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Open Access

Adulteration detection of plant protein beverages by UPLC-MS/MS based on signature peptide of allergen

Yawei Ninga, Zhuo Liua,b, Zheng Yanga,b, Junmei Mab, Yan Zhangb,c(), Qiang Lib()
College of Food Science and Biology, Hebei University of Science and Technology, Shijiazhuang 050018, China
Hebei Food Inspection and Research Institute, Hebei Food Safety Key Laboratory, Key Laboratory of Special Food Supervision Technology for State Market Regulation, Hebei Engineering Research Center for Special Food Safety and Health, Shijiazhuang 050000, China
Hebei Key Laboratory of Forensic Medicine, College of Forensic Medicine, Hebei Medical University, Shijiazhuang 050017, China

Peer review under responsibility of Tsinghua University Press.

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Abstract

Plant protein beverage adulteration occurs frequently, which may cause health problems for consumers due to the hidden allergens. Hence, a novel method was developed for authentication by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Almond, peanut, walnut and soybean were hydrolyzed, followed by separation by NanoLC-Triple TOF MS. The obtained fingerprints were identified by ProteinPilotTM combined with Uniprot, and 16 signature peptides were selected. Afterwards, plant protein beverages treated by trypsin hydrolysis were analyzed with UPLC-MS/MS. This method showed a good linear relationship with R2 > 0.99403. The limit of quantification (LOQ) were 0.015, 0.01, 0.5 and 0.05 g/L for almond, peanut, walnut and soybean, respectively. Mean recoveries ranged from 84.77% to 110.44% with RSDs < 15%. The developed method was successfully applied to the adulteration detection of 31 plant protein beverages to reveal adulteration and false labeling. Conclusively, this method could provide technical support for authentication of plant protein beverages to protect the rights and health of consumers.

Keywords

AllergenAdulteration detectionPlant protein beverageSignature peptideUPLC-MS/MS

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Food Science and Human Wellness
Volume 13 Issue 6,
November 2024
Pages 3371-3380
DOI: 10.26599/FSHW.2023.9250022
Cite this article:
Ning Y, Liu Z, Yang Z, et al. Adulteration detection of plant protein beverages by UPLC-MS/MS based on signature peptide of allergen. Food Science and Human Wellness, 2024, 13(6): 3371-3380. https://doi.org/10.26599/FSHW.2023.9250022
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Peptide-derived allergen protein, signature peptides and MRM parameters.

Material Entry identifier of UniProtKB Allergen Peptide Precursor (m/z) Product ions (m/z) Fragmentor (V) CE (V)
Note: * Quantitative peptides and product ions.
Almond E3SH28 Prunin 1 GNLDFVQPPR* 572.1 369.3* 75 23
497.3 25
NGLHLPSYSNAPQL1YIVQGR 781.3 735.3 120 36
226.1 47
E3SH29 Prunin 2 ALPDEVLQNAFR* 687.2 748.4* 110 33
342.4 44
VQGQLDFVSPFSR 740.7 593.3 90 31
1067.6 35
Peanut P43238 Ara h 1 DLAFPGSGEQVEK* 688.9 299.9*80 30
930.1 25
NTLEAAFNAEFNEIR 870.0 1139.6 130 35
992.4 35
082580 Ara h 3 RPFYSNAPQE1FIQQGR* 684.6 748.3* 75 27
836.3 30
TANDLNLLILR 628.4 741.6 60 25
854.3 30
Walnut Q2TPW5 Jug r 4EGQLLT1PQNFAVVK*829.3 902.7* 90 37
1116.7 30
ALPEEVLATAFQIPR 828.3 1016.7 110 40
356.2 57
Q9SEW4 Jug r 2 DFLAGQNNIINQLER* 873.3 659.4* 100 36
772.5 44
VFSNDILVAALNTPR 815.8 742.4 125 37
954.6 37
SoybeanO22120 Glv m 5LITLA1PVNKPGR* 696.8 767.5* 85 40
286.2 45
QFPFPRPPHQK 690.2 859.5 110 45
1103.7 57
P04776 Glv m 6 SQSDNFEYVSFK* 726.1 216.1* 100 31
1235.8 27
FYLAGNQEQEFLK 794.2 283.2 120 42
1092.5 34

Regression equation, linear range, R2, LOD and LOQ of the signature peptides (n = 4).

