Page 83 - Application Notebook - Solution for Food Safety

LAAN-A-LM-E112

Application Liquid Chromatography Mass Spectrometry

News High Sensitivity Analysis of Peanut Allergen in

Cumin and Spice Mix [LCMS-8060]
No.C141

Food allergens are a major public health concern. MRM transitions for each clone was determined by
Among them, peanut allergy is one of the common using Skyline (MacCoss Lab Software). The transition
food allergies. To avoid unexpected contact with food list, which contained more than ten peptides for each
allergens, food labels are strictly used to indicate the clone, was reviewed by removing several peptides that
presence of specific allergens. With the increasing could be susceptible by post translational modification
awareness of food allergies, the presence of undeclared and Maillard reaction during food processing.
peanut in cumin lead to huge recalls in recent years. Finally, nine peptides including three common peptides
Although ELISA is the most commonly used technique to both clones were selected based on sensitivity. Three
to detect allergens, its false-positive rate is a major transitions were set for each peptide.
concern due to its cross-reactivity. We developed a
method with high specificity and sensitivity to overcome Protein Amino Acid Sequences.
this issue by using a high sensitivity triple quadrupole DTHKSEIAHRFKDLGEEHFKGLVLIA
FSQYLQQCPFDEHVKLVNELTEFAK
mass spectrometer to detect peanut allergen Ara h1 TCVAD......................
(Fig.1) in commercially available spices and seasonings. Search for transitions by MRM analysis of
peanut digest.
(×10,000,000)
2.00
1.75
Predicting digested peptides. 1.50 1.25
Calculating precursor m/z. Method file 1.00 0.75
Predicting CE. 0.50
Selecting product ion candidates. 0.00 0.25
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 min
Data file
Collision energy optimization based on MRM
Select transitions based on analysis.
(×1,000,000)
2.00
observed chromatograms. 1.75
Preparing for collision energy Method file 1.50
1.25
optimization. 1.00
0.75
0.50 0.25
Data file 0.00 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 min
Selecting optimal transitions and
CE for quantitation based on Optimized MRM methods
actually measured data as well. Method file 2.00 (x1,000,000)
1.75
1.50
1.25
1.00
0.75
0.50
0.25
0.00
1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 min
Fig. 2 Workflow of MRM Transition Optimization Using Skyline
Fig. 1 Structure of Ara h1 [3S7I] (68kDa) Vicilin Like Protein
(×10,000,000)
1.15
Q Sample Preparation 1.10 SSEN... SSDN...
1.05
1.00 GTGN... (×0.2)
Commercially available defatted peanut flour was 0.95 NNPF...
purchased and used for the initial development work. 0.90
0.85
The test samples were ground and protein content was 0.80
0.75
enriched by liquid-liquid extraction. Extracted proteins 0.70
0.65
were denatured, reduced and alkylated before 0.60 EGEQ... EGEQ... IPSG...
0.55
subjecting to tryptic digestion to obtain peptides that 0.50
0.45
were quantitated as proxies of original protein 0.40 GSEE...
0.35
abundance. 0.30 NNPF...
0.25
Cinnamon, cumin, chilli pepper, ginger, garlic, mustard 0.20
0.15
seed, nutmeg, oregano, rosemary, sage, turmeric and 0.10
0.05
thyme were selected as test food samples for evaluating -0.05
0.00
cross-reactivity and sensitivity of the developed method. 1.0 2.0 3.0 4.0 5.0 6.0 7.0 min
Food samples were pretreated as above with or without Fig. 3 AA Sequences of P17/P41B and Nine MRM
2 ppm peanut powder. Chromatograms
Q Selection of MRM Transitions Using Skyline
Ara h1 is known as is known as the sensitizing allergen
in 95 % of peanut allergy. Tryptic digest of protein
extracted from peanuts were analyzed by monitoring
theoretically calculated transitions of peptides based on
amino acid sequences of two clones P17 and P41B of
Ara h1.