Application of characteristic peptide of sweet potato sporamin protein in identifying authenticity of sweet potato products

Abstract

The application relates to application of a characteristic peptide segment of a sweet potato sporamin protein in identifying the authenticity of a sweet potato product, and belongs to the field of food detection. The sequence of the characteristic peptide segment is RPLGHDVQM. The application method is to detect whether the characteristic peptide segment is contained in a sample; if the characteristic peptide segment is contained, it is determined that the sample contains sweet potato; and if the characteristic peptide segment is not contained, it is determined that the sample does not contain sweet potato. The detection method of the content of the sweet potato sporamin protein provided by the application has high specificity, and can distinguish the sweet potato sporamin protein from cassava, corn, sweet potato, potato and taro protein.

Description

Technical Field

[0001] This invention belongs to the field of food testing, specifically relating to the application of characteristic peptides of sweet potato sporamin protein in identifying the authenticity of sweet potato products. Background Technology

[0002] Sporamin is a unique group of proteins found in the tuberous roots of sweet potato (Ipomoea batatas Lam.). Discovered by Maeshima et al. in 1985, it is mainly present in sweet potato tubers, accounting for 60%–80% of the soluble proteins in the tubers, while it is almost absent in other organs. It is a type of storage protein that provides nitrogen (N) to germinating seedlings during plant development, and also possesses certain enzymatic activities.

[0003] Sweet potato starch and its products are recognized for their excellent qualities such as resistance to overcooking and good taste, resulting in good economic benefits for sweet potato production and processing enterprises and individual businesses. However, due to the significant price differences between corn starch, cassava starch, and sweet potato starch in the market, some individuals and producers, driven by economic interests, adulterate sweet potato products, leading to a decline in product quality, and even substituting corn or cassava products for sweet potato products for sale. Summary of the Invention

[0004] This invention addresses the aforementioned technical problems by providing an application of characteristic peptides of sweet potato sporamin protein in identifying the authenticity of sweet potato products. This application, through the detection of characteristic peptides of sporamin protein, can effectively determine whether a sample contains sweet potato.

[0005] This invention is achieved through the following technical solution:

[0006] The application of characteristic peptides of sweet potato sporamin protein in identifying the authenticity of sweet potato products, wherein the sequence of the characteristic peptides is: RPLGHDVQM.

[0007] Furthermore, the application method involves detecting whether the sample contains the aforementioned characteristic peptide; if the sample contains the characteristic peptide, it is determined that the sample contains sweet potato; if the sample does not contain the characteristic peptide, it is determined that the sample does not contain sweet potato.

[0008] Furthermore, the application is to identify whether the sweet potato content in sweet potato products meets the stated sweet potato content.

[0009] Preferably, liquid chromatography-tandem mass spectrometry is used for the detection. The precursor ion of the detection signal generated by the characteristic peptide in the mass spectrometer has a mass-to-charge ratio of m / z 525.76110; the daughter ions include daughter ions with mass-to-charge ratios of m / z 798.36887 and m / z 1051.52275, with an allowable deviation within 5 ppm.

[0010] Preferably, the isotope internal standard method is used to quantitatively detect sweet potato sporamin protein, wherein the internal standard peptide used is: RPLGHDVQM*; the parent ion of the detection signal generated by the internal standard peptide in mass spectrometry is m / z 530.76523; the daughter ions include daughter ions with mass-to-charge ratios of m / z 808.83658 and m / z 1060.94784, and the allowable deviation is within 5 ppm.

[0011] Furthermore, the concentration of the characteristic peptide is obtained by substituting the peak area ratio of the characteristic peptide to the stable isotope internal standard peptide into the formula, and then the content of the characteristic peptide RPLGHDVQM is obtained according to Equation 1.

