Food Control 102 (2019) 179–187
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Food Control
journal homepage: www.elsevier.com/locate/foodcont
A rapid immobilized trypsin digestion combined with liquid
chromatography – Tandem mass spectrometry for the detection of milk
allergens in baked food
T
Kailun Qia,b,1, Tong Liuc,1, Yunjia Yanga,b, Jing Zhanga,b, Jie Yina,b, Xiaojing Dinga,b,
Weijie Qinc,∗, Yi Yanga,b,∗∗
a
b
c
Beijing Key Laboratory of Diagnostic and Traceability Technologies for Food Poisoning, Beijing Center for Disease Prevention and Control, Beijing, 100013, China
Beijing Research Center for Preventive Medicine, Beijing, 100013, China
National Center for Protein Sciences Beijing, State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Lifeomics, Beijing, 102206, China
A R T I C LE I N FO
A B S T R A C T
Keywords:
PHMN-Trypsin
LC-MS
Milk
Allergen
Baked food
Cow's milk allergy is the most common food allergy, and caseins are the major allergens in milk. A quantitative
proteomics method based on liquid chromatography coupled to mass spectrometry has been widely used in
allergen analysis. However, the long period of digestion has limited its use in routine analysis. PHMN-Trypsin
(trypsin immobilized on hairy polymer-chain hybrid magnetic nanoparticles), a new type of immobilized
trypsin, was used to shorten the digestion time and enhance the digestion efficiency in this study. We developed
a rapid digestion method using PHMN-Trypsin to detect milk allergens in baked food by ultrahigh-performance
liquid chromatography – tandem mass spectrometry (UPLC-MS/MS). The PHMN-Trypsin digestion was completed within 15 min, with higher or equal sequence coverage compared to 12–16 h of conventional free trypsin
digestion. αs1-casein, αs2-casein, β-casein, and κ-casein were monitored as the allergen proteins. For each
target allergen, two or three specific and signature peptides were selected for the UPLC-MS/MS strategy in view
of the species specificity, mass response intensity as well as the stability. The limits of quantification of the four
proteins in baked foods were 0.38–0.83 μg/g, and their recoveries ranged from 65.2% to 86.1%, with relative
standard deviations of less than 11.0%. The method was applied to investigate allergens in commercial baked
foods, and the results indicate that this method would be helpful in food allergen screening.
1. Introduction
Food allergies are a global health issue and a research focus for food
safety scientists because nearly 5% of adults and at least 8% of children
worldwide suffered from a food allergy, and the incidence of food allergies continues to increase (Branum & Lukacs, 2008; Sicherer &
Sampson, 2014). The prevention of adverse events by food allergies
depends upon regulation of the government and food safety controls
throughout the food production chain, including manufacturers, distributors, and transporters, as well as retailers, restaurants, takeaways
and consumers themselves (Ajala et al., 2010; Dupuis et al., 2016;
Dzwolak, 2017; Soogali & Soon, 2018; Soon, 2018). Cow's milk is the
most common food allergy, especially in children, and it affects 2.5% of
infants during their first year of life (Sicherer & Sampson, 2010).
Caseins are the major allergens in milk, accounting for 80% of the total
milk protein (Docena, Fernandez, Chirdo, & Fossati, 1996). Caseins
consist of αs1-casein, αs2-casein, β-casein, and κ-casein, which account
for 32%, 10%, 28%, and 10% of the total milk protein, respectively
(Wal, 1998). Therefore, the concentration of casein in milk and dairy
products is an important indicator of milk allergens.
Enzyme-linked immunosorbent assay (ELISA) and the polymerase
chain reaction (PCR) are the most commonly used techniques for the
analysis of food allergens. However, the high false positive results of the
ELISA method and the indirect determination strategy of the PCR
method have been found to be insufficient in food allergen analysis (Ma
et al., 2010; Poms, Klein, & Anklam, 2004). Recently, quantitative
∗
Corresponding author.
Corresponding author. Beijing Key Laboratory of Diagnostic and Traceability Technologies for Food Poisoning, Beijing Center for Disease Control and Prevention,
Beijing, 100013, China.
E-mail addresses: aunp_dna@126.com (W. Qin), yybjcdc@163.com (Y. Yang).
1
These authors contributed equally to this work.
∗∗
https://doi.org/10.1016/j.foodcont.2019.03.017
Received 4 January 2019; Received in revised form 11 March 2019; Accepted 15 March 2019
Available online 26 March 2019
0956-7135/ © 2019 Elsevier Ltd. All rights reserved.
Food Control 102 (2019) 179–187
K. Qi, et al.
2.2. Stock solutions and the working solution
proteomic methods based on liquid chromatography coupled to mass
spectrometry (LC-MS) or tandem mass spectrometry (UPLC-MS/MS) are
becoming more commonly used in allergen analysis as a promising
analytical strategy and technology because of their sequence-specific
and protein-based properties (Chen et al., 2015; Heick, Fischer, &
Pöpping, 2011; Pilolli, De, & Monaci, 2017; Planque et al., 2016). Some
advantages have been achieved via quantitative proteomics in food
allergen detection, such as a higher sensitivity, specificity and accuracy.
Previous studies have established LC-MS/MS methods to detect the
proteins of allergens derived from eggs, milk, peanuts, soy and tree nuts
in processed food (Heick et al., 2011; Pilolli et al., 2017). Chen et al.
(2015) developed a LC-MS method to detect β-casein in baked foodstuffs with an isotope standards (IS) strategy, which can correct the loss
of analytes during the experiment. However, a barrier in the MS-based
quantitative proteomics method is the long period of digestion time,
usually ranging between twelve and sixteen hours.
