Standard characteristic peptide group for mass spectrometry identification of casein glycomacropeptide in peptide products

Abstract

The present invention discloses a standard characteristic polypeptide group for mass spectrometry identification of casein glycomacropeptide in polypeptide products, which belongs to the field of inspection and detection technology. Since casein glycomacropeptide has a variety of possible glycosylation modifications, its mass spectrometry identification is relatively difficult. After defatting and precipitating the protein of the sample, the present invention obtains a standard characteristic polypeptide group without glycosylation modification sites by enzymatic cleavage of casein glycomacropeptide, thereby avoiding the influence of glycosylation on the identification of casein glycomacropeptide with precise mass number. The standard characteristic polypeptide group for mass spectrometry identification of casein glycomacropeptide comprises the following three target peptide segments, whose amino acid sequences are shown as SEQ ID No.1, SEQ ID No.2, and SEQID No.3, respectively, and the three are close to the N-terminus, middle part and C-terminus of casein glycomacropeptide, respectively. In a polypeptide sample from which the intact protein has been removed, this standard characteristic polypeptide group can be used to qualitatively identify whether a complete casein glycomacropeptide is present.

Description

Technical Field

[0001] The present invention belongs to the field of inspection and detection technology, and specifically discloses a standard characteristic polypeptide group of casein glycomacropeptide in polypeptide products identified by mass spectrometry, and utilizes this polypeptide group in combination with mass spectrometry to qualitatively detect casein glycomacropeptide in polypeptide products. Background Art

[0002] Casein glycomacropeptide is a glycosylated peptide derived from the enzymatic hydrolysis of κ-casein. It contains no phenylalanine and its glycosyl groups are rich in sialic acid. It has antibacterial and anti-inflammatory properties, antimicrobial and detoxifying properties, and promotes brain development in infants and young children. Therefore, it is an ideal ingredient in special medical formulas for people with phenylketonuria and infant formula. Qualitative identification of casein glycomacropeptide in such products can determine whether vendors are selling inferior products as genuine products.

[0003] The amino acid sequence of casein glycomacropeptide consists of 64 amino acids, from methionine 106 to valine 169 in κ-casein, with 11 variants. Type A casein glycomacropeptide predominates in nature, with Thr at positions 121, 131, 133, 136, and 142 as potential glycosylation sites, all of which are acetylgalactosyloxyglycosidic bonds. The posterior glycosyl groups primarily contain galactose and sialic acid, and a single glycosyl group can have varying compositions, from monosaccharides to tetrasaccharides. The theoretical molecular weight of casein glycomacropeptide ranges from 7 to 11 kDa, but it can form multimers with varying apparent molecular weights at specific pH levels. Consequently, casein glycomacropeptide exhibits a large molecular weight, complex glycosylation, and diverse molecular forms, resulting in the lack of a robust method for accurate analysis.

[0004] Currently, the detection methods for casein glycomacropeptide are primarily indirect, such as the resorcinol hydrochloride method, which measures sialic acid content and indirectly reflects the amount of casein glycomacropeptide, and electrophoresis, which measures casein glycomacropeptide polymers. These methods have significant errors and are susceptible to interference, making them incapable of accurately detecting casein glycomacropeptide in casein glycomacropeptide-containing protein foods and reflecting their true content. Chinese invention patent application number CN201810316627.6, entitled "A Mass Spectrometry Method for Detecting A1 / A2 β-Casein," states that the critical analytical conditions of this method are not suitable for the analysis and detection of casein glycomacropeptide in peptide products such as casein hydrolysates. Chinese invention patent application number CN201810487863.4, entitled "A Characteristic Peptide and Method for Detecting A2 β-Casein Content in Dairy Products," utilizes liquid chromatography-mass spectrometry, requiring a process designed specifically for the specific amino acid fragments of A2 β-casein and the introduction of an internal standard peptide with a specific sequence. This method suffers from shortcomings such as long detection time, low sample size, and high testing costs. Furthermore, the key analytical method is not suitable for the analysis and detection of casein glycomacropeptides in peptide products such as casein hydrolysates. Chinese invention patent application number CN201911419245.7, entitled "Mass Spectrometry Detection of Standard Characteristic Peptides for A1 and A2 β-Casein in Dairy Products," also utilizes a method for extracting target peptides and key analytical conditions that are not suitable for the analysis and detection of casein glycomacropeptides in peptide products such as casein hydrolysates.

