METHOD FOR DETECTING CONTENTS OF WHEY PROTEIN AND CASEIN, AND/OR RATIO THEREOF IN MILK POWDER

Abstract

The present disclosure relates to a method for detecting the contents of whey protein and casein, and/or the ratio thereof in milk powder by liquid chromatography-mass spectrometry. According to the method of the present disclosure, the characteristic peptide segments of whey protein and casein are selected and used for correction, and thus the accuracy and stability of detection of peptide segments by mass spectrometry using external standards are improved; in addition, the method of the present disclosure has the following advantages: easy to pretreat samples, low cost, a wide range of applicability, enable to perform quantitative detection of whey protein at one time, not affected by protein denaturation in the production process, no need to synthesize isotope internal standard, and avoiding influence of ionization efficiency and matrix in mass spectrometry detection by means of their own multiple characteristic peptide segment ratios.

Claims

1. A method for detecting the contents of whey protein and casein, and/or the ratio thereof in milk powder by liquid chromatography-mass spectrometry, comprising: 1) preparing a standard solution of whey protein with the characteristic peptide segments of α-lactalbumin and β-lactoglobulin, in accordance with the α-lactalbumin and β-lactoglobulin accounting for 20% and 50% of the whey protein, respectively; 2) preparing a standard solution of casein with the characteristic peptide segments of α-casein and β-casein, in accordance with the α-casein and β-casein accounting for 50% and 40% of the casein, respectively; 3) mixing the standard solution of whey protein from step 1) with the standard solution of casein from step 2) to prepare a series of mixed protein standard solutions with a series of whey protein:casein ratios; 4) drawing standard curves with the ratio of whey protein to casein as abscissa and the peak area ratios of characteristic peptide segments of α-lactalbumin to β-casein and the peak area ratios of characteristic peptide segments of β-lactoglobulin to α-casein as ordinate; 5) detecting the milk powder to be tested for the peak area ratios of the characteristic peptide segments of β-lactoglobulin to α-casein and the peak area ratios of the characteristic peptide segments of α-lactalbumin to β-casein; 6) substituting the peak area ratio of the characteristic peptide segments of β-lactoglobulin to α-casein in the detected milk powder into the standard curve to determine the ratio M of whey protein to casein, and calculate the actual amounts of β-lactoglobulin and α-casein based on the ratio M thus determined; and, substituting the peak area ratio of the characteristic peptide segments of α-lactalbumin to β-casein in the detected milk powder into the standard curve to determine the ratio N of whey protein to casein, and calculate the actual amounts of α-lactalbumin to β-casein based on the ratio N thus determined; 7) acquiring the actual contents of whey protein and casein, and/or the actual ratio of whey protein/casein based on the actual amounts of α-lactalbumin, β-casein, β-lactoglobulin, and α-casein obtained from step 6).

2. The method according to claim 1, wherein the characteristic peptide segments of α-lactalbumin, β-casein, β-lactoglobulin, and α-casein are LDQWLCEK shown in SEQ ID.NO.1, VLPVPQK shown in SEQ ID.NO.2, TPEVDDEALEK shown in SEQ ID NO.3, and FALPQYLK shown in SEQ ID NO.4, respectively.

3. The method according to claim 1, wherein in step 3), the series of whey protein:casein ratios include whey protein:casein ratios of 0.25, 0.43, 0.67, 1, 1.5, and 2.33, respectively.

4. The method according to claim 1, wherein in step 6), the actual amounts of β-lactoglobulin and α-casein are calculated by the following formulas, respectively: $\beta -\mathrm{lactoglobulin}=\frac{M1}{M1+M2}X*50\%,\alpha -\mathrm{casein}=\frac{M2}{M1+M2}X*50\%;$ Wherein, M1 is the preceding term in the ratio M of whey protein:casein as determined based on the peak area ratio of characteristic peptide segments of β-lactoglobulin to α-casein, and M2 is the latter term in the ratio M of whey protein:casein as determined based on the peak area ratio of characteristic peptide segments of β-lactoglobulin to α-casein, with the provisions of: M=M1/M2, and M1+M2=1; and, wherein, X is the total protein concentration of the sample.

5. The method according to claim 1, wherein in step 6), the actual amounts of α-lactalbumin and β-casein are calculated by the following formulas, respectively: $\alpha -\mathrm{la}=\frac{N1}{N1+N2}X*20\%,\beta -\mathrm{cs}=\frac{N2}{N1+N2}X*40\%;$ wherein, N1 is the preceding term in the ratio N of whey protein:casein as determined based on the peak area ratio of characteristic peptide segments of α-lactalbumin to β-casein, and N2 is the latter term in the ratio N of whey protein:casein as determined based on the peak area ratio of characteristic peptide segments of α-lactalbumin to β-casein, with the provisions of: N=N1/N2, and N1+N2=1; and, wherein, X is the total protein concentration of the sample.