PHMN-Trypsin (trypsin immobilized on hairy polymer-chain hybrid
magnetic nanoparticles) is a new type of immobilized trypsin developed
by Qin et al. (2012). In this new type of immobilized trypsin, the trypsin
was immobilized on noncrosslinked polymer chains, which were fabricated on the surface of magnetic nanoparticles coated by silica (Qin
et al., 2012). Trypsin immobilized on different types of supports, such
as micro/nanoparticles, porous reactors, and membrane filters, has a
higher hydrolysis efficiency than traditional digestion (Bezstarosti,
Ghamari, Grosveld, & Demmers, 2010; Ibáñez, Muck, & Svatoš, 2007;
Krenkova, Lacher, & Svec, 2009; Liu, Bao, Zhang, & Chen, 2013; Ning &
Bruening, 2015). Trypsin immobilized on PHMN has a higher capacity
for trypsin than conventional magnetic nanoparticles or microspheres,
and it exhibits higher hydrolysis efficiency than free trypsin (Deng
et al., 2009; Krogh, Berg, & Højrup, 1999). Moreover, an additional
advantage is that PHMN-Trypsin can be reused.
In this work, we report the development of a rapid and specific
UPLC-MS/MS method for milk allergen detection in baked food using
PHMN-Trypsin digestion. The application of PHMN-Trypsin digestion
in food allergen detection significantly shortened the sample preparation time and enhanced the throughput.
Stock solutions of α-casein, β-casein and κ-casein were prepared by
dissolving 10 mg of the standard powder into 10 mL water. The stock
solutions were stored at −80 °C before use. Before analysis, the stock
solutions of the target proteins were combined in a mixed working
standard solution and further diluted with water.
Stock solutions of the signature peptides were prepared by dissolving 5 mg of the signature peptides in 5 mL water. Stock solutions were
stored at −20 °C. A working solution was prepared in water with stock
solutions of 100 μM. Before analysis, the working solutions of the signature peptides were combined in the mixed working standard solution
and further diluted with water.
Stock solutions of the isotope labeled peptides were prepared by
dissolving 5 mg of the signature peptides in 5 mL of water. Stock solutions were stored at −20 °C. A working solution was prepared in
water with the stock solutions of 100 μM. Before analysis, the working
solutions of the isotope labeled peptides were combined in mixed
working standard solutions and further diluted with water.
2.3. Selection of the signature peptides
The peptide mass fingerprinting (PMF) of the target proteins was
performed by MALDI-TOF MS analysis on an ultrafleXtreme time-offlight mass spectrometer (Bruker, Bremen, Germany). The spectra were
acquired in the MS-positive ion reflector mode with an accelerating
voltage of 20 kV in the m/z range of 700–3500. CHCA (7 mg/mL CHCA
in 60% ACN/H2O, 0.1% TFA) was used as the matrix in the experiments, and 1 μL of the matrix was spotted on the MALDI plate while the
1 μL enzymatic hydrolysate dried. The PMF was then performed by
searching the UniProt database using Mascot Daemon (version 2.3.0,
Matrix Science). Trypsin was selected as the proteolytic enzyme, and
two missed cleavages were allowed. The mass tolerance of the precursor ion was set to 0.2 Da. Oxidation (M) and carbamidomethyl (C)
were selected as the variable and fixed modification, respectively.
2.4. Preparation of the blank matrices, spiked matrices, model matrix and
no-baked matrix
2. Materials and methods
The blank matrix (milk allergen free) of baked food was prepared as
follows: 120 g of wheat flour, 5 g of olive oil, 50 g of sugar, 3 g of salt
and 100 mL of water were mixed in a bowl. After resting for 30 min, the
dough was cut into several pieces and then, baked at 160 °C for 20 min
in an oven. Spiked matrices were prepared in three independent replicates by adding target allergens to blank matrices to obtain a theoretical allergen protein concentration of 1, 2, 4 μg/g.
The model matrix was a baked matrix containing milk allergen,
which was prepared as a blank matrix with an extra addition of milk.
120 g of wheat flour, 5 g of olive oil, 50 g of sugar, 3 g of salt, 20 mL of
milk and 80 mL of water were mixed in a bowl. After resting for 30 min,
the dough was cut into several pieces and baked at 160 °C for 20 min in
an oven to obtain the model matrix. The raw material composition of
the no-baked matrix was same as the model matrix. However, after
resting for 30 min, the dough was air-dried at room temperature
without baking to prepare the unbaked matrix.
2.1. Chemicals and reagents
Ammonium bicarbonate (NH4HCO3), acetonitrile (ACN), dithiothreitol (DTT), trifluoroacetic acid (TFA), trypsin from porcine pancreas
(Proteomics Grade), α-casein (> 70%), β-casein (> 98%), κ-casein
(> 70%), TPCK-treated trypsin, glycidyl methacrylate (GMA), 2-bromoisobutyryl bromide, 3-aminopropyltriethoxysilane, triethylamine,
CuCl2, ethylenediamine, glutaraldehyde and N, N, N′, N’’, N’’-pentamethyl diethylenetriamine, cinnamic acid (CHCA) were purchased from
Sigma-Aldrich (St. Louis, MO, USA). Iodoacetamide (IAA) and formic
acid (FA) were purchased from Acros Organics (New Jersey, USA). The
1 M Tris-HCl was purchased from Beyotime Biotechnology (Shanghai,
China).
The signature peptides YLGYLEQLLR, FFVAPFPEVFGK, TTMPLW,
NAVPITPTLNR, FALPQYLK, GPFPIIV, AVPYPQR, EMPFPK, VLPVPQK,
YIPIQYVLSR, SPAQILQWQVLSNTVPAK and isotope labeled peptides
YLGYLEQL[13C-15N-L]R, FFVAPFPEV[13C-15N-F]GK, TTMP[13C-15N-L]
W, NAVPITPT[13C-15N-L]NR, [13C-15N-F]ALPQYLK, GPFPII[13C-15NV], A[13C-15N-V]PYPQR, EMP[13C-15N-F]PK, VLP[13C-15N-V]PQK, YIP
[13C-15N-I]QYVLSR, SPAQILQWQV[13C-15N-L]SNTVPAK were synthesized by Synpeptide Co., Ltd. (Shanghai, China) with a purity > 98%.
