Application of Proteomics in Food Authentication
Proteomics study is used for species authentication, breed identification, geographic origin, undeclared addition of plant/animal protein material, and type of material added (e.g., milk/milk proteins, blood/its constituents), production method (wild/farmed), technological processing, and proportion of ingredients used.
Milk and Dairy Products
Milk and milk products are expensive foodstuffs and their consumption is spread among the population because of their high nutritional value; for this reason, some dishonest producers who are tempted by the possibility to enlarge their profits, commit different frauds (Cozzolino et al., 2008). According to the legislation that regulates the production and marketing of milk and dairy products in different countries, the most common illegal practices are the selling of skimmed milk and semi-skimmed milk instead of whole milk, milk dilution by water, the addition of milk anhydrous products to liquid milk, and the mixing of high-quality milk, such as buffalo's, sheep's and goat's with cow's milk (De La Fuente and Juarez, 2005). Among the common illegal practices, the addition of powdered derivatives seems very difficult to detect because the adulterant materials have almost the same chemical composition like that of liquid milk. However, the high temperatures (180-200°C) used for milk powder production could imply the occurrence of some protein modifications (e.g., glycation, lactosylation, oxidation, deamidation, dehydration). The modified proteins or peptides could then be used as markers for the presence of powdered milk.
PAGE has been employed to analyze the individual protein group (Strange et al., 1992). Addition of bovine milk in ewe yoghurt (Kaminarides and Koukiassa, 2002) and goat milk (Tamime et al., 1999) have been quantified with PAGE. Cow milk adulteration in caprine milk has been quantified by HPLC/ESI-MS (Chen et al., 2004). This method identifies molecular masses to differentiate between proteins in the milk of cow and goat. MALDI-TOF protein profiling was used for the investigation of the addition of bovine milk to ewe and buffalo milk (Cozzolino et al., 2001) and in the adulteration of mozzarella cheese (Cozzolino et al., 2002), using whey proteins as biomarkers. The same technique has been used to reveal adulteration of donkey and goat milk with cow, ewe, and buffalo milk at levels down to 0.5% (Di Girolamo et al., 2014). Cozzolino et al. 2001 targeted whey proteins, a-lactalbumin and (3-lactoglobulin using MALDI-TOF-MS to determine the adulteration in raw ewe and water buffalo milk. Chen et al., 2004 analyzed adulteration in goat milk by HPLC/ESI-MS using /Mactoglobulin whey protein as biomarkers.
Chen et al., 2004 and Muller et al., 2008 used the protein p-lactoglobulin as a marker to detect and quantify bovine milk in goat, caprine or ovine milk by means of HPLC-ESI-MS or capillary electrophoresis-MS. The presence of cow milk was detected at levels not lower than 5 per cent. LC-MS/MS has been used frequently to detect melamine in milk and variety of infant formulas (Guelph, 2008; Sherri et al., 2008). Different types of mass spectroscopy have been employed to detect melamine in milk products, including LC-MS/MS, APCI-MS (Atmospheric Pressure Chemical Ionization-Mass Spectroscopy) and EESI-MS (Extractive Electrospray Ionization Mass Spectrometry) (Yang et al., 2009; Zhu et al., 2009).
Calvano et al. (2013) employed MALDI-TOF MS to analyze tryptic digests relevant to samples of raw liquid, commercial liquid and powdered cow's milk. Samples were subjected to two-dimensional gel electrophoresis; differences among liquid and powder milk were detected at this stage and eventually confirmed by MALDI analysis of the in gel-digested proteins. Sassi et al. (2015) used MALDI-TOF-MS to study adulteration in ovine, caprine, and buffalo milk by targeting casein, lactalbumin, and proteoso peptones; Lu et al. (2017) employed UPLC-quadrupole time-of-flight-mass spectrometry to detect soybean and pea proteins in raw milk; and Yang et al. (2019) used nano-HPLC-MS/MS combined with principal component analysis to detect the plant proteins (soy protein, pea protein, hydrolyzed wheat protein, hydrolyzed rice protein) in adulterated milk.
Adulteration of meat products not only misleads consumers but also has ethical and health implications. Consumers have the right to choose the correct meat species on the basis of religious or quality concerns. Ponce- Alquicira and Taylor (2000) used ESI-MS/MS on intact myoglobin extracted from beef and pork, and commercial proteins from horse and sheep to differentiate sheep and beef from each other and from horse and pork. However, the instrument resolution was not enough to differentiate horse and pork. This issue is likely to be overcome with current array of high- resolution mass spectrometers available.
Sentandreu et al. (2010) used a robust and simple method to study adulteration in meat. The method comprised of the extraction of myofibrillar proteins, enrichment of target proteins using OFFGEL isoelectric focusing, in-solution trypsin digestion of myosin light chain 3, and analysis of the generated peptides by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). Montowska and Pospiec (2012; 2013) found inter-species differences in 2-DE protein patterns between cattle, pig, chicken, turkey, duck, and goose, in both raw meat and processed products. Some of the proteins were stable during meat aging and resistant to thermal processing, and some of them could even be identified in highly processed products, such as fermented sausages, and were therefore proposed as suitable markers. However, a validation study focusing on amino acid sequence information from these stable proteins would be necessary in order to establish a high-throughput targeted MS/MS-based method, such as SRM, for the differentiation of the species. This requirement is strengthened by the fact that for duck and goose there is still little protein sequence information in current databases.
Von Bargen et al. (2013) developed an SRM method for the detection of horse and pork in beef. After identification of the biomarker peptides by a shotgun MS/MS-based approach, peptides specific to horse and pig were included in an SRM assay capable of detecting as low as 0.55 per cent horse or pork contamination in a beef matrix, or 0.13 per cent pork contamination in beef when an MS3 method was used. Montowska et al. (2015) differentiated meat species by liquid extraction surface analysis mass spectrometry using markers derived from myofibrillar, sarcoplasmic, and milk proteins.
Orduna et al. (2017) examined meat adulteration using a well-defined proteogenomic annotation, carefully selecting surrogate tryptic peptides, and high-resolution mass spectrometry. Selected mammalian meat samples were homogenized and the proteins extracted and digested with trypsin followed by chromatographic separation. Accurate mass was obtained by full-scan high-resolution mass spectrometry involving DIA scan that allowed the detection and identification of very specific tryptic peptides for targeted proteins from horse, beef, lamb, and chicken.
Pan et al. (2018) used Parallel Reaction Monitoring (PRM) mass spectrometry approach for detection of trace pork in meat mixtures (chicken, sheep, and beef). Specific peptides were identified and screened by a shotgun proteomic approach based on tryptic digests of certain protein. Five surrogate peptides from myosin were screened and then used for pork detection by PRM of Orbitrap MS (Fig. 2).
Figure 2. Tlie workflow of pork authentication in meat mixtures (adapted from Pan et al., 2018).