[0005] The paper "Quantitative determination of bovine k-casein macropeptide in dairy products by Liquid chromatography / Electrospray coupled to mass spectrometry (LC-ESI / MS) and Liquid chromatography / Electrospray coupled to total mass spectrometry (LC-ESI / MS / MS)" uses RP-HPLC-ESI-MS technology to detect the contents of type A, type B, and total casein glycomacropeptide. The advantages and disadvantages of UV, SIM, and MRM detection modes for quantifying total casein glycomacropeptide content are analyzed. However, this method does not perform qualitative analysis of casein glycomacropeptide, and only uses a single peptide segment (162-169) obtained by hydrolyzing casein glycomacropeptide to determine total casein glycomacropeptide content. Therefore, adulteration of casein glycomacropeptide products may not be detected, and the quantitative results may be biased.

[0006] In summary, the identification methods of casein glycomacropeptides still have the disadvantages of complex spectral information, difficult analysis, and low accuracy. Therefore, it is necessary to establish a casein glycomacropeptide identification method with simpler spectral information, easier analysis, and higher accuracy. Summary of the Invention

[0007] The present invention provides a standard characteristic polypeptide group for mass spectrometry identification of casein glycomacropeptide in polypeptide products and a method for identifying casein glycomacropeptide in polypeptide products using the standard characteristic polypeptide group. The method can perform qualitative identification of casein glycomacropeptide in polypeptide products and has the advantages of simple and rapid operation, low cost, and high throughput.

[0008] The first inventive principle of the present invention is to study the molecular weight characteristics of casein glycomacropeptide at the whole protein level, and then select a protease to further enzymatically hydrolyze the casein glycomacropeptide based on the simulated enzyme cleavage results to obtain several non-glycosylated small peptide segments.

[0009] The second principle of the present invention is to analyze and evaluate the non-glycosylated small peptides obtained by the above enzymatic digestion through mass spectrometry results, and select three suitable peptides as qualitative peptides of casein glycomacropeptide.

[0010] The third principle of the present invention is to complete the qualitative identification of casein glycomacropeptide in polypeptide products such as casein hydrolysate by detecting qualitative peptide segments in polypeptide products such as casein hydrolysate based on the above principle.

[0011] Therefore, the first object of the present invention is to provide a standard characteristic polypeptide group of casein glycomacropeptide, wherein the standard characteristic polypeptide group of casein glycomacropeptide consists of target peptide segments with amino acid sequences as shown in SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3:

[0012] Target peptide 1: PPKKNQDKTEI,

[0013] Target peptide 2: EDSPEV,

[0014] Target peptide 3: ESPPEI.

[0015] The standard characteristic polypeptide sequence is used to qualitatively identify casein glycomacropeptide, and three target peptide segments of amino acid sequences such as SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3 are combined as a standard characteristic polypeptide group for qualitatively identifying casein glycomacropeptide.

[0016] The extraction of the standard characteristic peptide group is based on the use of pre-treatment conditions that selectively identify the sugar groups in casein glycomacropeptides, and the selective identification and separation of casein glycomacropeptides from actual samples.

[0017] In one embodiment, the target peptides 1 to 3 serve as a standard characteristic peptide group for qualitatively identifying casein glycomacropeptide, specifically: target peptide 1, target peptide 2, and target peptide 3 are located at the N-terminus, middle, and C-terminus of casein glycomacropeptide, respectively. When the mass spectrometry peaks of the above three peptides: single charge 1297.7 m / z (5‰) or double charge 649.3 m / z (5‰), single charge 675.3 m / z (5‰), and single charge 671.3 m / z (5‰) coexist, it indicates that the sample to be tested contains casein glycomacropeptide.

[0018] Specifically, the principle of using target peptides 1 to 3 as a standard characteristic peptide group for qualitative identification of casein glycomacropeptide is as follows:

[0019] (I) The mass spectrum peak of the characteristic polypeptide of the casein glycomacropeptide with an amino acid sequence as shown in SEQ ID No. 1 is a single charge 1297.7 m / z (5‰, indicating the mass-to-charge ratio deviation is allowed within ±5‰, the same below) or a doubly charged 649.3 m / z (5‰). When the single charge or doubly charged mass spectrum polypeptide peak appears in the ion current chromatogram and mass spectrum of the polypeptide segment of the sample to be tested, it indicates that the sample to be tested contains the target peptide segment 1, indicating that the sample to be tested contains casein glycomacropeptide;

