FOURIER TRANSFORM ION CYCLOTRON RESONANCE MASS SPECTROMETRY (FT-ICR-MS) FOR HIGH-RESOLUTION ANALYSIS OF FENNEL PROTEINS

In recent years, proteomic analyses have played an important role in analytical and food chemistry; in particular mass spectrometry (MS) based methods have been suggested as confirmatory tools for an accurate protein identification in the field of food quality and safety. Emphasis is placed on food processing, in the determination of possible contaminants like bacteria and fungi, and in allergen detection [1,2]. Among the different proteomic strategies, bottom-up analysis remains the workhorse for protein characterization; nevertheless, it results in a greatly increased complexity of the generated peptide mixture, requiring highly sensitive and efficient methods which can lead to correct identifications. A prominent technology for high throughput proteomic analysis is Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry, providing the highest resolving power and mass measurement accuracy. Moreover, the large dynamic range and unmatched sensitivity of FTICR-MS currently provides the highest quality data for protein identification [3,4]. In this study, a rapid and sensitive bottom-up method by FT-ICR is described for the identification of fennel proteins, without prior fractionation. The peptide-level method was previously validated on tryptic digests from ubiquitin standard protein. Ultra-high-resolution mass profiles were acquired using a 12 Tesla FT-ICR mass spectrometer in positive mode via electrospray ionization (ESI). A mass resolving power of around 250,000 was achieved for all spectra while collecting 50 scans per sample with a 4M transient. The method benefits from high resolution which allows to detect proteins in a mass range up to m/z 8000 in a few seconds. The most intense precursor ions were selected for collision-induced fragmentation. The acquired MS and MS/MS datasets were used in the database searching for protein identification. Few microliters of fennel extracts were analyzed after enzymatic digestion with trypsin. Although fennel (Foeniculum vulgare Mill.) has attracted attention as a medicinal plant with an enormous amount of health benefits [5], it is recently recognized as an allergenic source, especially in the Mediterranean area. Therefore, this work represents the starting point for allergen characterization in fennel samples, allowing the upgrade of the pattern of allergenic molecules in food products. References [1] Chen, C.-H. W.: Review of a current role of mass spectrometry for proteome research. Anal. Chim. Acta 2008, 624, 16–36. [2] Piras, C., Roncada, P., Rodrigues, P.M., Bonizzi, L., Soggiu, A: Proteomics in food: Quality, safety, microbes, and allergens. Proteomics 2016, 16, 799-815. [3] Marshall, A.G., Hendrickson, C.L., Jackson, G.S.: Fourier transform ion cyclotron resonance mass spectrometry: a primer. Mass Spectrom. Rev. 1998, 17, 1–35. [4] Page, J.S, Masselon, C.D, Smith, R.D: FTICR mass spectrometry for qualitative and quantitative bioanalyses. Curr. Opin. Biotechnol. 2004, 15, 3–11. [5] Badgujar S.B.; Patel, V.V.; Bandivdekar, A.H. Foeniculum vulgare Mill.: A review of its botany, phytochemistry, pharmacology, contemporary application, and toxicology. BioMed Res. Int. 2014, 2014, 842674.