The new type of immobilized trypsin, PHMN-Trypsin, was synthesized by Professor Qin at the National Center for Protein Sciences,
Beijing. The details of the synthesis and the characterization of PHMNTrypsin have been described in our previous work (Qin et al., 2012).
2.5. Sample preparation
2.5.1. Extraction
Approximate 200 g of bakery food was cut into small pieces
(3–4 mm) with a knife, and then was homogenized by a Buchi B-600
automated sample homogenizer (Buchi, Switzerland). Samples were put
into 500 mL prelabeled dark glass pots and stored at −20 °C awaiting
for analysis. The 0.2 g homogenized baked food sample was extracted
with 1 mL of 200 mM of Tris-HCl (pH 8) in a homogenizing tube (Lysing
Matrix C, 1.0 mm silica spheres) from MP (Santa Ana, USA) using a
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sample was then shook for 1 h at 37 °C and 50 °C respectively. As shown
in Fig. 1, in different extraction conditions (high-speed homogenate
1 min, 37 °C shaking 1 h, 50 °C shaking 1 h), the extraction efficiency of
the 200 mM of Tris-HCl (pH 8) buffer was significantly higher than the
50 mM of NH4HCO3 (pH 9) buffer. With respect to the extraction
method, the extract efficiency of the high-speed homogenate was
slightly higher than the 37 °C shaking for 1 h and equivalent to 50 °C
shaking for 1 h. However, the extraction time of high-speed homogenate was only one minute, which sharply shortened the extraction
time. Therefore, the high-speed homogenate with 200 mM of Tris-HCl
(pH 8) was the feasible extraction method adopted for further analysis.
homogenizer (MP, Santa Ana, USA) for 20 s with an ice-bath. The extraction was repeated 2 times. Then, the homogenate was centrifuged at
14000 rpm with a centrifuge (Eppendorf 5418R, Hamburg, Germany)
for 10 min at 4 °C. The total protein concentration of the extract supernatant was detected by the Bradford Protein Assay Kit according to
the manufacturer's recommended protocols with a microplate absorbance reader (Bio-Rad, Hercules, USA).
2.5.2. Enzymatic digestion
The 50 μL total protein extract was diluted with 50 μL of 50 mM of
NH4HCO3 and was then reduced by adding 1 μL of 1 M of DTT for
10 min at 95 °C. Subsequently, the proteins were alkylated with 2 μL of
1 M of IAA for 1 h in the dark at room temperature. According to our
previous study, the PHMN-Trypsin digestion procedure is as follows
(Qin et al., 2012): 250 μg of the immobilized trypsin and 100 μL of the
alkylated protein solution was mixed. The mixture was treated with
ultrasound for 15 min at 37 °C for digestion. Then, the digested sample
and immobilized trypsin were separated by a magnet. The supernatant
was transferred into another tube for mass spectrometry analysis.
For the free trypsin digestion procedure, the protein reduction and
alkylation steps were conducted as previously described. After those
steps, 1 μL of free trypsin (1 μg/μL) in 10 mM of HCl was added into
100 μL of the alkylated protein solution at an enzyme-to-protein ratio of
1:50 (w/w). After incubating at 37 °C for 12 h, 0.5 μL of TFA was added
to the solution to stop the reaction for 30 min at 37 °C. Then, the mixture was centrifuged at 10,000×g, and the supernatant was then collected for analysis.
3.2. Comparing of digestion efficiency for immobilized and free trypsin
The digestion efficiency of PHMN-Trypsin for 2 min and of the
common free trypsin overnight was compared and evaluated by the
digested amino acid sequence coverage. PMF (Fig. 2) was performed by
searching the UniProt database using Mascot Daemon, and the identified digested peptides are listed in Tables S1–S4. As shown as Fig. 3,
there were 17, 18 and 9 peptides, for αS1-casein, αS2-casein and βcasein, respectively, detected after PHMN-Trypsin digestion for 2 min,
while only 10, 12 and 6 peptides were detected after free digestion for
16 h for αS1-casein, αS2-casein and β-casein, respectively. In particular, the digested amino acid sequence coverage of αS1-casein and
αS2-casein were 58% and 73%, respectively, which was significantly
higher than that of free trypsin digestion (46% and 52%) (Fig. 2). The
digested amino acid sequence coverage for κ-casein showed no significant difference using these two digestion procedures. All of the
above results demonstrate that the PHMN-Trypsin method achieved a
higher efficiency than the free trypsin digestion method for the caseins,
taking into consideration that the digestion time of PHMN-Trypsin was
remarkably less than that of free trypsin digestion. (Chen et al., 2015;
Lamberti et al., 2016; Lutter, Parisod, & Weymuth, 2011; Planque et al.,
2016). These results suggest that PHMN-Trypsin digestion is not only
highly efficient and fast but also reliable and credible.
2.6. UPLC-MS/MS analysis
The signature peptides were separated by a LC-30A ultrahigh-performance liquid chromatography system (Shimazu, Japan). Digested
samples were loaded onto a Waters ACQUITY UPLC® Peptide CSH™ C18
column (130 Å, 1.7 μm, 2.1 mm × 100 mm) with a column temperature
at 55 °C. The injection volume was 10 μL. The mobile phase consisted of
(Solvent A) water with 0.1% FA and (Solvent B) ACN with 0.1% FA. A
binary solvent was run at 0.3 mL/min. The binary solvent gradient was
2% B for 1 min, then linearly from 2% to 30% B in 5.5 min, linearly
from 30% to 80% B for 3.5 min, held at 80% B for 2 min, decreased to
2% B for 0.1 min and held at 2% for 3 min.