[0020] (II) The mass spectrum peak of the characteristic polypeptide of the casein glycomacropeptide with the amino acid sequence as shown in SEQ ID No. 2 is a single charge 675.3 m / z (5‰). When the single charge mass spectrum peak of the polypeptide segment appears in the ion current chromatogram and mass spectrum of the sample to be tested, it indicates that the sample to be tested contains the target peptide segment 2, indicating that the sample to be tested contains casein glycomacropeptide;

[0021] (III) The mass spectrum peak of the characteristic polypeptide of the casein glycomacropeptide with an amino acid sequence as shown in SEQ ID No. 1 is a single-charged 671.3 m / z (5‰). When the single-charged mass spectrum polypeptide peak appears in the ion current chromatogram and mass spectrum of the polypeptide segment of the sample to be tested, it indicates that the sample to be tested contains the target peptide segment 3, indicating that the sample to be tested contains casein glycomacropeptide.

[0022] Similarly, when the single-charged 1297.7m / z (5‰) or doubly-charged 649.3m / z (5‰), single-charged 675.3m / z (5‰), and single-charged 671.3m / z (5‰) mass spectrum polypeptide peaks appear simultaneously in the ion current chromatogram and mass spectrum of the polypeptide segment of the sample to be tested, it means that the sample to be tested contains target peptide segments 1 to 3 at the same time. Then, this means that the sample to be tested contains complete casein glycomacropeptide.

[0023] In the above embodiment, since the characteristic polypeptide with the amino acid sequence as shown in SEQ ID No. 1 has two charged forms, single charge and double charge, the position of the double-charged polypeptide SEQ ID No. 1 in the mass spectrum is half that of the single-charged polypeptide with the same mass unit, that is, the mass-to-charge ratio of the two is half.

[0024] The second object of the present invention is to provide a mass spectrometry model for identifying casein glycomacropeptide in polypeptide products, wherein the mass spectrometry model includes a standard characteristic polypeptide group of the casein glycomacropeptide.

[0025] In the mass spectrometry model, the standard characteristic polypeptide sequence is used to qualitatively identify casein glycomacropeptide, wherein target peptide 1, target peptide 2, and target peptide 3 are located at the N-terminus, middle, and C-terminus of casein glycomacropeptide, respectively. When the mass spectrometry peaks of the above three peptides: single charge 1297.7 m / z (5‰) or double charge 649.3 m / z (5‰), single charge 675.3 m / z (5‰), and single charge 671.3 m / z (5‰) coexist, it indicates that the sample to be tested contains casein glycomacropeptide.

[0026] The third object of the present invention is to provide a method for constructing the mass spectrometry model, comprising:

[0027] S1. Study the pure casein glycomacropeptide at the whole protein level. Through enzyme cleavage simulation, select a suitable enzyme (protease) to further enzymatically hydrolyze the casein glycomacropeptide to obtain several non-glycosylated small peptide segments and their polypeptide sequences.

[0028] S2. Perform mass spectrometry on the non-glycosylated small peptide fragments obtained by enzyme digestion to obtain a mass spectrum and collect data;

[0029] S3. Analyze and evaluate the obtained data, and screen out a standard characteristic peptide group that can be used for qualitative analysis. The standard characteristic peptide group has three characteristic peptides with the following mass-to-charge ratio peaks: the mass spectrum peak of target peptide 1 is single-charged 1297.7m / z (5‰) or doubly charged 649.3m / z (5‰), the mass spectrum peak of target peptide 2 is single-charged 675.3m / z (5‰), and the mass spectrum peak of target peptide 3 is single-charged 671.3m / z (5‰). According to the three mass-to-charge ratios of the target peptides 1, 2, and 3 of the polypeptide sequence, a qualitative mass spectrometry model of casein glycomacropeptide is established.

[0030] In one embodiment, the enzymatic hydrolysis method of step S1 includes removing oil and fat using a low-polarity organic solvent and removing whole protein using a protein precipitant, and then enzymatic hydrolysis using a protease to obtain a polypeptide solution.

[0031] In one embodiment, the low-polarity organic solvent comprises n-hexane.

[0032] In one embodiment, the protease used in the enzymatic hydrolysis method of step S1 includes proteinase K or endoproteinase Lys C.

[0033] In one embodiment, the analysis and evaluation in step S3 includes retaining mass spectrum data with a signal-to-noise ratio greater than 3, and performing evaluations based on four aspects: ion peak intensity, fragment peak intensity, specificity, and degree of enzymatic hydrolysis.

[0034] The fourth object of the present invention is to provide a method for qualitatively detecting casein glycomacropeptide, wherein the method utilizes mass spectrometry to detect a standard characteristic polypeptide group of the casein glycomacropeptide.

