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

Chen Q, Zhang J, Ke X, Lai S, Tao B, Yang J, Mo W, Ren Y - Food Addit Contam Part A Chem Anal Control Expo Risk Assess (2014)

Bottom Line: The peptide VLPVPQK was selected as the signature peptide for bovine β-casein because of the high sensitivity.A stable isotope-labelled internal standard was designed to adjust the instability of sample pre-treatment and ionisation caused by matrix effect.The UPLC-TQ-MS/MS method developed based on a tryptic signature peptide led to a reliable determination of bovine β-casein allergen in baked food matrices at a low quantitation level down to 500 μg kg(-1) with a satisfactory accuracy (< 8.9%) and recovery (98.8% ± 2.6% to 106.7% ± 3.0%).

View Article: PubMed Central - PubMed

Affiliation: a Zhejiang Provincial Center for Disease Control and Prevention , Hangzhou , China.

ABSTRACT
The quantification of allergens in food including baked food matrices is of great interest. The aim of the present study was to describe a non-immunologic method to quantify bovine β-casein using ultra-performance liquid chromatography tandem triple quadrupole mass spectrometry (UPLC-TQ-MS/MS) in multiple reaction monitoring (MRM) mode. Eight of 10 theoretical peptides from β-casein after tryptic digestion were compared and MRM methods were developed to determine five signature peptides. The peptide VLPVPQK was selected as the signature peptide for bovine β-casein because of the high sensitivity. A stable isotope-labelled internal standard was designed to adjust the instability of sample pre-treatment and ionisation caused by matrix effect. Using the present suspension digestion method, the native and denatured β-casein could be digested to release the signature peptide at the maximum extent. The UPLC-TQ-MS/MS method developed based on a tryptic signature peptide led to a reliable determination of bovine β-casein allergen in baked food matrices at a low quantitation level down to 500 μg kg(-1) with a satisfactory accuracy (< 8.9%) and recovery (98.8% ± 2.6% to 106.7% ± 3.0%).

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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.
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Figure 0001: 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.

Mentions: Based on the parameters obtained from the UPLC-TOF analysis, including the electronic charge, molecular weight and retention time of each peptide, the product ions of peptide candidates were searched using the UPLC-TQ-MS/MS with the daughter scan mode. Peptides 1 and 2 were not selected as they both showed poor chromatographic resolution because of long sequences (Table 1). Peptide 10 was not selected because the signal of its product ions was extremely low during UPLC-TQ-MS/MS analysis. After optimisation of the MS parameters (see the ‘UPLC-TQ-MS/MS conditions’ section), three product ions for each of the other five peptides (peptides 4 and 6–9 in Table 1) with the best sensitivity were selected for the establishment of MRM methods (Table 2). The specificity and selectivity of the five signature peptides were evaluated in silico and in vitro. The in silico evaluation was performed using the BLAST tool. However, BLAST can be carried out only with the peptides contain more than eight amino acid in their sequence such as peptide 4 in Table 1. The BLAST with peptides 6, 7 and 9 was performed after adding arginine or lysine before the N-terminal of the sequences. Otherwise, the signature peptides could not be produced after tryptic digestion. Peptide 8 could not be blasted due to the short sequences. Peptides 4, 6, 7 and 9 could be found only in β-casein from various mammalian species. The BLAST result showed that the four peptides would not exist in other proteins in the Uniprot. However, the current proteomic databases could not be considered exhaustive in terms of food ingredients. Therefore, an in vitro experiment was carried out with the often used ingredients in baked foods, such as wheat flour, soybean, coconut, cacao and chicken egg. The often-used ingredients in baked foods were pre-treated and analysed as described in the ‘Tryptic digestion and peptide extraction’ and ‘UPLC-TQ-MS/MS conditions’ sections. No chromatographic peaks of the peptides were identified and detected. However, the chromatographic peaks of these peptides appeared when a β-casein solution was also digested and analysed using the present method. The peptide VLPVPQK was finally selected as the signature peptide for β-casein in view of the signal intensity and signal-to-noise ratio (Figure 1). Other four peptides candidates were also qualitative analysed to enhance the confidence of the present of β-casein.Table 2.