6. The method according to claim 1, wherein, in step 7), the formula for calculating the actual content of whey protein is: $\frac{\frac{N1}{N1+N2}X*20\%+\frac{M1}{M1+M2}X*50\%}{0.7}$ the formula for calculating the actual content of casein is: $\frac{\frac{M1}{M1+M2}X*50\%+\frac{N2}{N1+N2}X*40\%}{0.9}$ the formula for calculating the actual ratio of whey protein/casein is: $\frac{\frac{N1}{N1+N2}X*20\%+\frac{M1}{M1+M2}X*50\%}{0.7}\text{⁠}/\frac{\frac{M2}{M1+M2}X*50\%+\frac{N2}{N1+N2}X*40\%}{0.9}=\frac{18N1+45M1}{35M2+28N2}=\frac{63\mathrm{MN}+18N+45M}{35N+28M+63};$ wherein, M1 is the preceding term in the ratio M of whey protein:casein as determined based on the peak area ratio of characteristic peptide segments of β-lactoglobulin to α-casein, and M2 is the latter term in the ratio M of whey protein:casein as determined based on the peak area ratio of characteristic peptide segments of β-lactoglobulin to α-casein, with the provisions of: M=M1/M2, and M1+M2=1; wherein, N1 is the preceding term in the ratio N of whey protein:casein as determined based on the peak area ratio of characteristic peptide segments of α-lactalbumin to β-casein, and N2 is the latter term in the ratio N of whey protein:casein as determined based on the peak area ratio of characteristic peptide segments of α-lactalbumin to β-casein, with the provisions of: N=N1/N2, and N1+N2=1; and, wherein, X is the total protein concentration of the sample.

7. The method according to claim 1, wherein, the detection in step 5) comprises the steps of sample treatment and enzyme digestion: dissolving the milk powder sample to be tested into a sample solution with a protein concentration of 0.1-0.4 mg/mL; subjecting the sample solution to rough filtration with a pure acetic acid filter membrane with a pore size of 0.45 μm; adding the roughly filtered sample solution into ammonium bicarbonate solution, followed by adding dithiothreitol solution, and standing in a water bath at 65-75° C. for 25-35 min; after cooling, adding iodoacetamide solution, and standing for 25-35 min in the dark; illuminating the mixture for 8-12 min, followed by adding calcium chloride solution; then adding trypsin solution to allow an enzyme digestion at 37° C. for 26-30 h; then adding acetic acid solution to stop the enzyme digestion; filtering the reaction mixture with polyethersulfone filter membrane, and then performing selective ion scanning analysis by mass spectrometry.

8. The method according to claim 1, wherein the upper detection limit of the method is at a total protein concentration of 0.4 mg/mL.

9. A combination of characteristic peptide segments for detecting the ratio of whey protein:casein by liquid chromatography-mass spectrometry, comprising: characteristic peptide segments of α-lactalbumin, β-casein, β-lactoglobulin, and α-casein with amino acid sequences as shown in SEQ ID.NO.1, SEQ ID.NO.2, SEQ ID.NO.3, and SEQ ID.NO.4, respectively.

10. The combination of characteristic peptide segments according to claim 9, which consists of the characteristic peptide segments of α-lactalbumin, β-casein, β-lactoglobulin, and α-casein with amino acid sequences as shown in SEQ ID.NO.1, SEQ ID.NO.2, SEQ ID.NO.3, and SEQ ID.NO.4, respectively.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0042] One or more examples are exemplified by the pictures in the accompanying drawings that correspond thereto and are not intended to be limiting of the embodiments. As used herein, the word “exemplary” means “serving as an example, embodiment, or illustrative”. Any embodiment described herein as “exemplary” is not necessarily to be construed as superior or better than other embodiments.

[0043] FIG. 1 is a full scanning mass spectrum of the mixed standard substance used in the Example;

[0044] FIG. 2 is a full scanning mass spectrum of the milk powder sample;

[0045] FIG. 3 is the mass spectrum of four characteristic peptide segments in PRM mode;

[0046] FIG. 4 is the full fragment ion diagram of the characteristic peptide segment (LDQWLCEK) shown in SEQ ID NO.1;

[0047] FIG. 5 is the full fragment ion diagram of the characteristic peptide segment (TPEVDDEALEK) shown in SEQ ID NO.3;

[0048] FIG. 6 is the full fragment ion diagram of the characteristic peptide segment (FALPQYLK) shown in SEQ ID NO.4;

[0049] FIG. 7 is the full fragment ion diagram of the characteristic peptide segment (VLPVPQK) shown in SEQ ID NO.2;

[0050] FIG. 8 is a quantitative daughter ion peak profile of four characteristic peptide segments;

[0051] FIG. 9 shows the change of peak area ratio of characteristic peptide segments LDQWLCEK(SEQ ID NO.1)/VLPVPQK(SEQ ID NO.2) at different enzyme concentrations and with different enzymolysis time, with the enzymolysis time as abscissa, and the peak area ratio of two characteristic peptide segments as ordinate;

[0052] FIG. 10 shows the change of peak area ratio of characteristic peptide segments TPEVDDEALEK(SEQ ID NO.3)/FALPQYLK(SEQ ID NO.4) at different enzyme concentrations and with different enzymolysis time, with the enzymolysis time as abscissa, and the peak area ratio of two characteristic peptide segments as ordinate;

[0053] FIG. 11 shows the impact of different substrate concentrations on the peak area ratio of characteristic peptide segments LDQWLCEK(SEQ ID NO.1)/VLPVPQK(SEQ ID NO.2) at an enzyme concentration of 1:20 with enzymolysis time of 28 h, with the substrate concentration as abscissa, and the peak area ratio of two characteristic peptide segments as ordinate;

[0054] FIG. 12 shows the impact of different substrate concentrations on the peak area ratio of characteristic peptide segments TPEVDDEALEK(SEQ ID NO.3)/FALPQYLK(SEQ ID NO.4) at an enzyme concentration of 1:20 with enzymolysis time of 28 h, with the substrate concentration as abscissa, and the peak area ratio of two characteristic peptide segments as ordinate;

[0055] FIG. 13 is a standard curve of peak area ratio vs concentration ratio of characteristic peptide segments TPEVDDEALEK(SEQ ID NO.3)/FALPQYLK(SEQ ID NO.4), with the concentration ratio of two characteristic peptide segments as abscissa, and the peak area ratio thereof as ordinate; and R2 of the standard curve regression equation is 0.9935;

[0056] FIG. 14 is a standard curve of peak area ratio vs concentration ratio of characteristic peptide segments LDQWLCEK(SEQ ID NO.1)/VLPVPQK(SEQ ID NO.2), with the concentration ratio of two characteristic peptide segments as abscissa, and the peak area ratio thereof as ordinate; and R2 of the standard curve regression equation is 0.992;

[0057] FIG. 15 is a primary mass spectrum of desalted whey powder;

[0058] FIG. 16 shows the composition of desalted whey powder protein.

