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A new HPLC-MS Method for the Detection of Lobster and Shrimp Allergens in Food Samples via MRM and MRM³ Robin Korte, Jean-Marc Monneuse, Elodie Gemrot, Isabelle Metton, Hans-Ulrich Humpf, and Jens Brockmeyer J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02620 • Publication Date (Web): 08 Jul 2016 Downloaded from http://pubs.acs.org on July 10, 2016
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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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Journal of Agricultural and Food Chemistry
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A new HPLC-MS Method for the Detection of Lobster and
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Shrimp Allergens in Food Samples via MRM and MRM³
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Robin Korte†, Jean-Marc Monneuse#, Elodie Gemrot#, Isabelle Metton#, Hans-Ulrich Humpf†,
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Jens Brockmeyer*†§
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†
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48149 Münster, Germany
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§
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Germany
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#
Institute of Food Chemistry, Westfälische Wilhelms-Universität Münster, Corrensstraße 45,
Analytical Food Chemistry, University of Stuttgart, Allmandring 5b, 70563 Stuttgart,
Phylogene, 62 Rn 113, 30620 Bernis, France
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*Corresponding author (Tel: +49 251 33392; Fax: +49 251 83 33396; E-mail: jbrockm@uni-
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muenster.de)
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ABSTRACT
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Crustacean shellfish allergy ranks among the most frequent and severe food allergies for
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adults, demanding rugged and sensitive analytical routine methods. The objective of this
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study was therefore to develop a mass spectrometric approach for the detection of
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contamination with shrimp and lobster, two economically important types of crustaceans, in
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complex food matrices. Following a biomarker approach, we identified proteotypic peptides
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and developed a multiple reaction monitoring (MRM) method allowing the identification and
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differentiation of shrimp and lobster in food matrix at concentrations down to 0.1%. To
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further enhance sensitivity we employed the MRM cubed (MRM³) mode which allowed us to
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detect crustaceans down to concentrations of 25 µg/g (crustacean/food, 0.0025%). We hereby
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present the first mass spectrometric method for the detection of shrimp and lobster in food
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matrices.
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Keywords: food allergens, crustacean allergy, mass spectrometry, MRM cubed, marker
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peptides
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INTRODUCTION
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Food allergy has emerged as a global health issue over the past decades and research on its
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prevalence and prevention has increasingly been attracting interest. Recent studies from
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westernized countries indicate growing prevalence rates which have reached 5% in adults and
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8% in young children.1 Allergies against crustacean shellfish are among the most severe food
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allergies with a particularly high prevalence of around 2% for adults, which is significantly
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higher compared to all other seafood allergies2,3, and rates of clinical cross reactivity are
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estimated to be around 75%.4 While tropomyosin was the first protein described as a shellfish
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allergen already in 19815, other allergens have been identified recently, including arginine
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kinase, sarcoplasmic calcium-binding protein and myosin light and heavy chain.6–8
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Due to the high risk they present to allergic consumers crustaceans are, along with milk, egg
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and wheat/gluten, among the most widely regulated allergenic foods worldwide.9 However, a
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recent study on products from the Japanese market showed that 9% of processed seafood
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products contained protein from shrimp or crab above the regulatory baseline of 10 µg/g10,
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most of them without advisory labeling. A recent recommendation by the Voluntary
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Incidental Trace Allergen Labeling (VITAL) expert panel suggests a maximum reference
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dose of 10 mg for total shrimp protein which should not be exceeded by allergic consumers.
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This reference dose might serve as a basis for the definition of future threshold values.11
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Enzyme-linked immunosorbent assays (ELISA) and polymerase chain reaction (PCR) are
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currently the most commonly used techniques for the analysis of food allergens12, although
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both show significant limitations: For ELISA a lack of reproducibility between assays from
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different vendors as well as a considerable effect of thermal processing on sensitivity has been
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described for the analysis of crustacean as well as for milk, egg and peanut allergens.13,14
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Beyond that ELISA assays are only of limited suitability for the differentiated analysis of
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such diverse groups of species as the Crustacea as the detected epitopes are usually unknown 3
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and assays often fail in discriminating between different crustacean subgroups (shrimps,
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lobsters, crabs, krill etc.).15 PCR methods are capable of distinguishing different crustaceans
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by specific DNA markers16 but provide only an indirect allergen detection. This might lead to
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false negative results in cases where processing has a stronger effect on DNA than on
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allergenic proteins17 as was found for lobster after boiling under acidic conditions.18 In this
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context the development of liquid chromatography coupled mass spectrometry (LC-MS)
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methods represents a promising trend in allergen analysis constituting a sequence-specific,
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protein-based and thus more rugged approach. Several LC-MS methods have been published
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for the trace analysis of egg, milk, peanut and tree nut allergens in different food matrices
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over the last few years13,19–24 and the principle suitability of LC-MS for the specific detection
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and differentiation of shrimps and crab has also been demonstrated recently.14,25
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In this study we developed the first LC-MS method for the detection of crustacean allergens
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from shrimp and lobster with applicability demonstrated at trace concentrations in a complex
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food matrix. Using tandem mass spectrometry in MRM mode we provide a method that suits
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the equipment of routine laboratories involved in food analysis. Furthermore, we employed
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the MRM cubed (MRM³) technology which has already been demonstrated to increase
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sensitivity for the targeted analysis of food proteins for the differentiation of mammalian
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meat26,27, to further increase sensitivity and specificity of the method.