The quantification data was acquired on a mass spectrometer LCMS8060 (Shimadzu, Kyoto, Japan) with an electrospray ion under positive
mode (ESI+). The conditions of the MS/MS were set as follows: interface voltage, 1.5 kV; nebulizing gas (N2), 2.0 L/min; heating gas
(air), 15 L/min; drying gas (N2), 5 L/min; collision gas, Ar; interface
temperature, 300 °C; DL temperature, 150 °C; heat block temperature,
250 °C; the detailed parameters are shown in Table 1.
3.3. Selection of signature peptides
The selection of a proper signature peptide, which should fulfill
specific requirements to be considered reliable markers for the
target allergen proteins, is an important step for an analytical method of
a food allergen detection-based mass spectrum. We developed an analytical method to detect 2 or 3 signature peptides for each allergen
protein with respect to their abundance, mass response intensity, stability, specificity, and modifications to improve the method sensitivity
and specificity. There were ten peptides selected as target peptides:
TTMPLW, YLGYLEQLLR and FFVAPFPEVFGK for αS1-casein; FALPQYLK and NAVPITPTLNR for αS2-casein; EMPFPK, GPFPIIV and
AVPYPQR for β-casein; and SPAQILQWQVLSNTVPAK and YIPIQYVLSR
for κ-casein. A BLAST analysis of the target peptide sequences in the
UniProt database was checked for interspecies homology (Table S-5).
Unfortunately, there was high homology between the milk proteins of
the bovine and caprine species. For example, according to the UniProt
database, the β-casein of Bos Taurus (Bos d 11) shares more than 90%
similarity to that of Capra hircus (goat) and Ovis aries (sheep). In a
previous research, YIPIQYVLSR was selected as a quantitative peptide
used to detect κ-casein in milk, but our results suggest that YIPIQYVLSR
is weakly specific since it derived from a variety species, not only from
Bubalus bubalis or Bos but also from Giraffa Camelopardalis, Ovis aries, or
Rupicapra rupicapra (Lamberti et al., 2016). Furthermore, some other
nonspecific peptides, including VLPVPQK (β-casein), EMPFPK (βcasein), and YLGYLEQLLR (αS1-casein), were also used as quantitative
peptides during the allergen analysis (Chen et al., 2015; Ji et al., 2017;
Lamberti et al., 2016). In the present study, four exclusive characteristic
peptides were identified, including FFVAPFPEVFGK (αS1-casein),
NAVPITPTLNR
(αS2-casein),
AVPYPQR
(β-casein)
and
3. Results and discussion
3.1. Optimization of the allergens proteins extraction
The best conditions for the efficient extraction of the total proteins
from baked food were investigated, and the total protein content was
estimated by a Bradford assay. A high-speed bead mill homogenizer was
the new sample extraction method. Because of the high efficiency and
speed, this extraction method is increasingly applied in food analysis
and biological sample analysis (Bury, Jelen, & Kalab, 2001). Therefore,
we compared the traditional shaking extraction method and this new
extraction method. According to previous research, the common extraction buffers are 50 mM of NH4HCO3 (pH 9) and 200 mM of Tris-HCl
(pH 8). In this study, the extraction efficiency of these two buffers were
compared simultaneously. For the high-speed bead mill homogenizer
extraction method, the procedure is found in the sample preparation
section. For the traditional shaking extraction method, the procedure
was as follows: 5 mL of buffer was added into 1 g of the sample and the
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Table 1
The MS parameters of allergy protein-targeted peptides.
Protein
α-S1-casein
Peptides
Precursor ion(m/z)
a
692.9(2+)
15
FFVAPFPEV[ C- N-F]GK
TTMPLW
697.9(2+)
748.4(+)
TTMP[13C-15N-L]W
YLGYLEQLLR
755.4(+)
634.4(2+)
YLGYLEQL[13C-15N-L]R
NAVPITPTLNR a
637.9(2+)
598.4(2+)
NAVPITPT[13C-15N-L]NR
FALPQYLK
601.9(2+)
490.3(2+)
[13C-15N-F]ALPQYLK
AVPYPQR a
495.3(2+)
415.8(2+)
A[13C-15N-V]PYPQR
EMPFPK
418.8(2+)
374.7(2+)
EMP[13C-15N-F]PK
GPFPIIV
379.7(2+)
742.5(+)
FFVAPFPEVFGK
13
α-S2-casein
β-casein
κ-casein
a
b
GPFPII[13C-15N-V]
SPAQILQWQVLSNTVPAK
a
++
460.8(y8
)
496.3(y9++)
++
465.8(y8
)
415.3(y3+)
546.3(y4+)
422.3(y3+)
249.1(b4++)
552.8(y9++)
556.4(y9++)
911.6(y8+) b
456.3(y8++)
918.6(y8+)
648.4(y5+)
219.1(b2+)
381.3(y6++)
330.7(y5++)
660.4(y5+)
330.7(y5++)
244.2(y2+)
488.3(y4+)
498.3(y4+)
441.3(y4+)
625.4(b6+)
447.3(y4+)
315.2(y3+) b
526.8(b9++)
738.4(b7+)
488.3(y8++)
975.6(y8+)
491.8(y8++)
748.5(+)
660.7(3+)
SPAQILQWQV[13C-15N-L]SNTVPAK
YIPIQYVLSR
663.1(3+)
626.4(2+)
YIP[13C-15N-I]QYVLSR
629.9(2+)
Product ion(m/z)
b
b
Q1(V)
CE(V)
Q3(V)
−22
−22
−22
−24
−24
−24
−20
−20
−20
−32
−32
−24
−16
−16
−16
−13
−13
−11
−13
−13
−13
−20
−20
−24
−20
−20
−38
−20
−20
−26
−23
−22
−23
−24
−25
−25
−25
−21
−21
−18
−18
−19
−19
−19
−17
−12
−14
−13
−16
−14
−14
−26
−25
−28
−16
−18
−21
−19
−19
−19
−23
−14
−23
−21
−28
−21
−29
−28
−28
−36
−23
−36
−34
−24
−19
−24
−36
−24
−27
−25
−26
−22
−24
−23
−16
−20
−22
−24
−38
−24
Quantitative peptide.