DETAILED DESCRIPTION OF THE INVENTION

[0059] In order to make the purpose, technical solutions, and advantages of the embodiments of the invention clearer, the technical solutions in the embodiments of the present disclosure will be described clearly and completely, obviously, the described embodiments are some of the embodiments of the present disclosure, but not all of them. Based on the embodiments of the present disclosure, all other embodiments obtained by those of ordinary skill in the art without creative work are within the scope of the present invention.

[0060] In addition, in order to better explain the present invention, a lot of specific details are given in the following embodiments. It will be understood by those skilled in the art that the present invention may be practiced without certain specific details. In some embodiments, materials, elements, methods, means, etc., well known to those skilled in the art, are not described in detail so as to highlight the spirit of the present invention.

[0061] Throughout the specification and claims, the term “comprising” or variations thereof, such as “including” or “containing” and the like, will be understood to include the stated components and not to exclude other elements or other components, unless expressly indicated otherwise.

[0062] In the following examples, the reagents used were as follows:

[0063] Dithiothreitol (sigma, ≥98% HPLC); iodoacetamide (sigma, ≥99% NMR); α-lactalbumin standard substance (sigma, ≥85%); β-lactoglobulin standard substance (sigma, ≥90%); α-casein standard substance (sigma, ≥70%); β-casein standard substance (sigma, ≥98%); bovine basic trypsin (sigma, ≥10,000 BAEE); Calcium chloride (AR); Ammonium bicarbonate (AR); Acetic acid (AR).

[0064] In the following examples, nano-high-performance liquid chromatography-orbitrap high resolution mass spectrometry (NanoLC-Orbitrap MS) was used for selective ion scanning analysis under the following chromatographic conditions:

[0065] Nano-high-performance liquid UltiMate 3000 (ACCELA)

[0066] Column: Acclaim® PepMap RSLC (75 μm×15 cm, nanoViper C18, 2 μm, 100 A Thermo Fisher Scientific); Acclaim PepMap™ 100 (75 μm×2 cm, nanoViper C18, 3 μm, 100 A Thermo Fisher Scientific)

[0067] Column temperature: 50° C.

[0068] Mobile phase A: 2% acetonitrile+98% water+0.1% formic acid

[0069] Mobile phase B: 80% acetonitrile+20% water+0.1% formic acid

[0070] Loading: 2% acetonitrile+98% water

[0071] Flow rate: NC: 0.25 μL/min, loading flow rate: 0.3 μL/min

[0072] Injection volume: 1 μL

[0073] Gradient elution conditions

[0074] Gradient elution

TABLE-US-00002 Elution time (min) Flow rate (μL/min) A % B % 0 0.25 96 4 5 0.25 96 4 65 0.25 78 22 70 0.25 10 90 75 0.25 96 4 76 0.25 96 4

[0075] In the following examples, the characteristic peptide segments were subjected to a mass spectrometry analysis in PRM mode under the following mass spectrometry conditions:

[0076] Q Exactive mass spectrometer (Thermo Fisher Scientific)

[0077] PRM mode

[0078] Running time: 5-75 min

[0079] Polarity: positive

[0080] Resolution: 17500

[0081] AGC target: 1e5

[0082] Maximum IT: 100 ms

[0083] Separation window: 1.0 m/z

[0084] Initial mass-to-charge ratio: Fixed first mass

[0085] NCE: 27%, 28%.

Example 1: Selection of Characteristic Peptide Segments

[0086] A mixed standard substance of α-lactalbumin, β-lactoglobulin, α-casein, and β-casein and a milk powder sample were subjected to a full-scanning by mass spectrometry, and the mass spectra thereof were shown in FIG. 1 and FIG. 2, respectively.

[0087] By using ExPASy PeptideMass website, four proteins obtained by retrieval in NCBI's database, α-lactalbumin, β-lactoglobulin, α-casein, and β-casein, were subjected to a simulated enzyme digestion (for the peptide segments produced by the simulated digestion, please see Table 2 below), and a comparation. The compared peptide segments were substituted to NCBI website for BLAST alignment analysis, and peptide segments with high response value and a sequence length of 5-25 amino acids were selected and finally determined as the specific peptide segments, which were:

[0088] characteristic peptide segment of α-lactalbumin (hereinafter also referred to as α-1a)-LDQWLCEK (as shown in SEQ ID NO.1),

[0089] characteristic peptide segment of β-lactoglobulin (hereinafter also referred to as β-1g)-TPEVDDEALEK (as shown in SEQ ID NO.3),

[0090] characteristic peptide segment of α-casein (hereinafter also referred to as α-cs)-FALPQYLK (as shown in SEQ ID NO.4),

[0091] characteristic peptide segment of β-casein (hereinafter also referred to as β-cs)-VLPVPQK (as shown in SEQ ID NO.2), respectively.