[0012] X = (Ф*c*V) / m Equation 1;

[0013] Where X (ng / g) is the content of the characteristic peptide RPLGHDVQM in the sweet potato sample, Ф is the proportion of the enzymatically digested protein volume to the total sample volume, c (ng / mL) is the concentration of the characteristic peptide in the trypsin digest, V (mL) is the volume of the trypsin digest, and m (g) is the mass of the sweet potato sample. This method aims to quantify the characteristic peptide RPLGHDVQM in the sweet potato sample.

[0014] Only when a sample meets the above specificity in terms of both precise m / z value and characteristic fragment ions can the sporamin protein content in the sample be considered reliable, and the quality of the sample can be identified based on the level of this content.

[0015] Preferably, the sample pretreatment includes: extracting proteins from the sample to be tested and enzymatically digesting the proteins using trypsin.

[0016] Preferably, UHPLC-Q Exactive plus or triple quadrupole mass spectrometry is used for liquid chromatography-tandem mass spectrometry detection.

[0017] The advantages of this invention compared to existing technologies are as follows: This invention utilizes the characteristic peptides of sweet potato sporamin protein to identify the authenticity of sweet potato products. Based on the content of these characteristic peptides in the sample, the content of sweet potato is obtained, and the proportion of sweet potato in the sample can be further calculated. The method for detecting sweet potato sporamin protein content provided by this invention has high specificity, providing methodological support for consumers to make informed choices about sweet potato products and protecting consumer rights. Attached Figure Description

[0018] Figure 1 Ion flow chromatogram of characteristic peptides in sweet potato samples;

[0019] Figure 2Mass spectra of characteristic peptides from sweet potato samples;

[0020] Figure 3 This is a secondary fragment mass spectrum of characteristic peptides from a sweet potato sample.

[0021] Figure 4 The ion chromatogram shows the peptide fragment of the internal standard (IS) in the sweet potato sample.

[0022] Figure 5 This is the mass spectrum of the internal standard (IS) peptide fragment in the sweet potato sample;

[0023] Figure 6 This is a secondary fragment mass spectrum of the internal standard (IS) peptide fragment in a sweet potato sample. Detailed Implementation

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0025] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0026] Ultra-high performance liquid chromatography-tandem quadrupole / high resolution electrostatic orbital trap mass spectrometer (UHPLC-Q ExactivePlus), high-speed centrifuge, microporous filter membrane, Sephadex G-75 gel chromatography column, ultrafiltration cup, EAE-52 ion exchange chromatography column, NaHSO3, Tris-HCl buffer solution, ammonium sulfate, EDTA, NaCl, iodoacetamide (IAA), dithiothreitol (DTT), NH4HCO3, and mass spectrometry-grade trypsin;

[0027] 40mM NH4HCO3 solution: Weigh 0.316g NH4HCO3, dilute to 100mL with ultrapure water, and store at 4℃ for later use.

[0028] 100mM DTT solution: Weigh 0.308 g of DTT, dissolve and mix it with 40mM NH4HCO3 solution, and make up to 20mL. Store at -20℃ until needed.

[0029] 100mM IAA solution: Weigh 0.39g of IAA, dilute to 20mL with 40mM NH4HCO3 solution, and store at -20℃ for later use.

[0030] 50 mmol·L -1 Tris-HCl buffer solution: Weigh 6.055g of Tris and place it in a 1L beaker. Add about 800mL of deionized water, stir thoroughly to dissolve, add concentrated hydrochloric acid to adjust the pH value as needed, and bring the solution to a final volume of 1L.

[0031] Example 1: Discovery of characteristic peptides of sweet potato sporamin protein.

[0032] Step 1: Select fresh sweet potatoes, wash, weigh, and chop them, then add 0.1% NaHSO3 at 50 mmol·L⁻¹. - 1 Soak in Tris-HCl buffer solution (pH 7.5) (1 L·kg⁻¹) -1 (Fresh weight), homogenize, centrifuge the slurry at 3500 r / min for 15 min, take the supernatant, add ammonium sulfate to 60% saturation under ice bath, let stand, centrifuge at 3500 r / min for 35 min, dissolve the precipitate, freeze dry, and obtain crude protein powder.

