# MALDI-TOF Mass Spectrometry Applications for Food Fraud Detection
## 2 MALDI Applications
### Fish and Meat
The best results in terms of repeatability and spectral resolution were obtained by boiling samples in 0.1 M trifluoracetic acid (TFA) for 5 min, permitting to discriminate between different fish samples, when combined with similarity coefficient-based analysis for their mass spectra. MALDI also turned out to be a good system for assessing fish freshness. For instance, the effect of storage time on fish muscle proteins was investigated using 2-DE coupled to MALDI [91], permitting the identification of three altered proteins. More recently [92], the same approach was involved in the analysis of fish samples, both fresh and stored in a refrigerator for different days, looking for protein markers for its freshness. It was found that the comparison of three specific proteins, l-lactate dehydrogenase, adenylate kinase isoenzyme 1, and myosin heavy chain, permitted to trace the freshness of the products. In a different study, the vitreous fluid of the eyes was taken from fishes subjected to different days of post-mortem storage, which was immediately analyzed by MALDI-TOF to evaluate fish spoilage [93]. Software spectra processing allowed the identification of four \(m/z\) ions able to differentiate between the tested days of storage, although a limited applicability was shown within the end of the tested period. A further study [94] of a comparison of different protein extraction protocols from seafood (marine mussel) prior to 2-DE was conducted by [PERSON] and co-workers. Then, MALDI-TOF/TOF analysis of the gels was performed and proteins with several functions, such as energy metabolism, cell signaling and regulation, and stress response, were identified. The method was a precious tool to investigate the protein expression in the species under investigation and potentially in other affiliated species. Several analytical methods are used in the meat industry for detecting contamination, adulteration, and authenticity, to safeguard safety, and to reassure consumers, who are increasingly aware and attentive to the quality of the food they consume. Two MALDI methods to determine the origin of raw and processed meat (pork, beef, horse, veal, and chicken) and of gelatin (pork or beef), respectively, were developed by [PERSON] and colleagues [95]. In the first method, intact proteins were determined in linear mode prior to cluster analysis, allowing the separation of meat into distinct mass spectra clusters depend ing on the origin. In the second method, gelatin was digested with trypsin and analyzed in reflectron mode. The relevant spectra showed specific profiles that permitted to distinguish pork from bovine gelatin (1% of gelatin in spiked candies and 20% of pork gelatin in beef gelatin). Although less sensitive compared to other approaches, these methods are fast and easy to perform and can be conveniently used for fast screening controls. An approach to reveal the differences in protein expression between young and old buffalo meat looking for biological markers of tenderness was performed by 2-DE coupled to MALDI-TOF/TOF [96]. Structural proteins with expression levels associated with meat tenderness were successfully identified through digestion and MS/MS analysis of selected gel spots. Moreover, the study demonstrated that through the ageing process, it was possible to reduce the variation in tenderness between young and old buffalo meat that, consequently, requires different processing strategies for their effective utilization. The 2-DE-MALDI combination was also exploited [97] to compare meat quality traits and to identify different protein expression between low and high pH pig muscles. Fourteen proteins that differed in spot density between the two groups were identified and nine of them involved in meat quality attributes significantly increased in high-pH muscles. The whole results obtained in the work provided useful information to understand the molecular mechanism at the base of meat quality.