Shotgun Aanalysis of fennel protein extracts using Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

A rapid shotgun method using Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR-MS) was developed for the characterization of fennel proteins. Following enzymatic digestion with trypsin, small volumes of the extract were analyzed via direct infusion in positive ion mode. For the molecular identification, a dedicated bioinformatic strategy was implemented, starting from the publicly available NCBI protein database. From the 231 entries originally listed under the Foeniculum vulgare organism, only the unique, non-redundant protein sequences were retained, eliminating partial or duplicate entries that appear in the database under different identifiers. This refinement produced a curated dataset of 92 specific fennel proteins. In consideration of possible allergenic cross-reactivity—especially relevant to individuals with spice-mugwort-allergy syndrome—this protein list was expanded by adding 10 known allergenic proteins from botanically related species such as celery, parsley, carrot, birch, and mugwort. Thus, a final database of 102 protein sequences was constructed. All proteins were then in-silico digested to simulate tryptic peptide fragmentation, producing a theoretical list of m/z values. These predicted values were compared to the experimental mass spectra acquired from FT-ICR-MS using a custom-developed MATLAB algorithm, designed to match signals with high accuracy. Finally, database searching in Peptide Mass Fingerprint mode was performed by using the matched m/z values as input data. A total of 70 proteins were successfully identified in the fennel extract, including 61 specific to fennel and 9 allergenic proteins from the additional reference sources. This method proves to be both time-efficient and robust for complex plant protein analysis.