[0033] Step 2: Weigh an appropriate amount of sweet potato crude protein powder, add 100 mL of distilled water to dissolve it, and centrifuge at 10000 r / min for 45 min. Filter the supernatant through a microporous membrane and then purify it using a DEAE-52 ion exchange chromatography column containing 1 mmol·L⁻¹. -1 EDTA and 0.2 mol·L -1 50 mmol·L⁻¹ NaCl -1 Elution with Tris-HCl buffer (pH 7.5) at a flow rate of 0.30 mL / min. -1 The absorbance of the eluent was measured at 280 nm and a curve was plotted. The preliminarily purified sporamin protein fraction was collected. Then, the preliminarily purified sporamin protein was concentrated using an ultrafiltration cup and further purified using a Sephadex G-75 gel chromatography column; 0.1 mol·L⁻¹ solution was used. -1 NaCl and 1 mmol·L -1 50 mmol·L of EDTA -1 Tris-HCl buffer (pH 7.5) was prepared at a temperature of 4°C and a flow rate of 0.45 ml / min. -1 Elution. The absorbance of the collected solution was measured at 280 nm to plot the elution curve. The pure sporamin protein was collected, lyophilized, and stored at -20°C.

[0034] Step 3: In an autoclave, treat the purified sporamin protein obtained in Step 2 at 127°C to remove its trypsin inhibitor activity. After returning to room temperature, weigh the purified sporamin protein and PBS solution in a 1:1 ratio, vortex thoroughly, and centrifuge at 4°C for 20 min. Collect the supernatant. Take 100 μL of the supernatant, add 400 μL of 40 mM NH4HCO3 solution, vortex and mix well, add 50 μL of 100 mM DTT solution, vortex thoroughly, react at room temperature for 60 min, then add 250 μL of 100 mM IAA solution and react in the dark at room temperature for 60 min.

[0035] Step 4: Add 2 μg excess trypsin to the final solution from step 3 and incubate overnight at 37°C. Upon completion of the reaction, add 1 μL of formic acid solution to inactivate the trypsin. Desalt the digested product using a C18 column, and then vacuum dry the desalted sample.

[0036] The samples obtained in steps five and four were dissolved in 0.1% formic acid solution, and the peptides were sequenced and identified by Full MS-ddMS2 in positive ion mode using UHPLC-Q Exactive plus.

[0037] Mass spectrometry data were collected and stored in Xcalibur software, and raw mass spectrometry data were imported into PEAKS 8.0 for qualitative analysis. Search parameters were set as follows: precursor mass tolerances of 15 ppm, fragment mass tolerances of 0.05 Da, enzyme: Trypsin digestion, maximum missed cleavage sites of 2; variable modification: oxidation (M, +15.99), fixed modification: carbamidomethyl (C, +57.02). All search results were analyzed using a positive / negative library fusion algorithm to control the false positive rate (FDR) for proteins and peptides; FDR < 1%.

[0038] After qualitative analysis using PEAKS 8.0, the peptide data of the matched sweet potato sporamin protein were analyzed and processed. Peptides that were present in different sweet potato sporamin proteins, had high response, were unmodified, had no enzyme cleavage sites, and were between 8 and 20 μm in length were selected.

[0039] Through the processing and analysis steps described above, several characteristic peptide segments of sweet potato sporamin protein were obtained, and their sequences are as follows:

[0040] 1)RPLGHDVQM (SEQ ID No. 1);

[0041] 2) RPHHHHAGDG (SEQ ID No. 2);

[0042] 3)RPESTVVMASTYQTF (SEQ ID No. 3);

[0043] 4)RLALSSYPFFFVIK (SEQ ID No. 4).

[0044] Although there are multiple characteristic peptides to choose from, considering factors such as the uniqueness, stability, and ion response of the peptides on mass spectrometry, only the optimal peptide can be selected for quantitative analysis.