[0092] Specific information on the above four characteristic peptide segments was shown in Table 3, and their mass spectra in PRM mode were shown in FIG. 3.

[0093] The respective daughter ion information maps of the above four characteristic peptide segments were shown in FIGS. 4, 5, 6, and 7, respectively, from which it can be seen that the quantitative daughter ions selected in the experiment have higher response values in the detection process; FIG. 8 showed the daughter ion peaks of four peptide segments detected separately (here, they were combined into one diagram), to illustrate that they were independent peaks and did not interfere with each other during detection; in addition, the quantitative fragment ion information was shown in Table 4; and fragment ions with appropriate response value and mass number of each specific peptide segment were selected as quantitative ions.

[0094] Table 2 Peptide segments produced by simulated digestion of α-lactalbumin, β-lactoglobulin, α-casein, and β-casein

[0095] β-Lactoglobulin

[0096] Protein Sequence (as Shown in SEQ ID NO.5):

TABLE-US-00003         10         20         30         40  LIVTQTMKGL DIQKVAGTWY SLAMAASDIS LLDAQSAPLR          50         60         70         80 VYVEELKPTP EGDLEILLQK WENDECAQKk IIAEKtkIPA         90        100        110        120 VFKIDALNEN KVLVLDTDYK kYLLFCVENS AEPEQSLVCQ        130        140        150        160 CLVRTPEVDD EALEKFdkal kALPMHIRLS FNPTQLEEQC HI  

[0097] Peptide Fragments by Theoretical Digestion (as Shown in SEQ ID NO.6-18, Respectively):

TABLE-US-00004 artif. posi- # medifi- modifi- peptide mass tion MC cations(s) cations sequence 2707.3759  15-40 0 VAGTWYSLA MAASDISLL DA QSAPLR 2675.2336 102-124 0 Cys.CAM: 2846.2980 YLLFCMENS 106, 119 AEPEQSLVC 121 QC LVR 2313.2587  41-60 0 VYVEELKPT PEGDLEILL QK 1058.7843 149-162 0 Cys_CAM: 1715.8057 LSFNPTQLE 160 EQCHI 1245.5645 125-135 0 TPEVDDEAL EK 1122.4520  61-69 0 Cys_CAM: 1179.4735 WENDECAQK 66 1065.5626  92-100 0 VLVLDTDYK  933.5437   1-8 0 LIVTQTMK  916.4734  84-91 0 IDALNENK  837.4763 142-148 0 ALPMHIR  674.4235  78-83 0 IPAVFK  673.3879   9-14 0 GLDIDQK  573.3606  71-75 0 BAEK

[0098] F Chain of Bovine α-Lactalbumin, Crystal Structure

[0099] Protein sequence (as shown in SEQ ID NO.19):

TABLE-US-00005         10         20         30         40  EQLTKCEVFR elkdlkGYGG VSLPEWYCTT FHTSGYDTQA          50         60         70         80 IVQNNDSTEY GLFQINNKIW CKDDQNPHSS NICNISCDKF         90        100        110        120 LDDDLTDDIM CVKkildkVG INYWLAHKAL CSEKLDQWLC EKl

[0100] Peptide fragments by theoretical digestion (as shown in SEQ ID NO.20-28, respectively):

TABLE-US-00006 artif. posi- # modifi- modifi- peptide mass tion MC cations(s) cations sequence 4654.1467  17-58 0 Cys_CAM: 4711.1681 GYGGVSLPE 26 WVCTTFHTS  GY DTQAIV QNNDSTEYG LFQIN NK 1889.7752  63-79 0 Cys_CAM: 2003.8162 DDQNPHSSN 73, 77 ICNISCDK 1642.7338  80-93 0 Cys_CAM: 1699.7553 FLDDDLTDD 91 IMCVK 1200.6524  99-108 0 VGINYWLAH   K 1034.4975 115-122 0 Cys_CAM:  1091.5190 LDQWLCEK 120  653.3075   6-10 0 Cys_CAM:   710.3290 CEVFR 6  650.3178 109-114 0 Cys_CAM:  707.3392 ALCSEK 111  618.3457   1-58 0 EQLTK  549.2853  59-62 0 Cys_CAM:  606.3068 IWCK 61

[0101] α-S1-Casein, Partial [Bovine]

[0102] Protein sequence (as shown in SEQ ID NO.29):

TABLE-US-00007         10         20         30         40  mkLLILTCLV AVALARPKhp ikHQGLPQEV LNENLLRFFV          50         60         70         80 APFPEVFGKe kVNELSKDIG SESTEDQAME DIKQMEAESI         90        100        110        120 SSSEEIVPNS VEQKHIQKED VPSERYLGYL EQLLRlkkyk        130        140        150        160 VPQLEIVPNS AEERLHSMKE GIHAQQKEPM IGVNQELAYF YPE

[0103] Peptide fragments by theoretical digestion (as shown in SEQ ID NO.30-42, respectively):

TABLE-US-00008 artif. posi- # modifi- modifi- peptide mass tion MC cation(s) cations sequence 2321.0813  74-94 0 QMEAESISS  SEEIVPNSV EQ K 1899.8833 148-163 0 EPMIGVNQE LAYFYPE 1767.7589  58-73 0 DIGSESTED QAMEDIK 1759.9449  23-37 0 HQGLPQEVL NENLLR 1694.0760   3-18 0 Cys_CAM:   1751.0975 LLILTCLVA 8 VALARPK 1580.8278 121-134 0 VPQLEIVPN SAEER 1334.7299  38-49 0 FFVAPFPEV FGK 1267.7045 106-115 0 YLGYLEQLLR  910.4741 140-147 0 EGIHAQQK  831.3843  99-105 0 EDVPSER  689.3828  52-57 0 VNELSK  615.3283 135-139 0 LHSMK  525.3143  95-98 0 HIQK