[0035] In one embodiment, the method comprises the following steps:

[0036] (1) Pretreatment: After removing the oil and whole protein in the sample to be tested, extract the casein glycomacropeptide with sugar groups in the sample, hydrolyze it with protease in a buffer solution, and filter it to obtain the sample pretreatment solution;

[0037] (2) Detection of casein glycomacropeptide: The sample pretreatment solution is filtered through a filter membrane for mass spectrometry detection to obtain an ion current chromatogram and mass spectrum of the sample polypeptide to be detected;

[0038] (3) Data analysis: If the ion current chromatogram and mass spectrum contain mass spectrometry peaks of the standard characteristic peptide group of the casein glycomacropeptide, it is determined that the sample to be tested contains casein glycomacropeptide.

[0039] In one embodiment, in step (1), a low-polarity organic solvent is used to remove oil from the sample to be tested.

[0040] In one embodiment, the low-polarity organic solvent comprises n-hexane.

[0041] In one embodiment, in step (1), the final concentration of the protease is not less than 0.05 mg mL -1 .

[0042] In one embodiment, in step (1), the enzymatic hydrolysis condition is 55-65° C. for 8 hours.

[0043] In one embodiment, the protease comprises proteinase K or endoproteinase Lys C.

[0044] In one embodiment, the protease is proteinase K.

[0045] In one embodiment, in step (1), a boron affinity column is used to enrich and separate casein glycomacropeptides with sugar groups in the sample.

[0046] In one embodiment, in step (2), the mobile phase conditions of the mass spectrometry are: initial mobile phase A accounts for 100%, 40-45 minutes mobile phase A accounts for 70%, mobile phase B accounts for 30%; 45-50 minutes mobile phase A accounts for 20%, mobile phase B accounts for 80%; 50-55 minutes mobile phase B accounts for 100%, 55 minutes mobile phase A accounts for 100%;

[0047] Mobile phase A was 100% 0.1 formic acid, and mobile phase B was acetonitrile.

[0048] In one embodiment, the mobile phase flow rate is set to 0.1 to 0.5 mL min -1 ,

[0049] In one embodiment, the chromatographic column is BEH C18 2.1×120 mm 1.7 μm, and the column temperature is 35-45°C.

[0050] In one embodiment, the mass spectrometry detection conditions are: positive ion mode, scanning mode: MRM, declustering voltage: 30-40 V, inlet voltage: 8-15 V, ion source voltage: 4000-5000 V, ion source temperature: 550° C., collision energy: 20-50 V.

[0051] In one embodiment, in step (3), the mass spectrum peak of target peptide 1 is a single charge of 1297.7 m / z (5‰) or a double charge of 649.3 m / z (5‰), the mass spectrum peak of target peptide 2 is a single charge of 675.3 m / z (5‰), and the mass spectrum peak of target peptide 3 is a single charge of 671.3 m / z (5‰).

[0052] The fifth object of the present invention is to provide a detection kit for qualitatively detecting caseinoglycomacropeptide, wherein the kit contains polypeptides having amino acid sequences as shown in SEQ ID Nos. 1 to 3.

[0053] In one embodiment, the detection kit further contains a buffer, a low-polarity solvent, and a protease.

[0054] In one embodiment, the protease is proteinase K.

[0055] The sixth object of the present invention is to provide an application of the above-mentioned standard characteristic polypeptide group, or the above-mentioned mass spectrometry model, or the above-mentioned method in the detection of casein glycomacropeptide.

[0056] Beneficial effects

[0057] Compared with the prior art, the present invention has the following beneficial effects:

[0058] 1. Through the method of the present invention, a standard characteristic polypeptide group without glycosylation modification sites is obtained by enzymatic cleavage of casein glycomacropeptide, which avoids the influence of glycosylation on the identification of casein glycomacropeptide with precise mass number, and can accurately and qualitatively identify casein glycomacropeptide in polypeptide products.

[0059] 2. The present invention adopts the HPLC-ESI-QTOF-MS method and proposes to achieve the qualitative identification of casein glycomacropeptide in polypeptide products by identifying the characteristic polypeptide group mass spectrum peak of casein glycomacropeptide.

[0060] 3. This method has the advantages of simple and rapid operation, low cost and high throughput. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 (a) Extracted ion chromatogram of target peptide 1, (b) primary mass spectrum of corresponding retention time, (c) magnified image of 649.3 m / z ion, and (d) magnified image of 1297.6 m / z ion.