[0045] Example 2: Establishment and detection method of characteristic peptides of sweet potato sporamin protein.

[0046] Step 1: Screen all characteristic peptides from Example 1 and verify them on NCBI and Uniprot websites. Screen for characteristic peptides that exist only in sweet potato sporamin protein. Review the mass spectrometry data generated in Example 1 and select the specific peptide with high response and no influence from other substances as the final characteristic peptide, i.e., RPLGHDVQM.

[0047] The second step involves synthesizing the characteristic peptide RPLGHDVQM and the stable isotope internal standard (IS) peptide RPLGHDVQM*, with a purity exceeding 98%, and storing them at -20°C for later use.

[0048] Step 3: Construction of the standard curve: A series of characteristic peptide standards (1 ng / mL, 3 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 40 ng / mL, 80 ng / mL) were prepared in the initial mobile phase (water / acetonitrile containing 0.1% formic acid, volume ratio 97:3). Then, IS peptide was added to each prepared standard to a concentration of 10 ng / mL. The standard curve was constructed by calculating the ratio of the peak areas of the characteristic peptide and the IS peptide, and the corresponding concentration of the characteristic peptide.

[0049] Step 4: Pretreatment of sweet potato samples:

[0050] (1) Select fresh sweet potatoes, wash, weigh, and chop them, and add 0.1% NaHSO3 at 50 mmol·L⁻¹. -1 Soak in Tris-HCl buffer solution (pH 7.5) (1 L·kg⁻¹) -1(Fresh weight) homogenized, the slurry was centrifuged at 3500 r / min for 15 min, the supernatant was collected, ammonium sulfate was added to 60% saturation under ice bath, allowed to stand, centrifuged at 3500 r / min for 35 min, the precipitate was reconstituted, and freeze-dried to obtain crude protein powder. An appropriate amount of sweet potato crude protein powder was weighed, dissolved in 100 mL of distilled water, and centrifuged at 10000 r / min for 45 min. The supernatant was filtered through a microporous membrane and purified by a DEAE-52 ion exchange chromatography column. The solution was then purified using a 1 mmol·L⁻¹ solution. -1 EDTA and 0.2 mol·L -1 50 mmol·L⁻¹ NaCl -1 Elution with Tris-HCl buffer (pH 7.5) at a flow rate of 0.30 mL / min. -1 The absorbance of the eluent was measured at 280 nm and a curve was plotted. The preliminarily purified sporamin protein fraction was collected. Then, the preliminarily purified sporamin protein was concentrated using an ultrafiltration cup and further purified using a Sephadex G-75 gel chromatography column; 0.1 mol·L⁻¹ solution was used. -1 NaCl and 1 mmol·L -1 50 mmol·L of EDTA -1 Tris-HCl buffer (pH 7.5) was prepared at a temperature of 4°C and a flow rate of 0.45 ml / min. -1 Elution. The absorbance of the collected solution was measured at 280 nm to plot the elution curve. The pure sporamin protein was collected, lyophilized, and stored at -20°C.

[0051] (2) In an autoclave, treat the pure sporamin protein at 127°C to remove its trypsin inhibitor activity. After returning to room temperature, weigh the pure sporamin protein and PBS solution in a 1:1 ratio, vortex thoroughly, centrifuge at 4°C for 20 min, and collect the supernatant.

[0052] (3) Take 100 μL of supernatant, add 400 μL of 40 mM NH4HCO3 solution and vortex mix it, add 50 μL of 100 mM DTT solution, vortex mix thoroughly, react at room temperature for 60 min, then add 250 μL of 100 mM IAA solution and react in the dark at room temperature for 60 min.

[0053] (4) Add 2 μg of excess trypsin to the final solution from (3) and incubate overnight at 37°C. When the reaction is complete, add 1 μL of formic acid solution to inactivate the trypsin. Desalt the digested product by passing it through a C18 column, and then vacuum dry the desalted sample.