[0104] α-S2-Casein Precursor [Bovine]

[0105] Protein sequence (as shown in SEQ ID NO.43):

TABLE-US-00009         10         20         30         40 mkFFIFTCLL AVALAKNTME HVSSSEESII SQETYKqekN         50         60         70         80 MAINPSKENL CSTFCKEVVR NANEEEYSIG SSSEESAEVA         90        100        110        120 TEEVKITVDD KHYQKALNEI NQFYQKFPQY LQYLYOGPIV        130        140        150        160 LNPWDQVKrN AVPITPTLNR EQLSTSEENS KkTVDMESTE        170        180        190        200 VFTKktkLTE EEKnrLNFLK kISQRyqkFA LPQYLKTVYQ        210        220 HQKAMKPWIQ PKtkVIPYVR yl

[0106] Peptide fragments by theoretical digestion (as shown in SEQ ID NO.44-63, respectively):

TABLE-US-00010 artif. modifi- posi- # ca- modifi- peptide mass tion MC tions(s) cations sequence 2709.4075 107-128 0    FPQYLQYLY QGPIVLNPW DQ VK 2688.1642  61-85 0   NANEEEYSI GSSSEESAE VA TEEVK 2299.0394  17-36 0   NTMEHVSSS EESIISQET YK 1556.8909   3-16 0 Cys_CAM: 1613.9123 FFIFTCLLA 8 VALAK 1386.6487 153-164 0    TVDMESTEV FTK 1367.6954  96-106 0    ALNEINQFY QK 1251.5699 141-151 0   EQLSTSEEN SK 1195.6793 130-140 0    NAVPITPTL NR 1098.6128 204-212 0    AMKPWIQPK 1044.4489  48-56 0 Cys_CAM: 1158.    ENLCSTFCK 51, 55 4918  978.5611 189-196 0    FALPQYLK  903.4683 197-203 0    TVYQHQK  874.4451  40-47 0    NMAINPSK  748.3723 168-173 0    LTEEEK  746.4559 215-220 0    VIPYVR  690.3668  86-91 0   ITVDDK  634.3022 176-180 0    LNFLK  575.2936  92-95 0   HYQK  503.2936 182-185 0    ISQR  502.2983  57-60 0    EVVR

[0107] β-Casein

[0108] Protein sequence (as shown in SEQ ID NO.64):

TABLE-US-00011         10         20         30         40 mkVLILACLV ALALARELEE LNVPGEIVES LSSSEESITR          50         60         70         80 inkkiekFQS EEQQQTEDEL QDKIHPFAQT QSLVYPFPGP         90        100        110        120 IHNSLPQNIP PLTQTPVYVP PFLQPEVMGV SKvkEAMAPK        130        140        150        160 hkEMPFPKYP VEPFTESQSL TLTDVENLHL PLPLLQSWNH        170        180        190        200 QPHQPLPPTV MFPPQSVLSL SQSKVLPYPQ KAVPYPQRDM        210        220 PIQAFLLYGE PVLGPVRGPF PIIV

[0109] Peptide fragments by theoretical digestion (as shown in SEQ ID NO.65-75, respectively):

TABLE-US-00012 artif.       modifi- ca- posi- # tions modifi- peptide mass tion MC (s) cations sequence 6359.2558 129-184 0   YPVEPFTES QSLTLTDVE NL HLPLPL LQSWMHQPH QPLPPTVMF PPQSVLSLS QSK 5356.8594  64-112 0   IHPFAQTQS LVYPFPGPI  MNSLPQNIP PLTQTPVVV PPFLQPEVM GVSK 2646.2992  17-40 0   ELEELNVPG EIVESLSSS  EE SITR 2186.1678 199-217 0   DMPIQAFLL YQEPVLGPV R 1981.8621  48-63 0   FQSEEQQQT EDELQDK 1436.9177   3-16 0   VLILACLVA LALAR  830.4519 192-196 0 Cys.  1495.9392 AVPYPQR CAM: 8  780.4978 185-191 0   VLPVPQK  746.3698 123-128 0   EMPFPK  742.4497 218-224 0 GPFPIIV  646.3228 115-120 0   EAMAPK

TABLE-US-00013 TABLE 3 Information on characteristic peptide  segments   Mole- Pro- Peptide cular tein segment weight Amino acid chain β-lg TPEVDDEALEK 623.29 Thr-Pro-Glu-Val-Asp- (SEQ ID  Asp-Glu-Ala-Leu-Glu- NO. 3) Lys α-la LDQWLCEK 546.23 Leu-Asp-Gln-Trp-Leu- (SEQ ID  Cys-Glu-Lys NO. 1) α-cs FALPQYLK 490.19 Phe-Ala-Leu-Pro-Gln- (SEQ ID  Tyr-Leu-Lys NO. 4) β-cs VLPVPQK 390.75 Val-Leu-Pro-Val-Pro- (SEQ ID  Gln-Lys NO. 2)