[0062] Figure 2 (a) Fragment ions generated by peptide chain cleavage and (b) MS / MS spectrum of target peptide 1.

[0063] Figure 3 (a) Extracted ion chromatogram of target peptide 2, (b) primary mass spectrum of corresponding retention time, and (c) magnified image of 675.3 m / z ion.

[0064] Figure 4 (a) Fragment ions generated by peptide chain cleavage and (b) MS / MS spectrum of target peptide 2.

[0065] Figure 5 (a) Extracted ion chromatogram of target peptide 3, (b) primary mass spectrum of corresponding retention time, and (c) magnified image of 671.3 m / z ion.

[0066] Figure 6 (a) Fragment ions generated by peptide chain cleavage and (b) MS / MS spectrum of target peptide 3.

[0067] Figure 7 (a) extracted ion chromatogram and (b) primary mass spectrum of the corresponding retention times of target peptide 1, target peptide 2, and target peptide 3 in the enzymatic hydrolysis sample of bovine milk powder to be tested in Example 5 (from bottom to top: target peptide 1, target peptide 2, target peptide 3).

[0068] Figure 8 This is the extracted ion current chromatogram of the enzymatic hydrolysis sample of goat milk powder to be tested in Example 6 (from bottom to top: target peptide 1, target peptide 2, target peptide 3). DETAILED DESCRIPTION

[0069] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0070] Example 1. Enzymatic peptide simulation

[0071] 1. Enzymatic peptide simulation

[0072] The PeptideMass program was used to simulate protease cleavage and mass spectrometry corresponding to mass-to-charge ratios. Simulation conditions included obtaining monoisotopic molecular weights, no cysteine treatment, and displaying peptides with masses greater than 500 Da.

[0073] 2. Results Analysis

[0074] PeptideMass was used to simulate the enzymatic hydrolysis of casein glycomacropeptide by common endoproteinases, and the enzymatic hydrolysis results were analyzed, as shown in Table 1:

[0075] Table 1. Fragments obtained by enzymatic hydrolysis of casein glycomacropeptide

[0076]

[0077]

[0078] As can be seen from Table 1, proteinase K can effectively hydrolyze casein glycomacropeptide into small peptide fragments. Proteinase K is inexpensive, has mild hydrolysis conditions, and is easy to terminate the reaction and separate. Therefore, it is an ideal enzyme for the hydrolysis of casein glycomacropeptide. Proteinase K was used for hydrolysis in the following examples.

[0079] Example 2. Proteinase K enzymatic hydrolysis of casein glycomacropeptide and target peptide screening

[0080] 1. Proteinase K enzymatic hydrolysis of casein glycomacropeptide

[0081] a) Prepare 20 mg mL -1 Proteinase K stock solution: Weigh 20 mg of proteinase K, dissolve in 1 mL of purified water, shake gently until completely dissolved, aliquot into 50 μL tubes, and store at -20°C.

[0082] b) Preparation of 50 mM pH 7.5 Tris-HCl, 10 mM CaCl2 buffer: Weigh 6.06 g Tris and 1.11 g CaCl2 and dissolve them in 900 mL of purified water. Add concentrated HCl dropwise with constant stirring to adjust the pH to 7.5. Add water to make up to 1000 mL.

[0083] c) Dissolve 10 mg of lyophilized pure casein glycomacropeptide in 10 mL of the above buffer, add 25 μL of proteinase K stock solution, and incubate at 58°C for 8 h.

[0084] d) After enzymatic hydrolysis, the enzyme was inactivated at 95°C for 10 min and centrifuged at 8000 rpm for 10 min.

[0085] e) Dialyze the sample using a 300 Da dialysis bag for 2 days, retain the dialysate, and store at 4°C until testing.

[0086] 2. Target peptide screening

[0087] Based on the site of proteinase K enzymatic hydrolysis of casein glycomacropeptide, peptides containing five or more amino acid residues were selected. Four peptides containing more than five amino acid residues were generated by enzymatic hydrolysis, as shown in Table 2: the 11-peptide PPKKNQDKTEI located at positions 109-119, the 12-peptide SGEPTSTPTTEA located at positions 127-138, the 6-peptide EDSPEV located at positions 147-152, and the 6-peptide ESPPEI located at positions 154-159. The 12-peptide located at positions 127-138 is not an ideal peptide for quantification because it contains a glycosylation site. Therefore, the 11-peptide PPKKNQDKTEI located at positions 109-119 (SEQ ID No. 1), the 6-peptide EDSPEV located at positions 147-152 (SEQ ID No. 2), and the 6-peptide ESPPEI located at positions 154-159 (SEQ ID No. 3) were selected as the three standard characteristic peptides. According to its site characteristics, it can be seen that the three peptide segments shown in amino acid sequences such as SEQ ID No. 1, SEQ ID No. 2, and SEQ ID No. 3 are located at the N-terminus, middle, and C-terminus of the casein glycomacropeptide, respectively.

