Session: Session 5

Characterization of peptides via Capillary Electrophoresis coupled with Ion Mobility Mass Spectrometry

Wallonia Region’s Peptiboost project focuses on the valorization of protein-rich byproducts from the agri-food industry as a source of bioactive peptides for food and feed. The characterization of the generated peptides after enzymatic digestion in native conditions without non-compliant additives in regard of food safety, or from fermentation requires novel analytical approaches. Capillary electrophoresis (CE) coupled with ion mobility spectrometry (IMS) and mass spectrometry (MS) was investigated as an alternative technique to liquid chromatography (LC)-MS for the comprehensive characterization of peptides in terms of peptides sequences and higher order structures.

The higher order structures of peptides in solution under physiologically relevant conditions determine their biological activities. In contrast to LC-MS, CE achieves separation of ionizable compounds in denaturing or non-denaturing and physiologically relevant conditions based on their average charge in solution (depending on the pH of the background electrolytes) and their hydrodynamic radius (that is shape dependent). The separation of highly hydrophobic and hydrophilic compounds is achievable by CE while providing insights into the structural characteristics of analytes in solution.

Ion mobility coupled to mass spectrometry (IM-MS) detection operates in the gas phase, providing additional dimensions of separation to the upstream separation methods, including LC or CE. Ion mobility provide structural parameters in the form of the Collision Cross Section (CCS) and mass spectrometry (MS and MSMS) provides m/z and stoichiometry, sequence of peptides, resilience to the fragmentation (breakdown curves) and the resilience of the structure in the gas phase under soft collision activation (collision induced unfolding).

CE was hyphenated in-line with ion mobility mass spectrometer using sheath liquid or sheathless interfaces. Additionally, orthogonality of CE-IM-MS was evaluated under different pH conditions.