TABLE-US-00014 TABLE 4 Information on quantitative fragment  ions Daughter Re- ion mass tention Parent number time particle (KDa) (min) TPEVDDEALEK 199.10836 27.5 (SEQ ID NO. 3) LDQWLCEK 268.16794 55.2 (SEQ ID NO. 1) FALPQYLK 120.08216 57.8 (SEQ ID NO. 4) VLPVPQK 372.2257 23.7 (SEQ ID NO. 2)

Example 2: Optimization of Enzymolysis Conditions, Selection of Substrate Concentration, as Well as Selection of Filter Membrane

[0110] Optimization of Enzymolysis Conditions:

[0111] In this experiment, milk powder samples under the brand names Sanyuan and Ailiyou (with a protein concentration of 11.6 g/100 g milk powder sample) were used as substrates, and subjected to enzymolysis at enzyme concentrations of 1:20, 1:40, 1:60, 1:80, and 1:100, respectively, according to the information provided by the product suppliers. As a result, an enzyme concentration of 1:20 and enzymolysis time of 28 h were selected based on variations in the peak area ratio of the characteristic peptide segments LDQWLCEK(SEQ ID NO.1)/VLPVPQK(SEQ ID NO.2) and variations in the peak area ratio of the characteristic peptide segments TPEVDDEALEK(SEQ ID NO.3)/FALPQYLK(SEQ ID NO.4) in the process of enzymolysis, both of which tended to be stable after enzymolysis for 28 h at the enzyme concentration of 1:20 (See FIG. 9 and FIG. 10 for details).

[0112] Selection of Substrate Concentration:

[0113] Milk powder samples were dissolved into solutions with ten protein concentration gradients between 0.1-1.0 mg/mL, which were then filtered roughly with pure acetic acid filter membrane with a pore size of 0.45 μm. 250 μL of sample solution was added to 150 μL of ammonium bicarbonate solution, followed by adding 10 μL of dithiothreitol solution, and kept in a water bath at 70° C. for 30 min. After cooling, 30 μL of iodoacetamide solution was added thereto, followed by standing for 30 min in the dark. After illuminating for 10 min, 10 μL of calcium chloride solution was added, followed by adding 50 μL of Trypsin solution, and then enzyme digestion was performed at 37° C. for 28 h, followed by adding 10 μL of acetic acid solution, and standing for 15 min to stop digestion. After filtration with 0.22 μm of polyethersulfone filter membrane, selective ion scanning analysis was carried out by nanoliter liquid chromatography. The results showed that when the protein concentration was below 0.4 mg/mL, the ratio of two characteristic peptide segments remained relatively stable after enzymolysis, indicating a good detection effect (See FIGS. 11 and 12).

[0114] Selection of Filter Membrane:

[0115] The pure acetic acid filter membrane having a large pore size of 0.45 nm was used to filter the milk powder sample preliminarily, to remove some additives, so as to avoid the impact thereof on the subsequent experiments and ensure the stability of the method. 0.22 μm of polyethersulfone filter membrane with extremely low protein adsorption was selected for filtration before running on machine.

Example 3: Detection Procedure of the Method of the Present Present Disclosure

[0116] 1) Establishment of Standard Curve:

[0117] Four protein standard substances of α-lactalbumin, β-lactoglobulin, α-casein, and β-casein were used. Specifically, whey protein standard solution was prepared in accordance with α-lactalbumin and β-lactoglobulin accounting for 20% and 50% of whey protein, respectively (i.e., the contents of α-lactalbumin and β-lactoglobulin were 20% and 50%, respectively); and casein standard solution was prepared in accordance with α-casein and β-casein accounting for 50% and 40% of casein, respectively (i.e., the contents of α-casein and β-casein were 50% and 40%, respectively); and the prepared whey protein standard solution had the same concentration as the prepared casein standard solution.

[0118] Certain amounts of the whey protein standard solution and the casein standard solution prepared as above were mixed to prepare 1 mL of a mixed solution with a whey protein/casein ratio of 0.25, 0.43, 0.67, 1, 1.5, 2.33 (mass ratio), which was then detected for drawing standard curves; the standard curve of peak area ratio vs concentration ratio of characteristic peptide segments TPEVDDEALEK(SEQ ID NO.3)/FALPQYLK(SEQ ID NO.4) was shown in FIG. 13, and the standard curve of peak area ratio vs concentration ratio of characteristic peptide segments LDQWLCEK(SEQ ID NO.1)/VLPVPQK(SEQ ID NO.2) was shown in FIG. 14.

[0119] 2) Treatment and Enzyme Digestion of Milk Powder Samples to be Tested:

[0120] dissolving the milk powder sample to be tested into a sample solution with a protein concentration of 0.2 mg/ml; subjecting the sample solution to rough filtration with a pure acetic acid filter membrane with a pore size of 0.45 μm; adding the roughly filtered sample solution into ammonium bicarbonate solution, followed by adding dithiothreitol solution, and placing the mixture in a water bath at 70° C. for 30 min; after cooling, adding iodoacetamide solution thereto, and standing for 30 min in the dark; then illuminating for 10 min, followed by adding calcium chloride solution thereto; adding trypsin solution to allow an enzyme digestion at 37° C. for 26-30 h; adding acetic acid solution to stop the enzyme digestion;

[0121] 3) filtering the enzymolysis products from step 2) with polyethersulfone filter membrane, and then performing selective ion scanning analysis by mass spectrometry to detect the peak area ratios of characteristic peptide segments of β-lactoglobulin to α-casein and of α-lactalbumin to β-casein in the milk powder;

[0122] 4) substituting the peak area ratio of the characteristic peptide segments of β-lactoglobulin to α-casein in the milk powder as detected from step 3) into the standard curve to determine the ratio M of whey protein to casein, and calculating the actual amounts of β-lactoglobulin and α-casein based on the ratio M thus determined;