Quantitative ion.
NAVPITPTLNR (αS2-casein), AVPYPQR (β-casein), and SPAQILQWQVLSNTVPAK (κ-casein) were selected as specific peptides and quantitative peptides. Finally, the stability of the four quantitative peptides
was tested to ensure the accuracy of the analytical method, and the
results showed that the four quantitative peptides exhibited good stability at 4 °C in 10 h (Fig. 4).
Cow's milk allergy is primarily an IgE-mediated or IgG-mediated
hypersensitivity reaction, and IgE or IgG recognition of allergens is
primarily due to amino acids comprising linear epitopes (Sicherer &
Sampson, 2014; Platts-Mills, Schuyler, Erwin, Commins, & Woodfolk,
2016). However, the epitopes were not detected previously in the study
of milk allergens using LC-MS/MS. Herein the four quantitativepeptides
for the four caseins all included the IgE- or IgG-binding epitopes. The
amino acid residues 38–49 of αS1-casein (FFVAPFPEVFGK) were involved in the IgE-binding regions of αS1-casein in milk (Inmaculada
et al., 2008). For αS2-casein, the quantitative peptide NAVPITPTLNR
included the IgE-binding regions of the amino acid residues 132–140
(VPITPTLNR) (Busse, Järvinen, Vila, Beyer, & Sampson, 2002). The
quantitative peptide AVPYPQR was likewise the IgE-binding epitope for
β-casein (Chatchatee et al., 2001). The amino acid residues 90–99
(SPAQILQWQV) in the quantitative peptide SPAQILQWQVLSNTVPAK
of κ-casein were both an IgE-binding and an IgG-binding epitope
(Järvinen, Chatchatee, Bardina, Beyer, & Sampson, 2001).
3.4. Optimization of analysis conditions
Fig. 1. The affection of extract buffer and extract means for sample extraction.
(n = 6).
To define the optimal LC-MS/MS analysis conditions, synthetic
peptide standard solutions were directly injected into the mass spectrometer. The theoretical value of the mass spectrum parameters of the
target peptides, such as the precursor ion, product ion and collision
energy, were generated by Skyline software (University of Washington,
version 3.5.0.9319), a predictive analytics tool for multiple reaction
monitoring (MRM) mass spectrum assays. According to the results of
SPAQILQWQVLSNTVPAK (κ-casein), which only existed in allergy
proteins from Bubalus bubalis or Bos Taurus (Table S5). In addition,
sensitivity is another factor that should be considered when choosing
the quantitative peptide. Hence, FFVAPFPEVFGK (αS1-casein),
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Fig. 2. MALDI-TOF MS spectra and sequence coverage of target allergen proteins (1 mg/mL) after digestion with (a) PHMN-trypsin for 2 min, (b) free trypsin for 16 h.
When the chromatographic behaviors and the mass spectrometric responses of formic acid and trifluoroacetic acid for the target peptides
was compared, the results showed that the response values and peak
shapes of the target peptides in the mobile phase system containing
formic acid were significantly better than those in the trifluoroacetic
acid system. Therefore, 0.1% formic acid-water and 0.1% formic acidacetonitrile were selected as the mobile phase. The UPLC-MS/MS
chromatogram of the target peptides is shown in Fig. 5.
the Skyline software, the precursor ions of the peptide fragments were
confirmed under an optimized cone voltage, then the collision energy
parameters were optimized for further MS/MS analysis of the peptides.
Two stable and sensitive characteristic ions were selected from the
product ions fragmentation to establish the MRM-detecting ions. Under
the optimized MRM conditions, these peptides were detected for qualification and quantification of the milk allergens. Table 1 described the
optimized experimental parameters.
The chromatographic column chosen was the Waters ACQUITY
UPLC® Peptide CSH™ special column C18 130 Å Peptide analysis because the peak capacity, peak shape and sensitivity of the CSH™ column
are preferable than the BEH C18 300 column. Due to the use of the
positive ion mode scanning in mass spectrometry, the addition of a
small amount of acid to the mobile phase can favor the ionization.
3.5. Optimization of digestion procedure for real samples
For the baked food, the content of allergens was very low since there
are numerous components including flour, food additives and other
ingredients in the matrix. The question has been raised if these
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Fig. 3. Sequence coverage of αS1-casein, αS2-casein, β-casein, κ-casein digested by (a) PHMN-trypsin, (b) free trypsin. Matched amino acids are shown in red. (For
interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
upward trend at 2–15 min and tended to stabilize after 15 min (Fig. 6).
Thus, the digestion time was set as 15 min for the baked food. Planque
et al. (2016) developed a LC-MS/MS method for allergen detection in
foodstuffs with a 16 h trypsin digestion procedure. Chen et al. (2015)
also detected allergens in complex foods by a LC-MS method with
overnight digestion. Compared to these previous studies, our digestion
ingredients could affect the digestion of PHMN-Trypsin. Although the
digestion of 1 μg/μL of the standard proteins could be accomplished in
1–2 min by 250 μg of PHMN-Trypsin, a longer digestion time (2 min,
5 min, 10 min, 15 min, 20 min, and 30 min) was tested to ensure the
sufficient digestion in real baked samples. The results indicated that the
enzymatic hydrolysis efficiency of four quantitative peptides showed an
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K. Qi, et al.
Fig. 6. Time curves of the PHMN-Trypsin digestion efficiency of the four
quantitative peptides in the blank matrix (n = 6).
Fig. 4. The stability results of the four quantitative peptides (n = 6).
method is remarkably time-saving.
