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Review

Farm animal milk proteomics☆

Paola Roncadaa,⁎, Cristian Pirasb, Alessio Soggiuc, Romana Turkd,Andrea Urbanie, f, Luigi Bonizzic

aIstituto Sperimentale Italiano L. Spallanzani, Milano, ItalybDipartimento di Scienze Zootecniche, Facoltà di Agraria, Università Degli Studi di Sassari, Sassari, ItalycDipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria, Facoltà di Medicina Veterinaria, Università Degli Studi di Milano,Milano, ItalydDepartment of Pathophysiology, Faculty of Veterinary Medicine, University of Zagreb, Zagreb, CroatiaeDipartimento di medicina interna,Università Tor Vergata, Roma, ItalyfFondazione Santa Lucia—IRCCS, Rome, Italy

A R T I C L E I N F O

☆ This article is part of a Special Issue entit⁎ Corresponding author.

E-mail address: paola.roncada@guest.uni

1874-3919/$ – see front matter © 2012 Elseviedoi:10.1016/j.jprot.2012.05.028

A B S T R A C T

Article history:Received 25 January 2012Accepted 16 May 2012Available online 26 May 2012

Milk is one of the most important nutrients for humans during lifetime. Farm animal milkin all its products like cheese and other fermentation and transformation products is awidespread nutrient for the entire life of humans. Proteins are key molecules of the milkfunctional component repertoire and their investigation represents a major challenge.Proteins in milk, such as caseins, contribute to the formation of micelles that are differentfrom species to species in dimension and casein-type composition; they are an integral partof the MFGM (Milk Fat Globule Membrane) that has being exhaustively studied in recentyears. Milk proteins can act as enzymes or have an antimicrobial activity; they could act ashormones and, last but not least, they have a latent physiological activity encoded in theirprimary structure that turns active when the protein is cleaved by fermentation or digestionprocesses. In this review we report the last progress in proteomics, peptidomics andbioinformatics. These new approaches allow us to better characterize the milk proteome offarm animal species, to highlight specific PTMs, the peptidomic profile and even to predictthe potential nutraceutical properties of the analyzed proteins.This article is part of a Special Issue entitled: Farm animal proteomics.

© 2012 Elsevier B.V. All rights reserved.

Keywords:Farm animalsMilkProteomicsSafetyQualityDairy products

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42602. Milk proteomics: general strategies and analytical methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4260

2.1. Prefractionation methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42612.2. Electrophoretic separation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42612.3. Mass spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42622.4. Bioinformatic tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4263

led: Farm animal proteomics.

mi.it (P. Roncada).

r B.V. All rights reserved.

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3. Milk fractions: an overview in intra and inter specific differences in farm animals . . . . . . . . . . . . . . . . . . 42643.1. Caseomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42643.2. Milk fat globules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42653.3. Whey proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4265

4. Proteomic tools in milk safety and quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42664.1. Milk as a diagnostic fluid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42664.2. Peptidomics and nutraceutical properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42674.3. Milk adulteration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42684.4. Dairy products characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4268

5. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4269Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4269References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4269

1. Introduction

In the last two decades, proteomics have become a funda-mental research tool for life scientists through its use inprotein characterization and biomarker discovery. Moreover,diagnostics has emerged as a great promise of medicine. Thegreatest challenge of animal production is to better under-stand the etiology and pathogenesis of disease, to enhanceanimal welfare, to improve production and to enhance qualityand safety food. In last decade, great efforts have beenaddressed to increase the study of milk proteomics (especiallyin human and bovine), which remains a bioactive biologicalfluid of great interest. Because of the complexity andmultiplicity of milk components, different research tech-niques have been combined to explore genetic aspects,molecular pathways, and cellular functions involved in milkproduction, quality, and safety to gain a multifaceted pictureaddressing this complexity. The rapid evolution of high-throughput technologies allows generating large-scale dataon the DNA, RNA, and protein levels in milk. Sophisticatedcomputational tools help to integrate this data set to enhanceinformation and they are being increasingly used in compar-ative biology approach wherever (as in case of some farmanimals) complete genome is not completely sequenced. Milkis one of the most important nutrients for humans duringlifetime. It is consumed since the life beginning to the elderlyage. It could be considered one of the major feeding resourcesfor humans if considering all the milk products like cheese,fermentation and transformation products. In contrast tohuman milk, that is a nutrient only in the early life, animalmilk and dairy products are nutrients for the entire life ofhumans.

