Mass spectrometry is a key analytical tool used to address a wide range of questions in food and nutrition research. One area where the technique is particularly useful is in the structural characterisation of proteins. This article explores the application of mass spectrometry to three areas related to food safety; the detection of microorganisms, the determination of modifications induced by food processing, and the identification of protein components responsible for food allergy and intolerance.

Proteins and peptides are major constituents of food and play a decisive role in determining its nutritional and functional properties. Mass spectrometry (MS) has gained increasing popularity as part of the toolkit of techniques developed to investigate proteins at the proteome-wide scale. The application of mass spectrometry to protein analysis has been revolutionised in recent years by the development of soft ionisation techniques such as electrospray ionisation (ESI-MS) and matrix assisted laser desorption/ionisation (MALDI-TOF-MS), two techniques which have complementary advantages for protein characterisation [Table 1]. The mass spectrometer can also be used to generate de novo amino acid sequence information from tandem mass spectra (MS/MS).

Microbial contamination of food

Traditional means of controlling microbial spoilage and safety hazards in foods include freezing, blanching, sterilisation, curing and use of preservatives. However, the developing consumer trend for ‘naturalness’, as indicated by the strong growth in sales of organic and chilled food products, has resulted in a move towards milder food preservation techniques. This raises new challenges for the food industry.

Bacterial profiling through MALDI-TOF-MS and ESI-MS/MS fingerprinting of bacterial proteins has been developed.These techniques allow different species and, in some cases strains, to be identified. These profiling methods are ideal for fast and sensitive detection of pathogens or spoilage microorganisms that affect food quality and safety during processing and storage. A more accurate description of contaminating microorganisms has been achieved by integrating proteomics with methodologies that are able to provide either structural or quantitative identification of specific metabolites produced by the various spoilage microorganisms. Defining the mode of action of foodborne bacteria and the mechanisms that confer ‘stress resistance’ should enable a more rational design of food preservation techniques. In addition, this information can also be used to identify areas of the food chain that are the most vulnerable to microbial contamination. It can be foreseen that these methods will eventually be integrated into design sensitive sensors on a microchip surface in order to enable automated detection.

The risks associated with food contamination are not limited to the presence and virulence of a microorganism. Protein toxins can be released into food, which are able to survive for long periods of time even after the source of contamination has been removed. This is the case for many of the microbes which cause foodborne diseases, including

In a study funded by the Italian Ministry of Agriculture [2], a procedure combining a proteomic approach with immunochemical, chromatographic, electrophoretic and MS techniques was developed. The procedure monitored production and levels of Enterotoxin A (SEA) and B (SEB) of

Listeria monocytogenes, Staphylococcus aureus, Escherichia coli, Clostridium botulinum A and Salmonella species. These pathogenic bacteria excrete a variety of virulence factors into the extracellular medium and at the cell surface. This mode of action plays an essential role in the colonisation of the host cells, and thus reflects the degree of bacterial pathogenicity. These toxins, being heat-stable and resistant to proteases, can be a danger for the consumer. S. aureus and Shiga-like toxins produced by E. coli O157:H7 in typical Italian cheeses. By producing cheese samples using milk contaminated with bacteria, it was possible to monitor 10-100 ppb contamination levels, and analysis of randomly collected market samples allowed toxin contamination in the two cheese types to be excluded.

Modifications induced by food processing

Heat treatment of milk can lead to the denaturation of whey proteins. The complex series of covalent reactions that take place (known as Maillard reactions) produce a decrease in nutritional quality and the formation of possible toxic compounds. A well-controlled Maillard reaction can, however, also be induced to achieve specific benefits, such as aroma generation in bread and baked products, or to improve the physico-chemical properties of whey proteins. The novel compounds formed have been proposed as useful markers to demonstrate either uncorrected heat treatment or the presence of heated milk/milk powders added to fresh milk. Some markers of heated milk have been identified, including lysinoalanine (LAL), formed by the reaction of abundant lysine residues and dehydroalanine (produced by heat-induced dephosphorylation of the phosphoserine residues of casein). The main difficulty lies in detecting trace amounts of LAL in the presence of other dominating free amino acids. An analytical method involving detection by LC/ESI-MS without any sample pre-fractionation step has been developed recently, making LAL a useful marker for the detection of heat treated products in a variety of foodstuffs, ranging from milk for drinking to milk powders and dairy products.

