Towards Absolute Quantification of Allergenic Proteins in Food—Lysozyme in Wine as a Model System for Metrologically Traceable Mass Spectrometric Methods and Certified Reference Materials

Abstract

Current routine food allergen quantification methods, which are based on immunochemistry, offer high sensitivity but can suffer from issues of specificity and significant variability of results. MS approaches have been developed, but currently lack metrological traceability. A feasibility study on the application of metrologically traceable MS-based reference procedures was undertaken. A proof of concept involving proteolytic digestion and isotope dilution MS for quantification of protein allergens in a food matrix was undertaken using lysozyme in wine as a model system. A concentration of lysozyme in wine of 0.95 +/- 0.03 microg/g was calculated based on the concentrations of two peptides, confirming that this type of analysis is viable at allergenically meaningful concentrations. The challenges associated with this promising method were explored; these included peptide stability, chemical modification, enzymatic digestion, and sample cleanup. The method is suitable for the production of allergen in food certified reference materials, which together with the achieved understanding of the effects of sample preparation and of the matrix on the final results, will assist in addressing the bias of the techniques routinely used and improve measurement confidence. Confirmation of the feasibility of MS methods for absolute quantification of an allergenic protein in a food matrix with results traceable to the International System of Units is a step towards meaningful comparison of results for allergen proteins among laboratories. This approach will also underpin risk assessment and risk management of allergens in the food industry, and regulatory compliance of the use of thresholds or action levels when adopted.

Development of certified matrix-based reference material of genetically modified rice event TT51-1 for real-time PCR quantification

“…Although there has been rapid development of new detection techniques for specific proteins, nucleic acids, and metabolites in biological research, the development of new CRMs is lagging behind [2]. For instance, there have been shortages of protein CRMs for allergen detection, RNA/DNA marker CRMs for disease diagnosis, and protein/DNA CRMs for analysis of genetically modified (GM) contents [3][4][5].…”

A candidate liquid chromatography mass spectrometry reference method for the quantification of the cardiac marker 1-32 B-type natriuretic peptide

“…Each hydrolysis included a reagent blank (solvents only), natural amino acids control, labeled amino acids control, a recovery vial, two calibration blends and two sample blends. The hydrolyzed samples were lyophilized, reconstituted in 80 μL MSTFA and analyzed by gas chromatography-mass spectrometry (GC-MS) [18]. Three hydrolyses containing two sample blends, and two calibration blends were performed.…”

“…Quantification of peptides: The method developed for the SI-traceable quantification of BNP and BNP tryptic digest peptide standards by amino acid analysis was a modification of a method previously described [17,18]. The samples were hydrolyzed at 190 °C for 30 min in an ETHOZ EZ Microwave Digestion System (Milestone, Sorisole, Italy) equipped with a protein hydrolysis kit containing 30 mL of 6 M hydrochloric acid (HCl).…”

“…Tryptic digestion was optimized as described by Cryar et al [18]. Ultimately 250 μL of sample blends and calibration blends in 500 mM TRIS buffer and 10 mM Ca 2+ (pH 8.10) were reduced and alkylated with 200 nmol TCEP (37 °C, 60 min) and 400 nmol: S-methyl methanethiosulfonate (MMTS) (in the dark, 37 °C, 30 min).…”

“…Quantification of BNP using tryptic digestion was performed by isotope dilution mass spectrometry (IDMS) using isotopically labeled peptides as internal standards [17][18][19]. Tryptic digestion was optimized as described by Cryar et al [18].…”