[0088] Table 2 Proteinase K enzymatic peptides (pentapeptides and above)

[0089]

[0090] Example 3. Detection and analysis of target peptides

[0091] 1. HPLC-ESI-Q-TOF MS detection of enzymatic fragments

[0092] The mass spectrometer used in this embodiment is: QTRAP4500 liquid chromatography-mass spectrometer (Ab Sciex, USA).

[0093] The liquid phase conditions and mass spectrometry mode used in this example are as follows:

[0094] Liquid phase conditions: chromatographic column: BEH C18 2.1×120 mm 1.7 μm, mobile phase A: 100% 0.1% formic acid, mobile phase B: acetonitrile, gradient elution: initial 100% A, 40 min 70% A + 30% B, 45 min 20% A + 80% B, 50 min 100% B, 55 min 100% A. Flow rate: 0.3 mL / min -1 , column temperature 45℃, injection volume 5μL.

[0095] Mass spectrometry conditions: positive ion mode, capillary voltage 3.5 kV, cone voltage 30 V, ion source temperature: 100 °C, desolvation temperature: 400 °C, desolvation gas flow rate: 700 lit hr -1 , cone gas flow: 50lit hr -1 , collision energy: 6 / 20V, mass range 50-2000m / z, detector voltage: 1800V.

[0096] 2. Peptide sequence retrieval

[0097] The BLAST function of the protein database Uniprot was used to search for peptides. The database selected was UniprotKBreference proteomes plus Swiss-Prot, the E-threshold was 1000, the Matrix selected was Auto, the Filtering selected was None, the Gapped selected was yes, and the Hits selected was 1000. The search results are shown in Tables 3, 4, and 5.

[0098] Table 3 BLAST search results of target peptide 1

[0099]

[0100]

[0101] Table 4 BLAST search results of target peptide 2

[0102]

[0103] Table 5 BLAST search results of target peptide 3

[0104]

[0105] 3. Target peptide analysis and evaluation

[0106] according to Figures 1-6The mass spectra of target peptide 1, target peptide 2, and target peptide 3 shown in the figure, the BLAST peptide sequence search results shown in Tables 3-5, and the simulated enzyme digestion results shown in Table 1 were used to evaluate the three target peptides from four aspects: ion peak intensity, fragment peak intensity, specificity, and degree of enzymatic digestion. The evaluation results are shown in Table 6.

[0107] Table 6 Comparison of three peptides

[0108]

[0109] The main characteristic peaks of the tandem quadrupole time-of-flight mass spectrometry of the three target peptides are shown in Table 7.

[0110] Table 7 Main characteristic peaks of tandem quadrupole time-of-flight mass spectrometry

[0111] name Single charge / double charge m / z Allowable offset range Target peptide 1 Single charge 1297.9 Within ±5‰ Target peptide 1 Double charge 649.3 Within ±5‰ Target peptide 2 Single charge 675.3 Within ±5‰ Target peptide 3 Single charge 671.3 Within ±5‰

[0112] According to the comprehensive evaluation and comparison of the results in Tables 3 to 7, the target peptide 1 with an amino acid sequence as shown in SEQ ID No. 1, the target peptide 2 with an amino acid sequence as shown in SEQ ID No. 2, and the target peptide 3 with an amino acid sequence as shown in SEQ ID No. 3 are located at the N-terminus, middle, and C-terminus of the casein glycomacropeptide, respectively. Therefore, the combination of target peptide 1, target peptide 2, and target peptide 3 can be used as a basis for qualitative identification of whether the sample contains a complete casein glycomacropeptide.

[0113] Example 4. Qualitative identification of casein glycomacropeptide by HPLC-ESI-QqQ MS

[0114] 1. HPLC-ESI-QqQ MS Identification of Casein Glycomacropeptides

[0115] The mass spectrometer used in the present invention is a MALDI SYNAPT MS ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometer (Waters Corporation, USA).