It is important to be able to detect molecules whose production in foods might represent a serious health concern. In several heated foods, high levels of acrylamide (AA) have been found as the product of the Maillard reaction between amino acids (mainly Asn, but also Gln and Met) and reducing sugars (d-fructose, d-galactose, lactose, glucose) as a consequence of cooking and frying processes. Acrylamide is classified by the International Agency for research on Cancer (IARC) as a "probable human carcinogen" and has been detected in carbohydrate-rich fried or baked food samples since 2002. Methods for AA analysis in foods have been recently reviewed, in which the use of mass spectrometry plays an irreplaceable role [3]. GC-MS and LC-MS-based procedures have been developed for the ppb-level quantification of AA as brominated derivatives in foods [Figure 1]. Applications for these methods include the analysis of raw and treated materials, for instance hazelnuts or almonds (where roasting is usually carried out either to generate the typical flavours or to allow for storing and further transformation processes). The techniques have also very recently been used to optimise the effect of growth conditions on the level of free amino acids in wheat grain. This has a consequence on the final AA levels during flour processing for the preparation of baked goods.

Food allergy and intolerance

Food allergy is an increasingly important issue for the food industry. Mass spectrometry-driven identification of either genes for allergic diseases or allergenic proteins is currently underway. The systematic analysis of cereal and legume species, such as wheat, rice, pea, soy and peanut, using high resolution separation techniques (in combination with MS) is leading to the detection and identification of several previously uncharacterised allergenic proteins in seed samples. This demonstrates the potential of proteomic approaches for analysing food samples for the occurrence of allergens. In these studies, MS-based methods have been developed to provide good detection limits (1ppm) for almonds, pecan nuts, hazelnuts and walnut antigens in food ingredients such as soy, milk, chocolate and breakfast cereals.

Allergy to cow’s milk is one of the more prevailing food allergies in infants, and in several cases, is due to intolerance of the main milk proteins - casein and beta-lactoglobulin. Despite the enzymatic hydrolysis of major milk proteins being carried out in order to increase food tolerance, residual antigens in hydrolysed milk formulae have been reported. Research in Italy has lead to a high-tolerability of dairy products via limited proteolysis of milk proteins. This intervention removes protein sequences involved in an allergic reaction.

Gliadins are considered the main factor triggering Coeliac Disease (CD), a common enteropathy in genetically susceptible individuals induced by ingestion of wheat gliadin and related prolamins from oat (avenin), rye (secalin) and barley (hordein). The structural basis for gliadin toxicity in CD is not completely clear and the molecular basis of the toxicity is also poorly understood, due to the structural complexity of gliadins. The high percentage of proline residues makes gliadin resistant to gastric, pancreatic and intestinal digestive proteases. Long gliadin fragments can reach high concentration levels in the gut epithelium as a result. For this reason, during endoluminal digestion, a family of Pro- and Gln-rich polypeptides are released that are responsible for the inappropriate T-cell-mediated immune response.

At present, new therapeutic approaches are being sought, including the search for genetically modified wheat lacking toxic gluten peptides. Furthermore, two approaches are being tested to prevent or reduce gluten toxicity in wheat flour:the masking of gliadin epitopes (by use of reticulating agents such as the enzyme transglutaminase) or the proteolytic degradation of glutan peptides. In both these fields, MS is playing an important role in validating results. On the issue of degradation of toxic epitopes, the main difficulty is that the gluten-derived T cell epitopes are highly resistant to proteolytic degradation within the gastrointestinal tract. In individuals suffering from CD, the gastrointestinal tract does not produce the enzyme necessary to efficiently cleave proline-rich peptides, thus driving the abnormal immune response. For this reason, oral supplementation with exogenous prolyl-endopeptidases (produced for instance by