Milk is a complex body fluid designed as a useful nutrientfor all newborn mammals. For this reason milk containsmany secreted proteins with different functions: nutrients,antimicrobials, cytokines and chemokines. All these proteinscontribute to post-partum environmental challenges such asinfections [1,2]. Moreover, for the dairy industry, milk is a highbiological value resource that could be transformed intocheese and other dairy products. While the major proteincomponents of both human and bovine milk have beenbiochemically characterized two decades ago [3], the analysisof the less abundant milk proteins have only just recentlybeen reported for bovine [4–7] and swine milk [7]. Since 1982,

when the investigation of milk through 2-DE [8] started,important progress has been made. For instance, these wererecently performed: the characterization of PTMs as glycosyl-ation and phosphorylation; the identification of variations inthe protein profiles depending on the mammalian species oron the lactation period; the detection and identification ofnew proteins such as the ones present in milk fat globulemembrane (MFGM). However, these last years a significantincrease in the identification of the low-abundant milkproteins has been observed and these findings are useful forthe characterization of pathways and mechanisms that occurduring lactation and give information on the biologicalactivity and functionality of these important proteins.

An important task of proteomics is the investigation of majorproteins, including caseins (CNs) (αs1-, αs2-, β- and κ-casein) andwhey proteins (β-lactoglobulin, α-lactalbumin, bovine serumalbumin). The polymorphisms of caseins are key characteristicsto be specifically considered in the cheese-manufacturingindustry. Milk proteins are characterized by a great heterogeneityand the presence of several isoforms different in ruminants.Proteomics is in particular useful for finding different geneticvariants, changes in the phosphorylation or glycosylation patternand other PTMs. Moreover, milk contains a high number of lowabundance proteins, such as lactoferrin, immunoglobulins,glycoproteins, hormones and enzymes [9]. This review exem-plifies the use of proteomics to study milk proteins, fromprefractionation methods to bioinformatic tools with specialhighlights in animal pathology, food safety and quality, that aremilestones in animal production. Furthermore, advances inproteomic analysis of milk from farm animals to investigate thedifferences between milk of different species will be described.This review article focuses on the challenges to overcome whenstudying milk from farm animals and it summarizes andpresents new directions, means and a selection of recentapplications useful in livestock production.

2. Milk proteomics: general strategies andanalytical methods

This section summarizes the general strategies to studyproteomics of milk, starting from raw samples. Thesestrategies are suitable for every type of milk, either humanor from farm animals. Some strategies are described only for

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human and bovine, not for the minor species, however allmethods are applicable on all types of milk (Fig. 1).

2.1. Prefractionation methods

The main protein fraction of milk comprises of caseins withthe concentration around 25 g/L for bovine milk correspond-ing to 78% of the total milk proteins; the protein fraction inwhey reaches the concentration of 5.4 g/L in bovine milk: 17%total milk [10].

Milk also contains a large number of less abundantproteins which represent 5% of the total milk protein andare located in whey or in the MFGM. Because of the wide rangein concentration and subcellular location, no unique protocolyet exists to analyze milk proteome in its entirety.

For proteomics, as in any other biological fluids, it isimportant to remove most abundant proteins to enhancecharacterization and separation. In the case of milk, there aredifferent high abundant proteins as caseins. Prefractionationmethods, from centrifugation to the use of hexapeptidelibrary resins, are fundamental to better understand milkproteome at different levels.