Material Allergen Peptide Linear range (g shelled materials/L) Regression equation R2 LOD (g/L) LOQ (g/L)
Almond Prunin 1 GNLDFVQPPR 0.015–70 Y = 4.68705 × 106X + 2468.34633 0.99873 0.005 0.015
Prunin 2 ALPDEVLQNAFR 0.15–70 Y = 3.54718 × 106X + 9236.62083 0.99562 0.05 0.15
Peanut Ara h 1 DLAFPGSGEQVEK 0.01–70 Y = 1.61214 × 106X + 3704.50192 0.99791 0.005 0.01
Ara h 3 RPFYSNAPQEIFIQQGR 0.05–70 Y = 1.83473 × 106X + 1195.16284 0.99809 0.015 0.05
Walnut Jug r 4 EGQLLTIPQNFAVVK 0.50–70 Y = 3.58788 × 105X - 2950.20868 0.99403 0.20 0.50
Jug r 2 DFLAGQNNIINQLER 0.50–70 Y = 3.66364 × 105X + 559.20101 0.99748 0.20 0.50
Soybean Gly m 5 LITLAIPVNKPGR 0.05–70 Y = 8.58301 × 105X + 988.30049 0.99874 0.02 0.05
Gly m 6 SQSDNFEYVSFK 0.10–70 Y = 4.24989 × 105X + 1876.77887 0.99740 0.05 0.10

Spiked recoveries and RSDs of 4 plant protein materials (n = 6).

Material Peptide Spiked level (g/L) Recovery (%) RSD (%)
Almond 0.5 89.23 7.59
GNLDFVQPPR 10 107.28 2.60
30 101.25 2.45
0.5 85.43 4.99
ALPDEVLQNAFR 10 109.38 3.73
30 104.54 10.23
Peanut 0.5 97.97 0.85
DLAFPGSGEQVEK 10 102.28 14.20
30 108.33 8.39
0.5 98.53 3.15
RPFYSNAPQEIFIQQGR 10 110.44 10.02
30 100.71 6.53
Walnut 5 102.68 4.75
EGQLLTIPQNFAVVK 10 95.97 5.11
35 102.63 4.45
5 88.32 2.83
DFLAGQNNIINQLER 10 102.77 9.43
35 105.64 0.74
Soybean 0.5 84.77 4.16
LITLAIPVNKPGR 10 98.23 6.54
30 86.53 1.55
0.5 106.73 4.59
SQSDNFEYVSFK 10 103.55 0.53
30 89.22 8.18

Summarization of the LC-MS detections for allergens.

Material Target Protein Foodstuff LOD LOQ Reference
Note: -: The LOQ or LOD is not given in the reference.
Almond Prunin 1 Plant protein beverage 0.005 galmond/L 0.015 galmond/L This study
Peanut Ara h 1 0.005 gpeanut/L 0.01 gpeanut/L
Walnut Jug r 2 0.2 gwalnut/L 0.5 gwalnut/L
Soybean Gly m 5 0.02 gsoybean/L 0.05 gsoybean/L
Almond Prunin 1 Plant protein beverage 17 μg almond/g 58 μgalmond/g
Peanut Ara h 3 10 μgpeanut/g 37 μgpeanut/g [33]
Walnut Jug r 4 55 μgwalnut/g 180 μgwalnut/g
Almond Prunin 1 Bread 17 μgdefatted almond/g - [27]
Peanut Ara h 1 11 μgdefatted peanut/g -
Walnut Jug r 1 70 μgdefatted walnut/g -
Soybean Gly m 6 24 μgdefatted soy/g -
Soybean β-Conglycinin-α-chain Cookie 2 μgtotal proteins/g 7 μgtotal proteins/g14 μgtotal proteins/g [34]
Peanut Ara h 1 and 3 Tree nut mixture 4 μgtotal proteins/g26 μgdefatted peanut/g 88 μgdefatted peanut/g [35]
Soybean Gly m 5, 2S-albumin, β-conglycinin Cookie - 5 μgtotal proteins/g [24]
Peanut Ara h 1 and 3 - 2.5 μgtotal proteins/g
Almond Prunin 1 and 2 Vanilla ice cream 0.34–1.92 μgtotal proteins/g, 1.8–10.1 μgalmond/g - [25]
Peanut Ara h 1 and 3 0.26–2.08 μgtotal proteins/g, 1.0–8.1 μgalmond/g -
Walnut Jug r 1, 2 and 4 0.8–5.0 μgtotal proteins/g, 5.7–35.7 μgalmond/g -
Peanut Ara h 1 Home-made cookie 13 μgpeanut/g 40 μgpeanut/g [12]
Soybean Gly m 6 6 μgsoy flour/g 19 μgsoy flour/g
Peanut Ara h 3, conglutin Home-made cookie 24 μgpeanut/g 84 μgpeanut/g [32]
Almond Prunin 1 and 2 Chocolate 0.4–0.8 μgtotal proteins/g 1.3–2.6 μgtotal proteins/g [28]
Peanut Ara h 1 and 3 0.8–1.3 μgtotal proteins/g 2.6–4.3 μgtotal proteins/g
Walnut Jug r 4 0.6 μgtotal proteins/g 2 μgtotal proteins/g
Soybean β-Conglycinin, Gly m 6 0.4–1.2 μgtotal proteins/g 1.3–4.0 μgtotal proteins/g
Almond Prunin 1 and 2 Chocolate, compote, jam, - 5–25 μgtotal proteins/g [13]
Peanut Ara h 1, 2 and 3/4 ham, sauce, paprika spices, - 2.5–5.0 μgtotal proteins/g
Walnut Jug r 1and 2 cookie and - 5.0–12.5 μgtotal proteins/g
Soybean 2S albumin, Gly m 6 Mayonnaise - 5–10 μgtotal proteins/g