[0054] (5) Detection of peptides: The sample obtained in step (4) was dissolved in 0.1% formic acid solution and analyzed by liquid chromatography-tandem triple quadrupole mass spectrometry.

[0055] 4. Data Processing

[0056] The concentration of the characteristic peptide is obtained by substituting the peak area ratio of the characteristic peptide to the stable isotope internal standard peptide into the formula, and then the content of the characteristic peptide RPLGHDVQM is obtained according to Equation 1.

[0057] X=(ФcV) / m Equation 1

[0058] Where X (ng / g) is the content of the characteristic peptide RPLGHDVQM in the sweet potato sample, Ф is the proportion of the enzymatically digested protein volume to the total sample volume, c (ng / mL) is the concentration of the characteristic peptide in the trypsin digest, V (mL) is the volume of the trypsin digest, and m (g) is the mass of the sweet potato sample. This method aims to quantify the characteristic peptide RPLGHDVQM in the sweet potato sample.

[0059] After analyzing the sweet potato samples, the ion chromatograms, mass spectra, and secondary fragment mass spectra of the characteristic peptides are as follows: Figures 1-3 As shown, the ion chromatogram, mass spectrum, and secondary fragment mass spectrum of the internal standard (IS) peptide are as follows: Figures 4-6 As shown, the spectrum of the sweet potato sample should contain the precise mass number of the characteristic peptide RPLGHDVQM (m / z 525.76110); the spectrum of the sample should contain the precise mass number of the stable isotope internal standard peptide characteristic peptide RPLGHDVQM* (m / z 530.76523), with an allowable deviation of less than 5 ppm.

[0060] The MS / MS spectrum (diffusion ion spectrum) should contain characteristic fragment ions of the sweet potato characteristic peptide RPLGHDVQM at m / z 798.36887 and m / z 1051.52275. Correspondingly, the MS / MS spectrum (of the stable isotope internal standard peptide characteristic peptide RPLGHDVQM*) should contain fragment ions at m / z 808.83658 and m / z 1060.94784, and the error in their precise mass number should be less than 5 ppm. Only when the above characteristics are met simultaneously in terms of precise m / z value and characteristic fragment ions can the content of the characteristic peptide RPLGHDVQM in the sweet potato sample be considered reliable, and the quality of the sweet potato can be identified based on the content.

[0061] Example 3: Practical application of the characteristic peptide fragment of sporamin protein in sweet potato

[0062] We purchased samples of cassava, corn, sweet potato, potato, taro, etc. from legitimate markets for actual sample testing.

[0063] (1) Weigh the sample, wash, weigh, and chop it, and add 0.1% NaHSO3 at 50 mmol·L⁻¹. -1 Soak in Tris-HCl buffer solution (pH 7.5) (1 L·kg⁻¹) -1 (Fresh weight), homogenize, centrifuge the slurry at 3500 r / min for 15 min, collect the supernatant, add ammonium sulfate to 60% saturation under ice bath, let stand, centrifuge at 3500 r / min for 35 min, dissolve the precipitate, freeze dry to obtain crude protein powder. Weigh an appropriate amount of crude protein powder, add 100 mL of distilled water to dissolve it, centrifuge at 10000 r / min for 45 min. Filter the supernatant through a microporous membrane and then purify it through a DEAE-52 ion exchange chromatography column. Use a solution containing 1 mmol·L⁻¹ -1 EDTA and 0.2 mol·L -1 50 mmol·L⁻¹ NaCl -1 Elution with Tris-HCl buffer (pH 7.5) at a flow rate of 0.30 mL / min. -1 The absorbance of the eluent was measured at 280 nm and a curve was plotted. The preliminarily purified protein fraction was collected. Then, the preliminarily purified protein was concentrated using an ultrafiltration cup and further purified using a Sephadex G-75 gel chromatography column; 0.1 mol·L⁻¹ of precipitate was used. -1 NaCl and 1 mmol·L -1 50 mmol·L of EDTA -1 Tris-HCl buffer (pH 7.5) was prepared at a temperature of 4°C and a flow rate of 0.45 ml / min. -1 Elution. The absorbance of the collected solution was measured at 280 nm to plot the elution curve, and the purified protein was collected and lyophilized. The purified protein was treated in an autoclave at 127°C to remove its trypsin inhibitor activity. After returning to room temperature, the purified protein and PBS solution were weighed in a 1:1 ratio, vortexed thoroughly, and centrifuged at 4°C for 20 min. The supernatant was collected.

