# MALDI-TOF Mass Spectrometry Applications for Food Fraud Detection
## 2 MALDI Applications
### Fish and Meat  
Fish represents and is perceived by consumers as a healthy and nutritious food resource. However, a common problem in the fish processing industry is adding or replacing cheap fish instead of expensive ones, as an accidental event due to the lack of experience or as a deliberate fraud. An adequate level of protection is then required to ensure seafood quality and safety, making the development of rapid, effective, accurate, and reliable analytical methods the key to effectively supervising the market. This need has prompted the issue of directives and regulations for quality control and encouraged the development of innovative MALDI analytical methodologies, almost always based on proteomic techniques. Highly specific mass spectrometric profiles from 25 different fish species were obtained using a new MALDI method [83] meant for the fast assessment of authenticity and fraudulent substitutions. Specific protein signals at \(m/z\) values around 11 kDa were selected as markers to discriminate the various species, while a structural characterization permitted to identify some major fish allergens arising from parvalbumin. In a pilot study [84], three freshwater fish species were discriminated based on the cluster analyses of the overall profiles generated by the MALDI analyses of muscle and liver tissues, after a simple single-step extraction procedure. Each tissue provided species-specific profiles, even if muscle is more suitable for routine controls since fillets are more commonly commercialized. The authors pointed out that the clear discrimination of the species was possible even though the settings and statistical algorithms used were originally designed for the analysis and identification of bacteria and fungi. Then, they suggested that performing an optimization of mass spectral analysis and data processingtargeted on fish species could lead to exhaustive evaluation of the potential of MALDI analysis for studies on freshwater fishes. The analysis of muscles taken from two closely related fish species was undertaken by two-dimensional gel electrophoresis (2-DE) [85] and the interspecies differences between the protein spots, which could be useful for their differentiation, were visually identified. MALDI-TOF MS and/or LC-MS were then necessary to identify 19 proteins, some of which resulted to be specific for each species. Another MALDI method [86] for the profiling of protein extracted from fish muscle was reported in a book chapter by [PERSON] at al. for fish authentication purposes and to detect fraudulent substitutions. Since universal sample preparation protocols prior to MALDI analysis were available to other species but not to fish, [PERSON] at al. [87] tested the performances of five preparation protocols to verify the capability to produce reproducible and high-quality spectra dependent on storage conditions and food processing, and eventually differentiate between different fish species. After the optimization of the best protocol, it was concluded that MALDI could be used for the purpose as soon as a valid species database is available. This need was evidently shared by [PERSON] and co-workers, who almost simultaneously used MALDI to establish a database [88] of protein patterns from 54 fish species susceptible to fraud, in order to detect and prevent substitution, characterized by low intraspecies but high interspecies variability. MALDI-TOF combined with multivariate analysis was exploited [89] for the development of a method to directly analyze fish skin or muscle tissues surface, for authentication purposes. Samples were quickly discriminated, hinting at potential future developments for the authentication of seafood in general and/or other protein food products. Another study centered on the comparison of several one-step sample pre-treatment protocols of fish muscle tissues, prior to MALDI determination of the relevant extracts, was recently carried out [90] by [PERSON] and colleagues.