High-resolution tandem mass spectrometry for multi-allergen analysis of fining-related egg and milk proteins in red wines

Abstract

Trabajo presentado al 7th Worskhop CSIC-CNRS: "Micro- and Nano-Techniques for Analysis and Characterization of Proteins", celebrado en Madrid del 22 al 23 de julio de 2019. ; Casein and ovalbumin are used in the wine clarification process to promote interactions with undesirable compounds, such as polyphenols and tannins. This kind of proteins may trigger allergic reactions in susceptible individuals, therefore their presence in wines could became a human health risk, especially when not reported. For this reason, the European Union established that concentrations higher than 0.25 mg L-1 should be declared in the label. The objective of this work was to develop a tandem mass spectrometry method based on UHPLC coupled to quadrupole-time of flight tandem mass spectrometry (q-TOF-MS/MS) to accurately identify and quantify these allergenic proteins in Chilean wines. We focused our efforts on increasing the number of detectable peptides reported in literature in order to increase accuracy and reliability on the analysis. Triple quadrupole tandem mass spectrometry (QqQMS/SM) was also used to comparative evaluate the performance of q-TOF vs QqQ. Proteins were extracted combining the use of ultrafiltration membranes and precipitated with organic solvents. Then, proteins were digested with trypsin using ultrasounds energy. A face-centered central composite design with two central points was selected to optimize the enzymatic digestion, setting up a digestion time of 3 minutes and an enzyme/protein ratio of 1:10. Peptides separation was carried out on Phenomenex Kinetex XB Core-Shell C18 column (100 mm x 4.6 mm, 2.6 μm), at 35oC, using a mobile phase composed of ultrapure water and acetonitrile, both with 0.1 % (v/v) formic acid. MS analysis was carried out on an Agilent Q-TOF MS 6540 equipped with an orthogonal electrospray ionization (ESI) source (Agilent Jet Stream, AJS) and a Shimadzu (Kyoto, Japan) LC-MS-8030 triple quadrupole (TQ) mass spectrometer. Marker peptides for quantification were selected among the most abundant and stable. Quantification was performed in MRM mode, using an isotopically labeled peptide as internal standard. Q-TOF analysis based on full scan data and on product ion scan mode allowed the identification of a larger number of marker peptides than those reported for QQQ analysis. Thus, several precursor ion peptides for α-, β-casein and ovalbumin were identified, as well as characteristic peptides of k-casein that had not been previously reported in literature. LODs ranged from 4.7 to 8.5 μg L-1 working in MRM mode with QqQ; while slightly higher LODs (10-60 μg L-1) were obtained operating with q-TOF. The results show the advantage of using a q-TOF mass analyzer to detect and identify new marker peptides, while using QqQ for its exact quantification. Using the proposed methodology, some of the target peptides exhibited lower LODs than those reported in literature. Finally, sixty samples of Chilean wines were analyzed, finding 14 samples with higher levels of casein and ovalbumin than recommended by the International Organization of Vine and Wine. ; This study was funded by the National Fund for Scientific and Technological Development (FONDECYT) project No1171857 and by the Fund for Scientific and Technological Equipment (FONDEQUIP) project No 130209. G.A.R. would like to acknowledge MINECO for the "Juan de La Cierva-Formación" postdoctoral grant FJCI-2016-30902. ; Peer reviewed