Milk proteins are present in soluble form in the wheyfraction whereas the caseins are present in micellar form. Inaddition, a part of the total protein content is bound to the fatglobule membrane. To obtain different protein fractions fromraw milk, it is necessary to perform the first step of mildcentrifugation (about 3000 rpm). After this passage, thefraction in the upper layer is composed of lipids and MFGsand the bottom layer is composed of the skimmed milkfraction that includes caseins and whey proteins. A moresubtle fractionation of milk proteins could be obtained withan ultracentrifugation step where it is possible to collect wheyproteins separately from caseins. Fig. 1 shows a scheme of

Fig. 1 – Milk prefractionation steps and

milk processing necessary to obtain the different milkfractions.

In contrast, the analysis of less abundant proteins isdifficult to perform because of the presence of high abundantmilk proteins such as caseins, lactalbumin, lactoferrin andlactoglobulines.

For the analysis of less abundant proteins it is possible touse the approach described by Righetti and colleagues whoused combinatorial peptide ligand libraries, containing hex-apeptides terminating with a primary amine, or modified witha terminal carboxyl group [5]. This approach successfullyallowed discovering and identifying a large number ofpreviously unreported proteins in cow's whey (also forhuman) and could be useful for mapping the deep milkproteome of all species avoiding the problems linked to highabundant proteins.

2.2. Electrophoretic separation

For several years, SDS-PAGE and IEF monodimensionalseparation of milk proteins were the key tools in caseinanalysis, especially for the investigation of the geneticvariants, intra and interspecies. There is a considerablenumber of papers that describe alleles using 1‐D electropho-resis, to enhance species with different milk attitude to makecheese [11–14]. In the last decade, two dimensional electro-phoresis has contributed to better understand global milkproteome providing a direct separation technology of intactproteins in the light also of post translational modifications.Two dimensional electrophoresis is useful to optimize sepa-ration of proteins of similar molecular weight but differentisoelectric point, which is not resolved using 1-DE. It ispreferable to use 2‐DE because of its higher resolution but insome cases 1-DE is the best choice in particular if the

proteomic experimental strategies.

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proteomic analysis has to be done on membrane andhydrophobic proteins. A typical case of a problematic proteinsample to analyze through 2DE is represented by MFGMP, forthis reason some authors prefer to use 1‐DE [15].

2‐DE is extremely successful in detecting and to separatethe different phosphorylated or glycosylated proteins becauseit can resolve their shift in their isoelectric point. For a goodquality analysis of (whole) milk proteome through 2-DE, thelipids removal is required and this is achieved by a mildcentrifugation step of raw milk. Before sample loading, totalprotein quantification should be performed and only after-wards it is advisable to solubilize the milk protein in achaotropic buffer [16].

A study conducted by Claverol and colleagues in 2003 useda 2-DE approach coupled with MS and revealed how thesucessfully separated isoforms of a mixture of k-casein had adifferent phosphorylation and glycosylation pattern [17].Other studies performed in 2009 highlight αs1-casein isoformsin donkey milk using 2-DE coupled with MS [17,18]. Caseinsisoform pattern were also investigated in goat milk using a 2-DE approach [16].

In 2006 Holland et al. characterized multiple forms ofbovine κ-casein with 2-DE. Before performing 2-DE for theidentification of casein isoforms, the authors used an enrich-ment procedure. Authors used a cysteine-tagging enrichmentprocedure to identify multiple low abundance isoformsproduced by variable phosphorylation and glycosylation [19].These isoforms produced by PTMs are present at very lowlevels and are difficult to detect or resolve in whole milksamples, without a specific prefractionation step.