Adulteration detection of commercial plant protein beverages by UPLC-MS/MS.

Sample Beverage name Labeled plant protein ingredients UPLC-MS/MS (g/L)
Peanut Soybean Almond Walnut
UPLC-MS/MS UPLC-MS/MS UPLC-MS/MS UPLC-MS/MS
Note: ND means that no plant protein materials are detected.
1 Almond beverages Almond 0.370 ± 0.016 ND 23.13 ± 0.21 ND
2 Almond beverages Almond ND ND 25.040 ± 0.025 ND
3 Almond beverages Almond ND ND 32.59 ± 0.15 ND
4 Almond beverages Almond ND ND 30.21 ± 0.35 ND
5 Almond beverages Almond 0.130 ± 0.021 ND 29.65 ± 0.57 ND
6 Almond beverages Almond ND ND 18.64 ± 0.68 ND
7 Almond beverages Almond, peanut 34.93 ± 0.54 ND 4.780 ± 0.049 ND
8 Almond beverages Almond, peanut 15.65 ± 0.54 ND 3.68 ± 1.00 ND
9 Almond beverages Almond, peanut 16.83 ± 0.36 ND ND ND
10 Almond beverages Peanut, almond 20.12 ± 0.21 ND 1.120 ± 0.028 ND
11 Almond beverages Peanut, almond 26.54 ± 0.18 ND 3.33 ± 0.61 ND
12 Walnut beverages Walnut ND ND ND 28.32 ± 0.31
13 Walnut beverages Walnut ND ND ND 21.26 ± 0.33
14 Walnut beverages Walnut ND 3.70 ± 0.17 ND 24.27 ± 0.62
15 Walnut beverages Walnut ND ND ND 32.56 ± 0.37
16 Walnut beverages Walnut ND ND ND 21.87 ± 0.52
17 Walnut beverages Walnut ND ND ND 37.26 ± 0.95
18 Walnut beverages Walnut, jujube juice 14.63 ± 0.17 ND ND 5.60 ± 1.28
19 Walnut beverages Walnut, peanut 16.45 ± 0.38 ND ND ND
20 Walnut beverages Peanut, walnut 12.11 ± 0.57 ND ND ND
21 Walnut beverages Peanut, walnut 29.32 ± 0.99 ND ND 12.22 ± 0.64
22 Walnut beverages Peanut, walnut 15.22 ± 0.44 ND ND 6.79 ± 0.38
23 Mixed plant protein beverages Almond, peanut 15.74 ± 0.22 ND 3.38 ± 0.89 ND
24 Mixed plant protein beverages Almond, peanut 17.27 ± 0.36 ND 2.64 ± 0.32 ND
25 Mixed plant protein beverages Almond, peanut 5.35 ± 0.76 ND ND ND
26 Mixed plant protein beverages Almond, soybean ND 6.990 ± 0.098 25.62 ± 0.78 ND
27 Mixed plant protein beverages Almond ND ND 29.520 ± 0.055 ND
28 Mixed plant protein beverages Peanut, almond 18.57 ± 0.48 ND 0.88 ± 0.17 ND
29 Mixed plant protein beverages Walnut 7.27 ± 0.15 ND ND 4.55 ± 0.22
30 Peanut beverages Peanut 13.17 ± 0.15 ND ND ND
31 Peanut beverages Peanut 10.25 ± 0.29 ND ND ND