3.6. Influence of allergens by high temperature
The high temperature processing was a common procedure of baked
food. However, high temperature was one of the important factors to
induce the degradation of the protein. Although the thermostability of
allergen was higher than the general protein, high temperatures over
150 °C can also degrade allergens. Therefore, in this study the influence
of milk allergens by high temperature in baking process was investigated. The model matrix and no-baked matrix were prepared with
the same raw material composition and contained the same quantity of
milk allergens. The model matrix was treatment by high temperature,
while the no-baked matrix was no treatment by high temperature. The
target milk allergens of model matrix and no-baked matrix were detected using the developed method. To intuitively estimate the influence of milk allergens by high temperature, the ratio of concentration of
allergen of model matrix/concentration of allergen of no-baked matrix
was calculated. The value of this ratio was inversely proportional to the
Fig. 7. The ratio of concentration of allergen of model matrix/concentration of
allergen of no-baked matrix for target allergen. (n = 6).
Fig. 5. UPLC-MS/MS chromatogram of the
target peptides for αS1-casein, αS2-casein,
β-casein and κ-casein. The most sensitive
transition is given in black, it is also the
quantitative ion; the second is given in red,
and the quantitative peptides are marked
with an asterisk. (For interpretation of the
references to colour in this figure legend,
the reader is referred to the Web version of
this article.)
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K. Qi, et al.
Table 2
Linear range, linear equation and r2 of the signature peptides.
Protein
Peptides
Linear range (nmol/L)
Linear equation
r2
αS1-casein
FFVAPFPEVFGK a
TTMPLW
YLGYLEQLLR
NAVPITPTLNR a
FALPQYLK
AVPYPQR a
EMPFPK
GPFPIIV
SPAQILQWQVLSNTVPAK
YIPIQYVLSR
1–100
1–100
1–100
1–100
1–100
1–100
1–100
1–100
1–100
1–100
y = 1.0103x-0.7111
y = 1.0062x-0.429
y = 1.0125x-0.8887
y = 0.9909x+0.621
y = 0.9942x+0.3994
y = 0.985x+1.0396
y = 0.9918x+0.571
y = 0.9967x+0.2316
y = 0.9938x+0.431
y = 1.0091x-0.6336
0.9981
0.9988
0.9971
0.9968
0.9993
0.9980
0.9989
0.9978
0.9998
0.9970
αS2-casein
β-casein
κ-casein
a
a
Quantitative peptide.
Table 3
Recovery test of the UHPLC-MS methods (n = 6).
Protein
Spike level (μg/g)
Recovery (%)
RSD (%)
LOD (μg/g)
LOQ (μg/g)
αS1-casein (FFVAPFPEVFGK)
1
2
5
1
2
5
1
2
5
1
2
5
65.2
69.7
71.5
80.2
79.6
86.1
74.4
83.6
82.3
81.5
83.0
85.5
3.9
4.0
2.4
10.6
2.8
4.5
0.2
4.9
5.5
3.3
10.0
6.4
0.25
0.83
0.15
0.50
0.26
0.70
0.12
0.38
αS2-casein (NAVPITPTLNR)
β-casein (AVPYPQR)
κ-casein (SPAQILQWQVLSNTVPAK)
casein and β-casein are an important indicator that can imply whether
the baked food contains the milk allergen.
Table 4
Determination of the allergens in baked food (μg/g).
NO.
cookie 1
cookie 2
cookie 3
cookie 4
cookie 5
cookie 6
cookie 7
cookie 8
cookie 9
cookie 10
cookie 11
cookie 12
cookie 13
bread 1
bread 2
bread 3
bread 4
bread 5
bread 6
bread 7
bread 8
bread 9
bread 10
a
b
c
Label information
a
N
N
N
N
N
N
Yb
Y
Y
Y
Y
Y
Y
N
N
N
N
N
Y
Y
Y
Y
Y
αS1-casein
ND
1.1
ND
ND
23.8
10.4
11.8
77.8
559.3
8.0
2.8
1.2
ND
ND
97.1
ND
37.1
17.0
443.0
23.4
28.2
4.6
33.2
αS2-casein
c
ND
0.7
ND
ND
6.4
5.8
4.0
6.4
20.8
4.4
0.6
ND
ND
ND
3.4
ND
1.8
ND
26.1
2.0
2.2
1.7
2.9
β-casein
κ-casein
ND
1.8
ND
ND
20.7
2.7
20.6
99.4
1036.9
3.2
8.9
3.5
1.7
ND
193.7
ND
69.0
9.1
1109.4
351.2
88.4
11.3
86.9
ND
0.3
ND
ND
2.8
ND
4.3
2.5
19.1
0.2
0.2
ND
ND
ND
0.2
ND
ND
ND
21.1
0.8
2.2
ND
2.4
3.7. Method validation
The standard solution was diluted to prepare a series of molar
concentrations of 1, 2, 5, 10, 20, 50, and 100 nmol/L, and the internal
standard molar concentration was fixed at 20 nmol/L. The four proteins
showed good linear relationships over the molar concentrations of
1–100 nmol/L, with correlation coefficients (r2) higher than 0.995. The
chromatograms of each peptide and the internal standard are shown in
Fig. 4. The molar concentration of the quantitative peptide was calculated via the calibration curve, and then the target protein concentration was calculated based on the molar ratio of the specific peptide to
the target protein, 1:1. The formula is Cx = 10−10 × M × nx, where Cx
(μg/g) is the mass concentration of the target protein; M is the moles of
target protein; and nx (nmol/L) is the molar concentration of the target
peptide. With the target protein spiked in bread and biscuits, the limit
of detection (LOD) and the limit of quantification (LOQ) corresponded
to S/N = 3 and S/N = 10, respectively. The results of the LOD and LOQ
are given in Table 2. The recovery of the whole analytical method was
assessed by three levels (1, 2, and 5 μg/g) of the protein standards
spiked into the blank matrix of baked food. The recoveries of the
quantitative peptides were 35.2%–86.1%, with relative standard deviations (RSD) of less than 10.6%. The results of the recoveries and the
precision of the four proteins are shown in Table 3.