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MATERIALS AND METHODS
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Sample material. Raw crustaceans used for method development (lobster (Homarus
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americanus), giant tiger prawn (Penaeus monodon) and whiteleg shrimp (Litopenaeus
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vannamei)) were purchased as whole animals from local grocery stores. For the identification
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of crustacean biomarkers species authenticity was confirmed by sequencing part of 16S
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rDNA. Sequences were compared with GenBank database sequences using BLAST
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algorithm. Synthetic peptides were obtained from JPT Peptides Technologies (Berlin,
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Germany) in SpikeTides Quality (unpurified and not quantitated).
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Identification of crustacean biomarkers
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Sample preparation. After removal of the shell, crustacean samples were ground in a
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Grindomix GM200 (Retsch, Haan, Germany). 12 mL of 2 M urea buffer was added to a 2 g
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test portion of each sample, mixed thoroughly and incubated at 56 °C during 60 min under
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agitation (140 rpm). Afterwards samples were centrifuged (12000 g, 5 min) and supernatants
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were collected.
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200 µL of 1 M dithiothreitol (Roth, Karlsruhe, Germany) were added to 6 mL of each
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supernatant and incubated at 37 °C for 1 h. Then 2 mL of 0.5 M iodoacetamide (Sigma-
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Aldrich, St Louis, MO) were added and incubated at 37 °C during 30 min in the dark. 500 µL
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of 0.5 µg/µL trypsin solution (Sigma-Aldrich) were added and samples incubated overnight at
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37 °C. At the end of incubation samples were centrifuged (15000 g, 10 min), supernatants
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were filtered (0.45 µm) and acidified by adding 2 mL of 2% formic acid. Peptide solutions
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were desalted using Sep-Pak tC18 500 mg devices (Waters, Milford, MA). Sep-Pak syringes
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were rinsed successively with 2 mL of 70% acetonitrile / 2% formic acid and 2 mL of 2%
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formic acid solutions. Samples were loaded onto the solid phase which was rinsed twice with
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2 mL of 2% formic acid solution. Peptides were eluted with 1.2 mL of 70% acetonitrile / 2%
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formic acid and, after drying under a gentle stream of nitrogen gas, solubilized in 150 µL
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aqueous 0.1% formic acid solution.
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Non-targeted LC-HRMS/MS. 0.8 µg of peptides (peptide amount of digests determined
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using Pierce BCA enhanced protocol (Thermo Fisher, Waltham, MA) were injected in
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duplicate for each sample. Chromatography was performed using nanoLC ultra 2D+
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equipment on a 150 mm x 75 µm i.d., 3 µm, Chrom XP C18CL 120 Å Eksigent column
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(Sciex, Framingham, MA). A 5-40% acetonitrile/water gradient was applied at a flow rate of
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300 nL/min and data were acquired on a TripleTof 5600 mass spectrometer (Sciex). MS scan
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was performed with high resolution settings with an accumulation time of 250 ms. MS/MS
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scan was performed with high sensitivity settings on the 25 most intense ions of each cycle
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with an accumulation time of 100 ms. 2036 cycles were performed, thus an average of 11
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cycles per chromatographic peak.
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Data processing. DDA spectra processing and database searching was performed with
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ProteinPilot ver. 4.5 beta (Sciex) using the Paragon algorithm. The search parameters were as
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follows: sample type: identification; cys alkylation: iodoacetamide; digestion: trypsin;
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instrument: TripleTOF 5600; special factors: Urea denaturation. ID focus: biological
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modifications. The database was downloaded from Uniprot (March 2013), filtering for
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Gnathostomata, crustacean and Mollusca (2117981 entries). The resulting .group file was
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loaded into PeakView ver. 1.0 (Sciex) and XICs from DDA runs were extracted with a
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peptide confidence threshold of 99% and a global false discovery rate 3. In case of MRM three corresponding transitions with the same intensity ratio compared to the reference peptide were required **specific for S4-1 only ***specific for S4-2 only
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