[0123] and, substituting the peak area ratio of the characteristic peptide segments of α-lactalbumin to β-casein in the milk powder as detected from step 3) into the standard curve to determine the ratio N of whey protein to casein, and calculating the actual amounts of α-lactalbumin to β-casein based on the ratio N thus determined;

[0124] the actual amounts of β-lactoglobulin and α-casein are calculated by the following formulas, respectively:

[00006]$\beta -\mathrm{lactoglobulin}=\frac{M1}{M1+M2}X*50\%,\alpha -\mathrm{casein}=\frac{M2}{M1+M2}X*50\%;$

[0125] wherein, M1 was the preceding term in the ratio M of whey protein:casein as determined based on the peak area ratio of characteristic peptide segments of β-lactoglobulin to α-casein, and M2 was the latter term in the ratio M of whey protein:casein as determined based on the peak area ratio of characteristic peptide segments of β-lactoglobulin to α-casein, with the provisions of: M=M1/M2, and M1+M2=1; and X is the total protein concentration of the sample; the actual amounts of α-lactalbumin and β-casein are calculated by the following formulas, respectively:

[00007]$\alpha -\mathrm{la}=\frac{N1}{N1+N2}X*20\%,\beta -\mathrm{cs}=\frac{N2}{N1+N2}X*40\%;$

[0126] wherein, N1 was the preceding term in the ratio N of whey protein:casein as determined based on the peak area ratio of characteristic peptide segments of α-lactalbumin to β-casein, and N2 was the latter term in the ratio N of whey protein:casein as determined based on the peak area ratio of characteristic peptide segments of α-lactalbumin to β-casein, with the provisions of: N=N1/N2, and N1+N2=1; and X is the total protein concentration of the sample;

[0127] 5) acquiring the actual contents of whey protein and casein, and/or the actual ratio of whey protein/casein based on the actual amounts of α-lactalbumin, β-casein, β-lactoglobulin, and α-casein obtained from step 4);

[0128] the formula for calculating the actual content of whey protein was:

[00008]$\frac{\frac{N1}{N1+N2}X*20\%+\frac{M1}{M1+M2}X*50\%}{0.7}$

[0129] the formula for calculating the actual content of casein was:

[00009]$\frac{\frac{M1}{M1+M2}X*50\%+\frac{N2}{N1+N2}X*40\%}{0.9}$

[0130] the formula for calculating the actual ratio of whey protein/casein was:

[00010]$\frac{\frac{N1}{N1+N2}X*20\%+\frac{M1}{M1+M2}X*50\%}{0.7}\text{⁠}/\frac{\frac{M2}{M1+M2}X*50\%+\frac{N2}{N1+N2}X*40\%}{0.9}=\frac{18N1+45M1}{35M2+28N2}=\frac{63\mathrm{MN}+18N+45M}{35N+28M+63};$

[0131] wherein, M1 was the preceding term in the ratio M of whey protein:casein as determined based on the peak area ratio of characteristic peptide segments of β-lactoglobulin to α-casein, and M2 was the latter term in the ratio M of whey protein:casein as determined based on the peak area ratio of characteristic peptide segments of β-lactoglobulin to α-casein, with the provisions of: M=M1/M2, and M1+M2=1;

[0132] wherein, N1 was the preceding term in the ratio N of whey protein:casein as determined based on the peak area ratio of characteristic peptide segments of α-lactalbumin to β-casein, and N2 was the latter term in the ratio N of whey protein:casein as determined based on the peak area ratio of characteristic peptide segments of α-lactalbumin to β-casein, with the provisions of: N=N1/N2, and N1+N2=1; and, wherein, X is the total protein concentration of the sample.

Example 4: Methodology Validation on the Detection Method of the Present Disclosure by Using Desalted Whey Powder

[0133] Standard Addition Recovery Rate

[0134] Desalted whey powder was used as the standard sample, and was known to have a protein content of 12% according to its product annotation, which was almost made up of whey protein, with only a very little or negligible amount of casein, as detected by an instrument; the primary mass spectrum of the desalted whey powder was shown in FIG. 15, and analyzed to obtain its composition as shown in FIG. 16.

[0135] the detection method of the present disclosure was verified by using the desalted whey powder and the results were shown in Table 5 below.

TABLE-US-00015 TABLE 5 Added amount Theoretical Detection Recovery RSD (mg) value (mg) value (mg) rate (%) (%) Substrate 0 / 19.48 / 7.35 Desalted 3.60 23.08 23.03  98.63 0.84 whey 7.20 26.68 26.90 103.06 7.42 powder 10.80 30.28 31.72 113.33 1.83 14.40 33.88 34.11 101.62 0.84

[0136] According to Table 5, the standard addition recovery rate of the desalted whey powder detected by the detection method of the present disclosure was 98.63%-113.33%, and the corresponding RSD was 0.84%-7.42%.

Example 5: Methodology Validation on the Detection Method of the Present Disclosure by Using Commercial Formula Milk Powder

[0137] According to the detection method of the present disclosure described in Example 3, three brands of commercial formula milk powder were tested, in which: 6 groups of parallel tests were conducted once a day for 3 days to verify the repeatability and precision of the detection method of the present disclosure; the detection results of the three brands of commercial formula milk powder were shown in Tables 6-8, in which, LD represents the characteristic peptide segment of α-lactalbumin, LDQWLCEK (as shown in SEQ ID NO.1), TP represents the characteristic peptide segment of β-lactoglobulin, TPEVDDEALEK (as shown in SEQ ID NO.3), FAL represents the characteristic peptide segment of α-casein, FALPQYLK (as shown in SEQ ID NO.4), and VL represents the characteristic peptide segment of β-casein, VLPVPQK (as shown in SEQ ID NO.2).

