# MALDI-TOF Mass Spectrometry Applications for Food Fraud Detection
## 2 MALDI Applications
### Milk and Dairy Products
However, this approach can produce false positive results due to a water buffalo \(\beta\)-casein peptide, which is also formed after plasminolysis of water buffalo milk and comigrates in isoelectric focusing with bovine \(\gamma_{2}\)-casein. Thus, [PERSON] at al. have developed a proteomic approach [33] to obtain the unambiguous detection of bovine milk in water buffalo milk and derived products (LOD of 0.8% \(\gamma\)/\(\eta\)) based on the MALDI determination of specific bovine and water buffalo \(\beta\)-casein phosphopeptide markers. The procedure was proposed as an integrative/alternative to the reference method. Other studies were focused on the development of MALDI methods for the detection of the addition of sheep and goat milk with the less expensive bovine milk. Contrary to some of the above-mentioned works, which were focused on the identification of specific signals that can work as adulteration markers, one study, divided into two parts [34; 35], proposed a different approach. The authors suggested that, in the analysis of a complex matrix such as milk, the simultaneous use of many variables reveals more information compared to the use of single or few variables and proposed a method for quantitative and multivariate use of the whole MALDI mass spectra of milk samples to determine adulteration. The first part of the work [34] was focused on the optimization of sample preparation and instrument setup, the second on the quantitative determination of cow, goat, and sheep milk in mixed milk samples [35]. The results obtained seemed to confirm the original hypothesis since, with optimal parameter settings, multivariate regression on whole spectra allowed to determine the concentrations of milk in mixtures with good accuracy. A further study to detect the presence of cow milk in goat and sheep milk using whole MALDI mass spectra in combination with multivariate techniques was reported by [PERSON] at al. [36]. Binary and tertiary mixtures of cow, sheep, and goat milk were easily and rapidly analyzed and accurate information on the amount and type of adulteration were obtained, with typical errors in the range 2-10% for cow milk. The potential of proteomic approaches to fight illegal manipulations of milk was again demonstrated [37] by the detection of cow milk-specific peptide markers in sheep and goat milk-specific peptide markers in sheep milk, by analyzing whole milk samples mixtures subjected to in-solution tryptic digestion. Seven peptide markers of cow milk and two peptide markers of goat milk were identified by MALDI-TOF analysis and the approach was able to detect adulteration up to a 5% level. Moreover, the same markers were found in cheese samples, demonstrating the applicability of the procedure even to milk-derived products. The authors stated that the use of the \(\alpha\)-cyano-4-chlorocinamic acid matrix was essential to reach the high sensitivity observed for the marker peptides. Moreover, the results were found in good agreement with those obtained with a traditional approach based on SDS-PAGE/in-gel digestion. The same research group reported another application [38] for the extraction and MALDI determination of phospholipids in milk, using the same matrix. The method was able to provide peculiar milk phospholipid profiles that were used for the detection of cow milk in sheep and goat milk. The abundance ratio of specific ions (\(m/z\) 703 and 706) was found to be species-specific and was used for the identification of the adulteration. Very recently, liquid atmospheric pressure--MALDI mass spectrometry--was used [39] to optimize a very accurate approach to classify goat and sheep milk and sheep milk containing 10% of goat milk, to evaluate colostrum quality and postnatal stages, through the recording of the milk lipid/protein profiles or the detected orthologs of single proteins. Donkey milk is a safe alternative for individuals which are allergic to cow milk and is often the object of adulteration, the detection of which is of critical importance for the safety of the consumers.