Recently, Alonso-Fauste and colleagues performed anoptimized protein separation with 2D electrophoresis toanalyze whey from control and mastitic animals. Theexperiments were conducted with a conventional proteomicapproach using 2D electrophoresis as a choice of separationmethod, coupled by MALDI TOF analysis for identification[20]. Another interesting approach that could be used fordifferential analysis of milk proteome is represented by two-dimensional difference gel electrophoresis (2D DIGE). 2D DIGEenables multiple protein extracts from different samples to beseparated on the same 2D gel. This is possible by labeling eachextract using spectrally resolvable, size and charge-matchedfluorescent dyes known as CyDye DIGE fluorophores. Thisapproach is able to reduce the variability due to electropho-resis experiment because both control and ‘to be investigated’sample can run in the same gel labeled with differentfluorophores. To study milk proteome, this kind of approachwas already being used by Addis and colleagues who used 2DDIGE to evaluate theMilk fat globules (MFGs) proteome both incontrol and in Mycoplasma agalactiae infected sheep [21]. Thesame approach has been recently used by Xia and colleaguesto perform a proteomic analysis of plasma from cows affectedwith milk fever. Authors detected 23 differentially expressedprotein spots in comparison to control and eight of themweresuccessfully isolated and identified by MALDI-TOF-MS [22].This technique, that is very expensive, is particularly suitablefor low abundant samples. Moreover most authors performmass spectrometry identifications of protein spots frompreparative 2‐DE gel stained with Coomassie to enhanceprotein amount and to have a better identification.

2.3. Mass spectrometry

Mass spectrometry investigations in milk samples analysis isusually coupled with a prefractionation step. Still nowadays,the most powerful method for the separation of complexintact protein mixtures is represented, as previously de-scribed, by 2D electrophoresis. However several other separa-tion methods are currently used mostly applying LC orbidimensional (2D) nanoLC coupled with amass spectrometrydetection for proteolytic fragment analysis of cleaved pro-teins. This biochemical strategy can be considered orthogonalto 2-DE in the protein repertoire analysis of milk. In fact,shotgun proteomics analysis is based on the separation ofproteolytic peptides by nanoHPLC and/or nanoUPLC coupledto MS analysis. Since the analysis is based on isolatedpeptides, the information on the PTMs is leveled out giventhe higher number of un-modified peptides which could finda direct matching in database search. Moreover, the specific-ity for this kind of acquisition is very high, nonetheless, whendealing with complex mixtures, co-elution phenomena ofdifferent species commonly happens and the less abundantare not recorded. Nevertheless the time of data collecting canbe quite fast and the operator is not exposed to carcinogeniccompounds such as acrylamide, thus this experimental set-up is increasingly successfully applied especially whenprotein PTMs are not of specific interest.

In 2009, Mollé and colleagues used in parallel electrospray(ESI) and matrix-assisted laser desorption (MALDI) ionizationto enhance protein identification. A total of 39 bovine milkproteins were identified with a high degree of confidence.More hydrophobic peptides with larger masses were prefer-entially detected by ESI, whereas smaller and basic peptideswere favored by MALDI. Thus, mass spectrometers withdifferent ion sources and analyzers may yield complementaryproteome coverage [23]. Affolter and colleagues reported thequalitative and quantitative profiling of two MFGM-enrichedmilk fractions, a whey protein concentrate (WPC) and abuttermilk protein concentrate (BMP) using different analyt-ical workflows. Authors used an LC–MS/MS-based shotgunapproach that revealed 244 protein identities in WPC and 133in BMP respectively, and provided an extensive characteriza-tion of the protein content in those two fractions. Thereforelabel-free profiling resulted in rapid and efficient semi-quantitative comparison and yielded valuable protein finger-prints. Following these experimental design an absolutequantification of selected MFGM proteins was achieved bystable isotope dilution (SID)-MS, in combination with multiplereaction monitoring (MRM) detection of proteotypic transi-tions [24].

In 2010, Boehmer and collaborators described the applica-tions of LC–MS/MS for the identification of proteins incomplex mixtures, in this case bovine milk under normalconditions and during experimentally induced mastitis [25].

