Quantification of bovine β-casein allergen in baked foodstuffs based on ultra-performance liquid chromatography with tandem mass spectrometry

Figures & data

Table 1. Theoretical peptides of simulated tryptic digestion and detectable peptides using UPLC-TOF.

Number	Peptide	Position	Matched product ions	Peptides score^a
1	YPVEPFTESQSLTLTDVENLHLPLPLLQ SWMHQPHQPLPPTVMFPPQSVLSLSQSK	114–169	16	7.74
2	IHPFAQTQSLVYPFPGPIPNSLPQNIPPLT QTPVVVPPFLQPEVMGVSK	49–97	8	7.40
3	ELEELNVPGEIVESLSSSEESITR	2–25	Undetectable
4	DMPIQAFLLYQEPVLGPVR	184–202	31	8.58
5	FQSEEQQQTEDELQDK	33–48	Undetectable
6	AVPYPQR	177–183	2	6.91
7	VLPVPQK	170–176	7	7.79
8	EMPFPK	108–113	5	7.47
9	GPFPIIV	203–209	10	8.07
10	EAMAPK	100–105	5	7.03

Note: ^aPeptide scores were calculated by ProteinLynx Global Server Version 2.5 software.

Table 2. MRM parameters of the precursor ion, product ion, cone voltage, collision energy and type of fragment for each candidate signature peptide and ISs.

Peptide	Precursor ion (m/z)	Cone voltage (V)	Product ion (m/z)	Collision energy (eV)	Fragment
EMPFPK	374.9	15	146.1	25	y1
			243.2	15	y2
			487.3	10	y4
VLPVPQK	391.0	15	213.5	12	b2
			371.8	18	y3
			568.3	12	y5
AVPYPQR	416.9	15	175.1	26	y1
			330.7	10	y5
			400.2	18	y3
DMPIQAFLLYQEPVLGPVR	730.8	20	428.0	29	y4
			737.0	29	y7
			867.0	26	y8
GPFPIIV	742.9	40	441.3	27	y4
			512.3	27	b5
			625.4	23	b6
VL*PV*PQK (IS1)	397.5	15	220.3	12	b2
			371.8	18	y3
			574.3	12	y5
VMPVLK (IS3)	344.0	15	231.2	10	b2
			260.2	17	y2
			456.3	12	y4

Figure 1. MRM chromatograms of the quantitative product ion spectra from the selected precursors of the target peptides with the signal-to-noise ratio, from top to bottom: DMPIQAFLLYQEPVLGPVR, GPFPIIV, EMPFPK, VLPVPQK and AVPYPQR.

Figure 2. Comparison of the relative peak areas normalised to a standard of IS1, IS3 and a signature peptide in different food matrices (n = 3). A total of 10 µg ml^–1 bovine β-casein solution and 1:100 diluted human milk were digested respectively and combined with 0.25 µg ml^–1 IS1 solution at the ratio of 1:1:1 (v/v/v) to form a digest mixture. The negative sample and commonly used baking ingredients (wheat flour, sugar, salt, peanut oil, hen’s eggs, cacao and coconut) were treated identical with human and bovine β-casein with trypsin, and mixed in a ratio of 1:1 (v/v) with the digest mixture. The standard solution was prepared by mixing the digest mixture and water in the ratio of 1:1 (v/v), and its peak area was set as 100% abundance.

Figure 3. Relative peak areas normalised to a signature peptide standard and IS1, IS2 in different food matrices (n = 3). A total of 0.1 g negative sample and commonly used baking ingredients (wheat flour, sugar, salt, peanut oil, hen’s eggs, cacao and coconut) were weighed into the Eppendorf caps and mixed with 100 µl of 10 µg ml^–1 bovine β-casein. After spiking of 10 µl IS1 (0.25 µg ml^–1) and IS2 (1 µg ml^–1) into the caps, the mixtures were digested as described in the ‘Tryptic digestion and peptide extraction’ section. The solutions of bovine β-casein and ISs at the same concentration were digested as a standard, and its peak area was set as 100% abundance.

Figure 4. Recoveries of extraction and suspension digestion: black, suspension digestion; and white, extraction digestion (n = 3).

Table 3. Comparison of recovery obtained with the different extraction method.

	Buffer	Extraction method	Recovery^a	Reference
1	50 mM NH4HCO3, 1 M urea, pH 8.0	2 g sample added with 20 ml preheated (60°C) buffer, homogenised, shake for 15 min	24.4% ± 0.8%	Lutter et al. (2011)
2	Tris-buffer, pH 8.2	2 g sample added with 20 ml buffer, homogenised, incubated at 60°C for 3 h	21.5% ± 0.5%	Heick et al. (2011)
3	1 M guanidine hydrochloride, 20 mM DTT, 10 mM sodium citrate, pH 6.75	1 g sample added with 25 ml buffer, homogenised, shake for 30 min	35.9% ± 0.8%	Monaci et al. (2010)
4	4.3 mM Na2HPO4, 1.4 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4	2 g sample added with 20 ml buffer, homogenised, incubated at 4°C for 3 h^b	19.9% ± 0.8%	Kim et al. (2002)
5	Water	2 g sample added with 20 ml water, homogenised, shake for 3 h	23.1% ± 1.4%
6	Method described in the ‘Tryptic digestion and peptide extraction’ section		50.8% ± 1.6%

Notes: ^aRecovery = detected β-casein/spiked β-casein*100%.

^bThe ratio of sample to water was increased from 1:3 in the literature to 1:10.

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