TABLE-US-00016 TABLE 6 Repeatability and precision results of the detection of 1# brand formula milk powder (Sanyuan, Enbeiyi, Section 1) Days 1 2 3 TP/FAL LD/VL TP/FAL LD/VL TP/FAL LD/VL 5.661328 0.596471 5.943357 0.593253 5.854161 0.594823 6.698231 0.591934 6.182401 0.636411 6.215530 0.601842 5.833083 0.626097 6.463053 0.658798 6.251452 0.620248 6.070490 0.592644 5.611366 0.614108 5.950025 0.581455 6.191200 0.604171 5.853564 0.582013 6.072519 0.562768 6.120052 0.558423 6.115780 0.597187 6.182007 0.561523 Average 6.095731 0.594957 6.028253 0.613628 6.087616 0.587110 SD 0.355257 0.021937 0.293936 0.029136 0.158603 0.023038 RSD (%) 0.058280 0.036871 0.04876 0.047482 0.026053 0.039241 Average 6.070533 0.598565 between groups SD 0.036840 0.013623 RSD (%) 0.006069 0.022759

TABLE-US-00017 TABLE 7 Repeatability and precision results of the detection of 2# brand formula milk powder (Sanyuan, Aixinbao, Section 1) Days 1 2 3 TP/FAL LD/VL TP/FAL LD/VL TP/FAL LD/VL 6.255670 0.611835 5.629679 0.615912 5.666182 0.586044 5.760567 0.605571 5.619842 0.648726 5.724010 0.538855 6.331991 0.590981 5.304704 0.630166 5.634282 0.574862 6.156652 0.634878 5.836408 0.631432 5.471585 0.574179 5.802848 0.562343 5.592876 0.613239 5.762156 0.532703 5.901041 0.552298 5.636404 0.627743 5.465830 0.576933 Average 6.034795 0.592984 5.603319 0.627870 5.620674 0.563929 SD 0.244480 0.031200 0.170689 0.012728 0.125822 0.022300 RSD (%) 0.040512 0.052615 0.030462 0.020272 0.022386 0.039545 Average 5.834348 0.605599 between groups SD 0.244257 0.032015 RSD (%) 0.041865 0.052864

TABLE-US-00018 TABLE 8 Repeatability and precision results of the detection of 3# brand formula milk powder (Sanyuan, Ailiyou, Section 1) Days 1 2 3 TP/FAL LD/VL TP/FAL LD/VL TP/FAL LD/VL 7.453970 0.531694 7.704645 0.561678 6.723771 0.593539 7.259978 0.595509 7.603946 0.570701 8.520871 0.662325 7.114793 0.510083 7.885622 0.638107 8.433776 0.676738 7.299271 0.476919 7.420642 0.548663 8.070415 0.759728 7.429340 0.553053 7.749015 0.577207 8.319099 0.720309 7.765910 0.583946 7.761472 0.602464 7.881502 0.613721 Average 7.387210 0.541867 7.687557 0.583137 7.991573 0.671060 SD 0.222611 0.044956 0.159372 0.032354 0.664624 0.062759 RSD (%) 0.030135 0.082965 0.020731 0.055483 0.083166 0.093522 Average 7.688780 0.598688 between groups SD 0.302183 0.065985 RSD (%) 0.039302 0.110217

[0138] According to Tables 6-8, the RSD within groups for the above detections was in the range of 2.03%-9.35%, and the RSD between groups was in the range of 0.61%-11.02%, indicating that the detection method of the present disclosure had good repeatability and precision.

Example 6: Detection Example of the Detection Method of the Present Disclosure

[0139] According to the detection method of the present disclosure described in Example 3, the content of whey protein in commercial formula milk powder (Abbott, Classic Enmeili, Section 1) was detected, and the specific procedure was as follows:

[0140] To 0.2 g commercial formula milk powder (Abbott, Classic Enmeili Section 1), ultrapure water was added to a fixed volume of 200 mL. The obtained sample solution was then filtered roughly with pure acetic acid filter membrane with a pore size of 0.45 μm. Then 250 μL of the filtered sample solution was added to 150 μL of ammonium bicarbonate solution, followed by adding 10 μL of dithiothreitol solution, and kept in a water bath at 70° C. for 30 min. After cooling to room temperature, 30 μL of iodoacetamide solution was added thereto, and allowed to stand for 30 min in the dark. After illuminating for 10 min, 10 μL of calcium chloride solution was added thereto, followed by adding 50 μL of Trypsin solution to allow an enzyme digestion at 37° C. for 28 h, and further followed by adding 10 μL of acetic acid solution and standing for 15 min to stop digestion. Filtration was carried out with 0.22 μm of polyethersulfone filter membrane prior to detection on the machine. It was calculated that whey protein accounted for 61.77% of the total protein content in the sample.

[0141] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present disclosure, but not to limit it; although the present disclosure has been described in detail with reference to the foregoing embodiments, it will be understood by one of ordinary skill in the art that the technical solutions described in the foregoing embodiments can still be modified or some technical features can be equivalently substituted; however, these modifications or substitutions do not make the essence of the corresponding technical solutions departing from the spirit and scope of the technical solutions of various embodiments of the present disclosure.