Recently, MALDI-TOF MS was used in a linear mode tomeasure molecular weights of major proteins (α-lactalbumin,β-lactoglobulin, and α- and β-casein) in goat milk in compar-ison to cow's milk [26]. In this work authors showed thatMALDI TOF MS could be used for rapid determination of MWof milk proteins without prefractionation steps. Furthermore,capillary zone electrophoresis [27,28] and capillary isolelectric

Fig. 2 – Phylogenetic tree of average distance based of %homology of primary structure of kappa-casein in humanand farm animals.

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focusing [29] coupled to mass spectrometry were used incharacterization of bovine and buffalo milk proteins as a validalternative to 2D-PAGE-MS. CE-MS was also used as a rapidtool for evaluation of ovine and caprine milk adulterations[30]. In the past years, glycome of human and bovinemilk wasinvestigated in deep using a mass spectrometry experimentalstrategy. Wilson and colleagues investigated global N-linkedglycoproteins of human and bovine Milk Fat Globule Mem-branes. The presence of Lewis b epitope, a target for theHelicobacter pylori bacteria, was identified only in human milkfat globule membrane mucins and not in bovine mucins,supporting the evidence of a protective immune function ofhuman milk [31]. Using FTICR-MS and IRMPD-MS coupled toHPLC-Chip with porous graphited carbon Tao [32] and Nwosu[33] have investigated global glycome in bovine and humanmilk evidencing large differences in glycosylation patternsand a higher concentration of oligosaccharides in humanmilk. N-glycomics in early bovine lactation was also per-formed by chemoselective glycoblotting technique andMALDI-TOF⁄TOF MS analysis [34].

2.4. Bioinformatic tools

With the development of fast next-gen DNA sequencingtechnology, in the last years several genome projects havebeen pursuing farm animal sequencing. The genome se-quence was completed for chicken, rabbit, cow, sheep andpig. The sequencing of a number of other farm animals is stillongoing or under final annotation [35]. Bovine milk is a majorhuman food and a valuable farm product. Bovine milk proteinsequences in comparison to the milk of other farm animalspecies are important to possibly highlight different molecu-lar functions in nutrition and for health.

The inter-specific variability of milk proteome is a keytopic to be defined at the protein sequence level. In fact, incaseins, inter-specific sequence homology rapidly decreaseswith the phylogenetic distance between species [36,37] (Fig. 2).

A recent work from Khaldi [38] investigated with severalsequence alignment tools (BLASTP, TCOFFEE and CLUSTALX)the changes of the isoelectric point due to aminoacid variationsin nine major milk proteins in 13 mammals. κ-casein, lactad-herin, and muc1 have undergone the highest change in theisoelectric point during evolution, probably associated with theadaptive and functional changes.

Milk contains several types of components that providemany biological activities. Many of these are proteins that:protect individuals from exogenous stress, toxins, and path-ogens; encourage optimal growth, development, and adapta-tion to a chosen environment; and promote metabolicregulation for physical and intellectual performance. Most ofthe cited properties are accomplished by milk proteins or bypeptides of milk proteins through the mechanism of protein-protein interaction. For this reason, it is important to evaluateand to characterize the whole milk proteome as well as theprotein functions and their interaction network. Omics-oriented approaches are providing a much deeper evaluationof the proteome of milk and its fractions [39]. Most of bioactivefunctions of milk are not carried out by milk proteins in theirwhole conformation but by peptides belonging to cleavageprocesses of their primary structure. The digestion process