[0064] (2) Take 100 μL of supernatant and add 400 μL of 40 mM NH4HCO3 solution to mix with it by vortexing. Add 50 μL of 100 mM DTT solution and mix thoroughly by vortexing. React at room temperature for 60 min. Then add 250 μL of 100 mM IAA solution and react in the dark at room temperature for 60 min.

[0065] (3) Add 2 μg of excess trypsin to the final solution obtained in (2) and incubate overnight at 37°C. When the reaction is complete, add 1 μL of formic acid solution to inactivate the trypsin. Add IS peptide to each sample to ensure that its concentration remains at 10 ng / mL during subsequent reconstitution. Desalt the digested products and vacuum dry them. Dissolve the samples in 0.1% formic acid solution and analyze them by liquid chromatography-tandem triple quadrupole mass spectrometry.

[0066] As shown in Table 1, the characteristic peptide information was only detected in the sweet potato sample, and no characteristic peptide information was detected in other samples.

[0067] Table 1. Detection of sporamin protein in different varieties of samples

[0068]

[0069] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. The application of characteristic peptides of sweet potato sporamin protein in identifying the authenticity of sweet potato products, characterized in that, The sequence of the characteristic peptide is: RPLGHDVQM.

2. The application according to claim 1, characterized in that, The method of the application is to detect whether the sample contains the characteristic peptide; if the characteristic peptide is present, the sample is determined to contain sweet potato; if the characteristic peptide is not present, the sample is determined not to contain sweet potato.

3. The application according to claim 1, characterized in that, The application is to identify whether the sweet potato content in sweet potato products meets the stated sweet potato content.

4. The application according to claim 2, characterized in that, The detection was performed using liquid chromatography-tandem mass spectrometry. The precursor ion of the detection signal generated by the characteristic peptide in the mass spectrometer has a mass-to-charge ratio of m / z 525.76110; the daughter ions include daughter ions with mass-to-charge ratios of m / z 798.36887 and m / z 1051.52275, with an allowable deviation within 5 ppm.

5. The application according to claim 4, characterized in that, UHPLC-Q Exactive plus or triple quadrupole mass spectrometry was used for liquid chromatography-tandem mass spectrometry detection.

6. The application according to claim 1, characterized in that, The isotope internal standard method was used to quantitatively detect sweet potato sporamin protein. The internal standard peptide used was RPLGHDVQM*. The precursor ion of the detection signal generated by the internal standard peptide in mass spectrometry was m / z 530.76523. The daughter ions included daughter ions with mass-to-charge ratios of m / z 808.83658 and m / z 1060.94784, with an allowable deviation within 5 ppm.

7. The application according to claim 6, characterized in that, The concentration of the characteristic peptide is obtained by substituting the peak area ratio of the characteristic peptide to the stable isotope internal standard peptide into the formula, and then the content of the characteristic peptide RPLGHDVQM is obtained according to Equation 1. X = (Ф*c*V) / m Equation 1; Where X is the content of the characteristic peptide RPLGHDVQM in the sweet potato sample, Ф is the proportion of the enzymatically digested protein volume to the total sample volume, c is the concentration of the characteristic peptide in the trypsin digest, V is the volume of the trypsin digest, and m is the mass of the sweet potato sample.

8. The application according to claim 6, characterized in that, Sample pretreatment includes: extracting proteins from the sample to be tested and enzymatically digesting the proteins using trypsin.

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