with the best response for peptides. Construct the MRM with the parent ion, daughter ion, and daughter ion. 3 Ion pairs were ultimately used to establish MRMs for 13 ion pairs of 13 small molecule metabolites and 14 ion pairs of 7 peptides. 3 method; (9) Select 13 ion pairs of 13 small molecule metabolites and 14 ion pairs of 7 peptides from step (8) as target analytes. Use DI to directly introduce the test solution into the electrospray ionization (ESI) source equipped with the mass spectrometer for separation. Use MRM 3 Qualitative and quantitative analysis was performed using scanning.

2. The method for simultaneous and rapid analysis of characteristic polypeptides and small molecule metabolites in donkey-hide gelatin according to claim 1, characterized in that: In step (1), the test solution is prepared as follows: 0.1 g of donkey-hide gelatin sample is accurately weighed and added to 10 mL of aqueous solution containing 1% NH4HCO3. The sample is extracted by ultrasonication for 30 min. After centrifugation at 12000 rpm for 10 min, 10 µL of 1 µg / µL trypsin solution is added to every 90 µL of supernatant. The sample is then enzymatically hydrolyzed on a shaker at 37 °C and 200 r / min for 16 h. 0.1% formic acid is added to terminate the reaction. After centrifugation at 12000 rpm for 15 min, the sample is filtered through a 0.22 µm microporous membrane. The filtrate is then used to obtain the test solution.

3. The method for simultaneous and rapid analysis of characteristic polypeptides and small molecule metabolites in donkey-hide gelatin according to claim 1, characterized in that: In step (2), the elution program is 0–2 min, 0%B; 2–7 min, 0%–35%B; 7–8 min, 35%–90%B; 8–8.1 min, 90%–0%B; 8.1–11 min, 0%B.

4. The method for simultaneous and rapid analysis of characteristic polypeptides and small molecule metabolites in donkey-hide gelatin according to claim 1, characterized in that: In step (4), the elution program is 0–65 min, 5%–30%B; 65–75 min, 30%–50%B; 75–85 min, 50%–95%B.

5. The method for simultaneous and rapid analysis of characteristic polypeptides and small molecule metabolites in donkey-hide gelatin according to claim 1, characterized in that: In step (8), the 13 ion pairs of the 13 small molecule metabolites and the 14 ion pairs of the 7 polypeptides are as follows: 。 6. The method for simultaneous and rapid analysis of characteristic polypeptides and small molecule metabolites in donkey-hide gelatin according to claim 1, characterized in that: In step (9), the parameters of the electrospray ESI ion source are set as follows: collection in positive ion mode, atomizer gas GS1 is 14 psi, heater gas GS2 is 0 psi, curtain gas is 20 psi, ion spray needle voltage is 5500 V, collision-activated dissociation CAD gas is High, ionization temperature TEM is 100 ℃, and flow rate is set to 10 µL / min.

7. The method for simultaneous and rapid analysis of characteristic polypeptides and small molecule metabolites in donkey-hide gelatin according to claim 1, characterized in that: In step (9), MRM 3 The scan parameters were set as follows: center, window size ±2 Da; AF2 energy set to 0.005 V; CE value set to the online optimized OCE value for each ion pair; fixed time set to 100 ms; excitation time set to 50 ms; Q0 trap checked.

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