N means there is no allergen labeling.
Y means the food has allergen labeling.
ND means not detected.
3.8. Food sample analysis application
degree of the allergen's degradation induced by high temperature. As
shown in Fig. 7, the ratio of αS1-casein (0.73) was the highest, followed
by αS2-casein (0.65) and β-casein (0.54), and the ratio of κ-casein
(0.18) was the lowest. Therefore, it was suggested that for baked food,
the influence of high temperature on αS1-casein was weak. αS1-casein
and β-casein are the main components of casein and milk allergic
protein. Therefore, it was suggested that the concentrations of αS1-
To evaluate the allergens in the real samples, 13 cookies and 10
breads obtained from a local market were pretreated and analyzed
using this newly developed method. Six cookies and five breads had no
label regarding allergen information. However, the milk allergen was
detected in six of the eleven samples without allergen labeling, as
shown in Table 4. The UPLC-MS/MS chromatograms of the target
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K. Qi, et al.
peptides in a cookie and bread are shown in Figure S. For cookies, the
detection rates of milk allergen were 50% in samples without allergen
labeling, and for breads, the detection rates of milk allergen were 60%
in samples without allergen labeling. A previous study has suggested
that the reference dose of milk-based parametric modeling of minimal
eliciting doses from food-allergic populations is 0.1 mg of milk protein
(Taylor, Katrina, & Allen, 2014). For our results, the total concentrations of casein protein in the sample cookie 4, bread 2 and bread 4 were
745.5 μg/g, 294.4 μg/g and 1599.6 μg/g, respectively. This result
means that 0.13 g of cookie 4, 0.34 g of bread 2 or 0.06 g of bread could
elicit food allergy symptoms. It is clear that these foods without allergen labeling increased the risk of consumers.
In addition, according to the result of section 3.6 in this study, it
suggests that the concentration of αS1-casein and β-casein in baked
food was the main indicator that implies whether the baked food contains milk allergens. This hypothesis was verified by our detection result of a real sample in Table 4. Therefore, the αS1-casein and β-casein
could be good markers for milk allergens in baked food.
prevalence and hospitalizations. NCHS Data Brief, 10(10), 1–8.
Bury, D., Jelen, P., & Kalab, M. (2001). Disruption of lactobacillus delbrueckii ssp. bulgaricus 11842 cells for lactose hydrolysis in dairy products: A comparison of sonication, high-pressure homogenization and bead milling. Innovative Food Science &
Emerging Technologies, 2(1), 23–29.
Busse, P. J., Järvinen, K. M., Vila, L., Beyer, K., & Sampson, H. A. (2002). Identification of
sequential IgE-binding epitopes on bovine alpha(s2)-casein in cow's milk allergic
patients. International Archives of Allergy and Immunology, 129(1), 93–96.
Chatchatee, P., Järvinen, K. M., Bardina, L., Vila, L., Beyer, K., & Sampson, H. A. (2001).
Identification of IgE and IgG binding epitopes on beta- and kappa-casein in cow's milk
allergic patients. Clinical & Experimental Allergy, 31(8), 1256–1262.
Chen, Q., Zhang, J., Ke, X., Lai, S., Tao, B., Yang, J., et al. (2015). Quantification of bovine
β-casein allergen in baked foodstuffs based on ultra-performance liquid chromatography with tandem mass spectrometry. Food Additives and Contaminants Part A
Chemistry Analysis Control Exposure and Risk Assessment, 32(1), 25–34.
Deng, Y., Deng, C., Qi, D., Liu, C., Liu, J., Zhang, X., et al. (2009). Synthesis of core/shell
colloidal magnetic zeolite microspheres for the immobilization of trypsin. Advanced
Materials, 21(13), 1377–1382.
Docena, G. H., Fernandez, R., Chirdo, F. G., & Fossati, C. A. (1996). Identification of
casein as the major allergenic and antigenic protein of cow's milk. Allergy, 51(6),
412–416.
Dupuis, R., Meisel, Z., Grande, D., Strupp, E., Kounaves, S., Graves, A., et al. (2016). Food
allergy management among restaurant workers in a large U.S. city. Food Control,
63(5), 147–157.
Dzwolak, W. (2017). Assessment of food allergen management in small food facilities.
Food Control, 73(3), 323–331.
Heick, J., Fischer, M., & Pöpping, B. (2011). First screening method for the simultaneous
detection of seven allergens by liquid chromatography mass spectrometer. Journal of
Chromatography A, 1218, 938–943.
Ibáñez, J. A., Muck, A., & Svatoš, A. (2007). Metal-chelating plastic MALDI (pMALDI)
chips for the enhancement of phosphorylated-peptide/protein signals. Journal of
Proteome Research, 6(9), 3842–3848.
Inmaculada, C., Javier, Z., Wayne, G. S., Jing, L., Ludmilla, B., Carmen, D. M., et al.
(2008). Mapping of the IgE and IgG4 sequential epitopes of milk allergens with a
peptide microarray–based immunoassay. The Journal of Allergy and Clinical
Immunology, 122(2), 589–594.
Järvinen, K. M., Chatchatee, P., Bardina, L., Beyer, K., & Sampson, H. A. (2001). IgE and
IgG binding epitopes on alpha-lactalbumin and beta-lactoglobulin in cow's milk allergy. International Archives of Allergy and Immunology, 126(2), 111–118.
Ji, J., Zhu, P., Pi, F., Sun, C., Sun, J., Jia, M., et al. (2017). Development of a liquid
chromatography-tandem mass spectrometry method for simultaneous detection of
the main milk allergens. Food Control, 74, 79–88.
Krenkova, J., Lacher, N. A., & Svec, F. (2009). Highly efficient enzyme reactors containing
trypsin and endoproteinaselysc immobilized on porous polymer monolith coupled to
ms suitable for analysis of antibodies. Analytical Chemistry, 81(5), 2004–2012.