[0116] The liquid chromatography conditions and mass spectrometry conditions used in this example are as follows:

[0117] Liquid chromatography conditions: chromatographic column is AgilentAdvancePeptidemapping 2.1×150mm 2.7μm, mobile phase A is 100% formic acid, mobile phase B is acetonitrile, gradient elution, 0-5min 98% A + 2% B, 5-20min 70% A + 30% B, 20-25min 70% A + 30% B, 25-28min 98% A + 2% B, 28-30min 98% A + 2% B. Flow rate 0.3mLmin -1, column temperature 40℃, injection volume 10μL.

[0118] Mass spectrometry conditions: positive ion mode, scan mode: MRM, declustering voltage: 35 V, inlet voltage: 10 V, ion source voltage: 4500 V, ion source temperature: 550 °C, collision energy: 40 V, ion source gas 1: 60 psi, ion source gas 2: 40 psi.

[0119] (1) Sample preparation: Weigh 5-10 g of food sample and dissolve it in 30 mL of deionized water. Add 10 mL of n-hexane and shake to remove fat. Let it stand until it separates into layers. Then remove the organic phase and repeat three times. Use trichloroacetic acid to precipitate protein to further remove the whole protein. Use a boron affinity column to enrich and separate the sugar-containing casein glycomacropeptide in the sample. The final aqueous phase is pre-cooled and placed in a freeze dryer for lyophilization.

[0120] (2) Preparation of sample buffer: 50 mmol L -1 pH 7.5 Tris-HCl, 10 mmol L -1 Preparation of CaCl2 buffer: Weigh 6.06g Tris and 1.11g CaCl2 and dissolve them in 900mL of purified water. Add concentrated HCl dropwise while stirring continuously to adjust the pH to 7.5. Add water to make up to 1000mL.

[0121] (3) Proteinase K enzymatic hydrolysis: Dissolve 100 mg of the lyophilized sample in 5 mL of sample buffer, add 25 μL of proteinase K stock solution, and perform enzymatic hydrolysis at 58°C for 8 h. After the enzymatic hydrolysis, inactivate the enzyme at 95°C for 10 min, centrifuge at 4310 g for 10 min, retain the supernatant, dialyze with a 300 Da dialysis bag for 2 days, retain the dialyzed fluid, and store at 4°C for testing.

[0122] (4) The enzymatic hydrolyzate of the sample to be tested was filtered through a 0.22 μm aqueous filter membrane and then detected by HPLC-ESI-QqQ MS (the same as the HPLC-ESI-Q-TOF MS detection method in Example 3) to obtain the ion current chromatogram and mass spectrum of the polypeptide in the sample to be tested. The presence of casein glycomacropeptide was determined based on whether the ion current chromatogram and mass spectrum contained the mass peaks (mass-to-charge ratio) of the target peptide segments 1 to 3. The specific determination method was as follows:

[0123] (i) When a single-charged 1297.7 m / z (5‰) or a doubly-charged 649.3 m / z (5‰) mass spectrum peptide peak appears in the ion current chromatogram and mass spectrum of the peptide segment of the sample to be tested, it indicates that the sample to be tested contains the target peptide segment 1. In this case, it indicates that the sample to be tested may contain casein glycomacropeptide;

[0124] (ii) When a single-charged 675.3 m / z (5‰) mass spectrum peptide peak appears in the ion current chromatogram and mass spectrum of the peptide segment of the sample to be tested, it indicates that the sample to be tested contains the target peptide segment 2. In this case, it indicates that the sample to be tested may contain casein glycomacropeptide;

[0125] (iii) When a single-charged 671.3 m / z (5‰) mass spectrum peptide peak appears in the ion current chromatogram and mass spectrum of the peptide segment of the sample to be tested, it indicates that the sample to be tested contains the target peptide segment 3. In this case, it indicates that the sample to be tested may contain casein glycomacropeptide;

[0126] (iv) When the peaks of single-charged 1297.7 m / z (5‰) or doubly-charged 649.3 m / z (5‰), single-charged 675.3 m / z (5‰), and single-charged 671.3 m / z (5‰) appear simultaneously in the ion current chromatogram and mass spectrum of the polypeptide segment of the sample to be tested, it indicates that the sample to be tested contains target peptide segments 1 to 3 at the same time. In this case, it means that the sample to be tested contains complete casein glycomacropeptide.

[0127] Example 5 Qualitative experiment of casein glycomacropeptide in enzymatic hydrolysis sample of cow milk powder

[0128] Referring to the method described in Example 4, a qualitative experiment was conducted on casein glycomacropeptide in cow's milk powder samples.