Mass spectrometry is the only non-immunological method presently available to detect (with high specificity) gliadins and related prolamins in flours and in food samples. A first approach is based on the possibility of obtaining characteristic MALDI-TOF-MS profiles of unfractionated gliadin, hordein, secalin and avenin extracts. Based on these four distinguishable mass patterns, prolamins from different cereals can be differentiated and also identified when simultaneously present in foods. A remarkable application is in the analysis of the products of starch hydrolysis, such as glucose syrup, crystalline dextrose and maltodextrins (largely used as sweeteners, anti-crystallisers, and stabilising agents), obtained industrially through chemical and/or enzymatic methods. In these products, gluten determination by immunological tests is made unreliable by several factors, including the low amount of gluten to be detected being dispersed in a very high amount of substance (low and high mass sugars, other by-products of the process) which interfere with determination. Quantitative measurement of gluten in these products is made possible by combining procedures of extraction and isolation with MALDI-TOF-MS analysis. Low quantities of protein (estimated sensitivity 1-10 ppm) can be identified, thus allowing verification of whether these products exceed the 20ppm limit required for foods "rendered" gluten-free [4]. The pattern of proteins/peptides present in the samples has been found to vary either qualitatively or quantitatively, depending on the sample type. This means that the MS approach may allow the differences to be identified and the protein/peptide level in different industrial products of the same category to be quantified.

Aspergillus niger) that are able to digest gluten, has been proposed as an alternative treatment to the gluten-free diet. In vitro and in vivo studies driven by MS, have confirmed the effectiveness of such supplements. Alternative approaches for gluten degradation are based on gluten fermentation with different microbial media, including probiotic preparations or sourdough Lactobacilli.

Future perspectives

Nutrition plays a crucial role in health as well as disease. Despite being a relatively new field of research, food proteomics is already influencing multiple aspects of the food chain (agriculture, food production, food safety and quality assurance). The research field is also delivering economic benefits and improving aspects of human nutrition and health. Using the new technology, by dissecting the proteome and peptidome into their constituents, specific markers can be identified to trace complete production processes, from raw materials to end-products. Even food products originating from specific geographical areas can be characterised at the molecular level.

Food allergy and intolerance is another field in which MS has proven to be a key player. This includes the removal of epitopes from a certain food. The correct MS technique can provide the means to identify an immunodominant epitope in the target food. This is a starting point for designing antibodies against specific epitopes, and for standardising the quantitative detection of trace amounts of allergen by the development of protein chips [Figure 2]. The results of such technology include large savings and increased health benefits. Future developments in protein array technology, largely driven by MS-derived information on food proteins, will also lead to an improvement in food safety.

References

1. Han X, Aslanian A, Yates JR. Curr Opin Chem Biol 2008; 12: 483.

2. Ferranti P, Proceedings of the workshop on Quality, Salubrity and Safety of some Typical Italian Cheeses, National Research Council/Italian Ministry of University and Research. Rome, 5th May 2005.

3. Tareke F, Rydberg P, Karlsson P, Eriksson Tornqvist SM. Chem Res Toxicol 2007;
13: 517.

4. Ferranti P, Mamone G, Picariello G, Addeo F. J. Mass Spectrom 2007; 42: 1531.

The authors

Antonella Nasi and Pasquale Ferranti*

Department of Food Science

University of Naples "Federico II", Italy

*Corresponding author:

Department of Food Science

(Dipartimento di Scienza degli Alimenti)

University of Naples "Federico II"

Parco Gussone, I-80055, Portici, Italy.

E-mail: ferranti@unina.it

Proteins seperated by two-dimensional gel electrophoresis (2DE), and capillary HPLC-fractionated peptides, can be characterised by MS techniques. This is the case for even very complex samples, and the presence and expression levels of single components can be determined. This is crucial in the study of food proteins from sources such as meat, cereals, or milk.

MS-based strategies for food and nutrition proteomics are now capable of addressing a wide range of analytical questions. These include issues related to food safety, certification and traceability of products. In this article, using selected research case studies, we illustrate how MS and proteomic techniques can be used in the field of food safety. To date, MS has had two main applications; the detection of microorganisms, which may be the cause of food spoilage or be hazardous to human health, and the safety evaluation of food components. In the first application, MS is applied in order to control and detect foodborne microorganisms. In the second application, the presence of toxic or allergenic compounds are detected. Toxic compounds may originally be present in the raw material (and therefore need to be eliminated by the manufacturing process), or they may be generated during the production process. In the case of food-related allergy and intolerance, MS analysis is focused on compounds which are harmful for specific groups of consumers.

By Dr Antonella Nasi and Dr Pasquale Ferranti