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

Chen Q, Zhang J, Ke X, Lai S, Tao B, Yang J, Mo W, Ren Y - Food Addit Contam Part A Chem Anal Control Expo Risk Assess (2014)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4487529&req=5

Figure 0001: 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.
Mentions: Based on the parameters obtained from the UPLC-TOF analysis, including the electronic charge, molecular weight and retention time of each peptide, the product ions of peptide candidates were searched using the UPLC-TQ-MS/MS with the daughter scan mode. Peptides 1 and 2 were not selected as they both showed poor chromatographic resolution because of long sequences (Table 1). Peptide 10 was not selected because the signal of its product ions was extremely low during UPLC-TQ-MS/MS analysis. After optimisation of the MS parameters (see the ‘UPLC-TQ-MS/MS conditions’ section), three product ions for each of the other five peptides (peptides 4 and 6–9 in Table 1) with the best sensitivity were selected for the establishment of MRM methods (Table 2). The specificity and selectivity of the five signature peptides were evaluated in silico and in vitro. The in silico evaluation was performed using the BLAST tool. However, BLAST can be carried out only with the peptides contain more than eight amino acid in their sequence such as peptide 4 in Table 1. The BLAST with peptides 6, 7 and 9 was performed after adding arginine or lysine before the N-terminal of the sequences. Otherwise, the signature peptides could not be produced after tryptic digestion. Peptide 8 could not be blasted due to the short sequences. Peptides 4, 6, 7 and 9 could be found only in β-casein from various mammalian species. The BLAST result showed that the four peptides would not exist in other proteins in the Uniprot. However, the current proteomic databases could not be considered exhaustive in terms of food ingredients. Therefore, an in vitro experiment was carried out with the often used ingredients in baked foods, such as wheat flour, soybean, coconut, cacao and chicken egg. The often-used ingredients in baked foods were pre-treated and analysed as described in the ‘Tryptic digestion and peptide extraction’ and ‘UPLC-TQ-MS/MS conditions’ sections. No chromatographic peaks of the peptides were identified and detected. However, the chromatographic peaks of these peptides appeared when a β-casein solution was also digested and analysed using the present method. The peptide VLPVPQK was finally selected as the signature peptide for β-casein in view of the signal intensity and signal-to-noise ratio (Figure 1). Other four peptides candidates were also qualitative analysed to enhance the confidence of the present of β-casein.Table 2.

Bottom Line: The peptide VLPVPQK was selected as the signature peptide for bovine β-casein because of the high sensitivity.A stable isotope-labelled internal standard was designed to adjust the instability of sample pre-treatment and ionisation caused by matrix effect.The UPLC-TQ-MS/MS method developed based on a tryptic signature peptide led to a reliable determination of bovine β-casein allergen in baked food matrices at a low quantitation level down to 500 μg kg(-1) with a satisfactory accuracy (< 8.9%) and recovery (98.8% ± 2.6% to 106.7% ± 3.0%).

View Article: PubMed Central - PubMed

Affiliation: a Zhejiang Provincial Center for Disease Control and Prevention , Hangzhou , China.

ABSTRACT
The quantification of allergens in food including baked food matrices is of great interest. The aim of the present study was to describe a non-immunologic method to quantify bovine β-casein using ultra-performance liquid chromatography tandem triple quadrupole mass spectrometry (UPLC-TQ-MS/MS) in multiple reaction monitoring (MRM) mode. Eight of 10 theoretical peptides from β-casein after tryptic digestion were compared and MRM methods were developed to determine five signature peptides. The peptide VLPVPQK was selected as the signature peptide for bovine β-casein because of the high sensitivity. A stable isotope-labelled internal standard was designed to adjust the instability of sample pre-treatment and ionisation caused by matrix effect. Using the present suspension digestion method, the native and denatured β-casein could be digested to release the signature peptide at the maximum extent. The UPLC-TQ-MS/MS method developed based on a tryptic signature peptide led to a reliable determination of bovine β-casein allergen in baked food matrices at a low quantitation level down to 500 μg kg(-1) with a satisfactory accuracy (< 8.9%) and recovery (98.8% ± 2.6% to 106.7% ± 3.0%).

Show MeSH