Differentiation of fish species based on O-acetylated N-glycan fragments using LC-IM-MS to combat seafood adulteration

• Species-specific O -acetylated sialic acid ( O -Ac-Sias) on N -glycans were identified. • O -Ac-Sia signatures in muscle tissues are fish species authentication biomarkers. • Ion Mobility - Mass Spectrometry delineates multiple isoforms of O -Ac-Sias on N-glycans are present in fish. Food fraud poses a serious safety risk and affects the economy, with seafood being particularly vulnerable due to high species diversity and complex global supply chains. Accurate fish species identification is crucial for sustainable fishery management, food safety and protecting consumers with species-specific fish allergies who are at risk of life-threatening anaphylaxis. While some methods such as DNA-based PCR are well-established, they have limitations for processed foods and are costly, complex and time-consuming. Glycan markers are highly stable and have recently emerged as a tool for food authentication due to their unique species-specific characteristics. This study introduces N -glycan profiling as a novel technique for fish species authentication and addresses the need for reliable methods applicable to processed seafood products. By employing liquid chromatography ion mobility-mass spectrometry analysis, we examined N -glycan profiles of raw and heated fish muscle tissues from three fish species, which represent widely consumed seabass and snapper as well as their potential counterfeit substitute, tilapia from markets and restaurants. N -glycan structures containing different degrees of O -acetylated sialic acids ( O -Ac-Sias) were identified as species-specific markers and clustering based on their percentage abundance enabled species classification. This study provides the foundation for the development of a rapid, species-specific authentication tool, which could be employed throughout the seafood supply chain, from harvest to retail, improving traceability and reducing mislabelling in markets and restaurants.