Krogh, T. N., Berg, T., & Højrup, P. (1999). Protein analysis using enzymes immobilized to
paramagnetic beads. Analytical Biochemistry, 274(2), 153–162.
Lamberti, C., Acquadro, E., Corpillo, D., Giribaldi, M., Decastelli, L., Garino, C., et al.
(2016). Validation of a mass spectrometry-based method for milk traces detection in
baked food. Food Chemistry, 199, 119–127.
Liu, S., Bao, H., Zhang, L., & Chen, G. (2013). Efficient proteolysis strategies based on
microchip bioreactors. Journal of Proteomics, 82(8), 1–13.
Lutter, P., Parisod, V., & Weymuth, H. (2011). Development and validation of a method
for the quantification of milk proteins in food products based on liquid chromatography with mass spectrometric detection. Journal of AOAC International, 94(4),
1043–1059.
Ma, X., Sun, P., He, P., Han, P., Wang, J., Qiao, S., et al. (2010). Development of
monoclonal antibodies and a competitive elisa detection method for glycinin, an
allergen in soybean. Food Chemistry, 121(2), 546–551.
Ning, W., & Bruening, M. L. (2015). Rapid protein digestion and purification with
membranes attached to pipet tips. Analytical Chemistry, 87(24), 11984–11989.
Pilolli, R., De, A. E., & Monaci, L. (2017). Streamlining the analytical workflow for
multiplex MS/MS allergen detection in processed foods. Food Chemistry, 221,
1747–1753.
Planque, M., Arnould, T., Dieu, M., Delahaut, P., Renard, P., & Gillard, N. (2016).
Advances in ultra-high performance liquid chromatography coupled to tandem mass
spectrometry for sensitive detection of several food allergens in complex and processed foodstuffs. Journal of Chromatography A, 1464, 115–123.
Platts-Mills, T. A. E., Schuyler, A. J., Erwin, E. A., Commins, S. P., & Woodfolk, J. A.
(2016). IgE in the diagnosis and treatment of allergic disease. The Journal of Allergy
and Clinical Immunology, 137(6) 1662-1170.
Poms, R. E., Klein, C. L., & Anklam, E. (2004). Methods for allergen analysis in food: A
review. Food Additives and Contaminants, 21(1), 1–31.
Qin, W. J., Song, Z., Fan, C., Zhang, W., Cai, Y., Zhang, Y., et al. (2012). Trypsin immobilization on hairy polymer chains hybrid magnetic nanoparticles for ultrafast,
highly efficient proteome digestion, facile 18O labeling and absolute protein quantification. Analytical Chemistry, 84(7), 3138–3144.
Sicherer, S. H., & Sampson, H. A. (2010). Food allergy. The Journal of Allergy and Clinical
Immunology, 125(2), s116–s125.
Sicherer, S. H., & Sampson, H. A. (2014). Food allergy: Epidemiology, pathogenesis, diagnosis, and treatment. The Journal of Allergy and Clinical Immunology, 133(2),
291–307.
Soogali, N. B., & Soon, J. M. (2018). Food allergies and perceptions towards food allergen
labelling in Mauritius. Food Control, 93(11), 144–149.
Soon, J. M. (2018). ‘No nuts please’: Food allergen management in takeaways. Food
Control, 91(9), 349–356.
Taylor, S. B., Katrina, J., & Allen, K. J. (2014). Establishment of reference doses for residues of allergenic foods: Report of the VITAL expert panel. Food & Chemical
Toxicology, 63(1), 9–17.
Wal, J. M. (1998). Cow's milk allergens. Allergy, 53(11), 1013–1022.
4. Conclusion
In this work, a rapid method for milk allergen detection that was
based on immobilized trypsin and ultrahigh-performance liquid chromatography – tandem isotope dilution mass spectrometry was developed. The MS/MS methods of ten candidates of signature peptides were
established, and four exclusive peptides (FFVAPFPEVFGK, NAVPITPTLNR, AVPYPQR and SPAQILQWQVLSNTVPAK) were finally selected as
the quantitative peptides of target allergens due to the high specificity,
sensitivity and stability. αs1-casein, αs2-casein, β-casein, and κ-casein
in baked food were detected within a limit of quantification as low as
0.38 μg/g.
Notably, the most important point of this work was the enhancement of the digestion efficiency. The PHMN-trypsin immobilized higher
amounts of trypsin than the conventional magnetic nanoparticles or the
microspheres. The time of digestion was reduced to 15 min from 12 to
16 h of conventional in-solution digestion. This verified new method
truly realized the rapid detection of allergens in baked goods. It is more
economical and environmental-friendly due to its reagent-saving and
time-saving procedure compared with the conventional trypsin digestion method. With the commercialization of PHMN-Trypsin, this
method will be especially suitable for the quality control of food processing enterprises. This will improve the quality of food and reduce the
risk of food safety accidents, resulting in longterm economic benefits.
This is the first report of immobilized trypsin applied in food allergen
detection, which could provide technical support for food allergen
monitoring and supervision.
Acknowledgements
This work was supported by the Beijing Municipal Science and
Technology Project, China (No. Z181100009318009) and the Beijing
Municipal Natural Science Foundation, China (No. 7162088).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.foodcont.2019.03.017.
References
Ajala, A. R., Cruz, A. G., Faria, J. A. F., Walter, E. H. M., Granato, D., & SantAna, A. S.
(2010). Food allergens: Knowledge and practices of food handlers in restaurants. Food
Control, 21(10), 1318–1321.
Bezstarosti, K., Ghamari, A., Grosveld, F. G., & Demmers, J. A. (2010). Differential proteomics based on 18O labeling to determine the cyclin dependent kinase 9 interactome. Journal of Proteome Research, 9(9), 4464–4475.
Branum, A. M., & Lukacs, S. L. (2008). Food allergy among U.S. Children: Trends in
187