Development and Interlaboratory Evaluation of an LC–MS/MS Method for the Quantification of Lysozyme in Wine Across Independent Instrument Platforms

Abstract

Background Various processing aids and fining agents are used in winemaking to help improve sensory characteristics. Some of these materials may contain or be derived from allergenic foods, such as eggs. In order to ensure food safety and that products meet regulatory compliance, it is essential to have robust and effective analytical methods to verify the removal of allergenic proteins following their use. Current methods include immunoassays (ELISA) and mass spectrometry methods, which can target either whole foods or individual proteins, and provide either quantitative data or qualitative confirmation of proteins. Mass spectrometry methods offer the potential to test for multiple proteins within a single assay to improve cost and efficiency, whereas ELISA methods typically analyze for a single protein per assay. Objectiv This study focuses on the development of a LC-MS/MS quantitative method for lysozyme in white wine and compares performance across two laboratories utilizing two different instrument platforms. Methods Lysozyme target peptides were selected by conducting bottom-up discovery proteomics. Candidate targets were evaluated using parallel reaction monitoring (PRM) or selected reaction monitoring (SRM) LC-MS/MS, depending on the instrument in each laboratory. Quantification of lysozyme was conducted using internal, stable-isotope-labeled synthetic peptide standards. Results Three of eight candidate target peptides showed performance suitable for the final quantitative method. White wine spiked with 0.1 and 0.5 ppm lysozyme demonstrated quantitative recovery of 70–120%. While the PRM method delivered better repeatability, the SRM method gave higher quantitative recovery values. Conclusion A targeted LC-MS/MS method for quantification of lysozyme in white wine has been developed and deployed on two different MS instrument platforms in two laboratories. Highlights Both SRM and PRM targeted LC-MS/MS methodologies can be used for quantification of lysozyme in white wine. This study is among the first to evaluate an MS method for food allergen quantification in multiple laboratories.

Proteomics and glycoproteomics of beer and wine

“…Animal‐ and plant‐based fining agents are commonly used for high‐quality wine, leading to potential risks for consumers who are allergic to proteins from those sources [74]. Although ELISA is the current standard method used for monitoring fining agent residues in wine, LC‐MS/MS offers several advantages including the ability to measure many proteins in a single assay, robustness to the folding status of the protein and even higher sensitivity [75–77]. Methods developed for monitoring residual fining proteins tend to use targeted analyses with parallel reaction monitoring (PRM) or selected reaction monitoring [75, 76, 78], and quantification using stable isotope dilution analysis has also been reported [79].…”

“…or selected reaction monitoring [75,76,78], and quantification using stable isotope dilution analysis has also been reported [79]. The protease digestion step is a bottleneck in standard proteomic workflows, normally taking several hours.…”

“…Although ELISA is the current standard method used for monitoring fining agent residues in wine, LC‐MS/MS offers several advantages including the ability to measure many proteins in a single assay, robustness to the folding status of the protein and even higher sensitivity [75–77]. Methods developed for monitoring residual fining proteins tend to use targeted analyses with parallel reaction monitoring (PRM) or selected reaction monitoring [75, 76, 78], and quantification using stable isotope dilution analysis has also been reported [79]. The protease digestion step is a bottleneck in standard proteomic workflows, normally taking several hours.…”

“…Although ELISA is the current standard method used for monitoring fining agent residues in wine, LC-MS/MS offers several advantages including the ability to measure many proteins in a single assay, robustness to the folding status of the protein and even higher sensitivity [75][76][77]. Methods developed for monitoring residual fining proteins tend to use targeted analyses with parallel reaction monitoring (PRM)…”