New High-Performance Liquid Chromatography Coupled Mass

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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

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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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ACS Paragon Plus Environment


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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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Table of Contents Graphic

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New High-Performance Liquid Chromatography Coupled Mass

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