[0129] The source of the cow's milk powder sample in this embodiment is: Xinnong Tianshang Tianshan adult whole milk powder.

[0130] The ion chromatogram and mass spectrum of the enzymatic hydrolysis sample of milk powder to be tested are as follows: Figure 7 As shown, according to its ion current chromatogram and mass spectrum, it can be seen that the mass spectrum peptide peaks of single charge 1297.7m / z (5‰) and doubly charged 649.3m / z (5‰), single charge 675.3m / z (5‰), and single charge 671.3m / z (5‰) appear simultaneously in the figure, indicating that the target peptides 1 to 3 are contained in the enzymatic hydrolysis sample of the cow's milk powder to be tested. Then, at this time, it means that the enzymatic hydrolysis sample of the cow's milk powder to be tested contains complete casein glycomacropeptide, thereby completing the qualitative detection of casein glycomacropeptide in the enzymatic hydrolysis sample of the cow's milk powder to be tested.

[0131] Example 6 Qualitative experiment of casein glycomacropeptide in enzymatic hydrolysis sample of goat milk powder

[0132] Referring to the method described in Example 4, qualitative and quantitative experiments were performed on casein glycomacropeptide in goat milk powder samples.

[0133] The source of the goat milk powder sample in this embodiment is: Youaibet pure goat milk powder.

[0134] The ion chromatogram of the enzymatic hydrolysis sample of goat milk powder to be tested is as follows: Figure 8 As shown, according to its ion current chromatogram and mass spectrum, it can be seen that the chromatographic peak signals of single charge 1297.7m / z (5‰) and doubly charged 649.3m / z (5‰), single charge 675.3m / z (5‰), and single charge 671.3m / z (5‰) in the figure are extremely low, which can be judged as baseline signals, indicating that the enzymatic hydrolysis sample of goat milk powder to be tested does not contain target peptides 1 to 3. Then, at this time, it means that the enzymatic hydrolysis sample of goat milk powder to be tested does not contain complete casein glycomacropeptide, thereby completing the qualitative detection of casein glycomacropeptide in the enzymatic hydrolysis sample of goat milk powder to be tested.

[0135] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for identifying casein glycomacropeptide in a polypeptide product by mass spectrometry, characterized in that The following steps are involved: (1) Pretreatment: After removing oil and whole protein from the sample to be tested, it is enzymatically hydrolyzed in a buffer solution using a protease to obtain a sample pretreatment solution; (2) Detection of casein glycomacropeptide: The sample pretreatment solution is filtered through a filter membrane and then detected by liquid chromatography-mass spectrometry to obtain an ion current chromatogram and mass spectrum of the sample polypeptide to be detected; (3) Data analysis: If the ion current chromatogram and mass spectrum simultaneously contain the mass spectrometry peaks of the three peptides of the standard characteristic peptide group of casein glycomacropeptide, it is determined that the sample to be tested contains casein glycomacropeptide; The standard characteristic polypeptide group of the casein glycomacropeptide is composed of three polypeptides, the amino acid sequences of the three polypeptides are shown in SEQ ID No.1, SEQ ID No.2, and SEQ ID No.

3. The mass spectrometry peak of the polypeptide shown in SEQ ID No.1 is a single charge of 1297.7m / z (5‰) or a double charge of 649.3m / z (5‰), the mass spectrometry peak of the polypeptide shown in SEQ ID No.2 is a single charge of 675.3m / z (5‰), and the mass spectrometry peak of the polypeptide shown in SEQ ID No.3 is a single charge of 671.3m / z (5‰).

2. The method according to claim 1, characterized in that The protease in step (1) includes proteinase K or endoproteinase Lys C, and the final concentration of the protease is not less than 0.05 mg mL -1 .

3. The method according to claim 1, characterized in that The mobile phase conditions of the chromatography in step (2) are as follows: initial mobile phase A accounts for 100%, mobile phase A accounts for 70% and mobile phase B accounts for 30% during 40-45 minutes; mobile phase A accounts for 20% and mobile phase B accounts for 80% during 45-50 minutes; mobile phase B accounts for 100% during 50-55 minutes and mobile phase A accounts for 100% during 55 minutes; mobile phase A is 100% 0.1 formic acid and mobile phase B is acetonitrile.

4. The method according to claim 1, wherein The mass spectrometry detection conditions in step (2) are: positive ion mode, scan mode: MRM, declustering voltage: 30-40 V, inlet voltage: 8-15 V, ion source voltage: 4000-5000 V, ion source temperature: 550° C., collision energy: 20-50 V.

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