produces several types of bioactive peptides characteristic ofeach protein and animal species. To investigate the putativenutraceutical properties of milk proteins, several methodshave been proposed. As it will be described afterwards, in theparagraph about bioactive peptides, Minkiwicz and colleaguesdeveloped a database (BIOPEP) with more than 2000 peptidesclassified according to their type of bioactivity useful fordiscovering potential bioactive peptides/protein fragments[40]. New approaches in the investigation of milk proteinmolecular functions using functional enrichment of geneontologies (GOs) are also reported in literature. In a recentwork D'Alessandro reported FatiGO functional enrichment ofgene ontology (GOs) and a hierarchical clustering analysis ofcow's milk proteins [41]. Another strategy to evaluate theputative functions of milk proteins is to analyze the protein–protein interactions. It is well known that proteins are themain actors of most cellular activities in a complex synergicrelationship, and functionally similar proteins are oftenrelated in the same molecular clusters [42]. Such a relation-ship network can be extracted using specific software basedon a semantic search of database information such asIngenuity Pathway Analysis (IPA) or STRING. Using IPA,D'Alessandro and colleagues have built up a preliminarymap of the human [1] and bovine milk [41] proteinsinteractome. This approach provided a preliminary importantnetwork of protein interactions of human and bovine milk.Recently, Lemay and colleagues used bioinformatic approacheson high coverage genomic data from Bos taurus and theyshowed evolutionary insights into the bovine milk genomeand proteome [43]. Moreover Ibeagha-Awemu showed biologi-cal processes, functions, pathways, and molecular networksthat were significantly enriched by proteins that emergedduring E. coli or S. aureus mastitis [44]. Currently generation ofhigh confidence network is possible only in case of the humanand cattle interactome. Unfortunately, it is still difficult, due tothe lack of data, to obtain the same level of confidence ofphysical and functional interactions for mare and sow at thepresent time. Currently available information does not allow usto draw milk interactome of goat, sheep and buffalo. However,an initial interactome of MFGM proteins for sheep was recentlydescribed [15].

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3. Milk fractions: an overview in intra and interspecific differences in farm animals

In this section, a focus on each fraction of milk proteome issummarized. In particular, caseomics, whey proteins, milk fatglobule and peptides are discussed in separated ways andthey are presented in terms of comparison of data in literatureof farm animals.

3.1. Caseomics

CNs are organized as macromolecular aggregates with min-erals (micelles). Their amount is very variable among species(80% in bovine milk, about 35% in human [45] and 50% inequine [46,47]. Caseomics studies are important especially forthe implication in dairy industry. An interesting recent paperfrom Larsen and coworkers described the potential difficultiesin cheese making related to casein phosphorylation [48].Authors investigated the causes of non coagulating milk incows with proteomics techniques and demonstrating that itcould be due to a low expression of κ-casein.

Bramanti and colleagues analyzed the different type ofCNs (Fig. 3) and their concentrations in cows, goats, sheep andbuffalo [49]. Miranda and collaborators in 2004 analyzed thedifferent types of CNs in equine milk [50] concluding that it isthe most similar to human milk, and could be considered agood substitute of cow's milk for many children with a severeIgE-mediated cow milk protein allergy.

Caseins (Fig. 4) could be well resolved using 2D electropho-resis [51]. Holland and colleagues in 2004 used 2-DE withnarrow IPGs to analyze bovine milk proteins. Separation anddetection of 10 different κ-casein forms ranging from isoelec-tric point values 4.47 to 5.81 was possible with the creation ofa linear immobilized pH gradient (IPG) 4–7 that was used forthe first dimension [52].

The same method and IPG was used to compare qualita-tively and quantitatively analyzed β- and κ-casein in milksamples from normal and transgenic cattle. This studydemonstrated the possibility to obtain, by a transgenicapproach, a line of cows that is able to produce milk withincreased casein levels [54].

Fig. 3 – Casein composition of cow, goat, sheep, buffalo, mareand human milk samples. From [49,50].

There are several inter-specific differences in the caseinamino acids sequence, the highest similarity has beenobserved among ruminants (Table 1).

Caseins family presents a great heterogeneity due to PTMs,in particular they show a different phosphorylation patternon serine/threonine residues. Phosphorylation stoichiometryof bovine beta-casein and alpha-casein using inductivelycoupled plasma mass spectrometry (ICP-MS) was reported byCiavardelli [56] and recently Matéos and colleagues identifiedphosphorylation sites of equine alpha s1 [57] and beta-caseinby nESI-MS/MS [58]. In this case micro-heterogeneity wasevaluated by 2-dimensional electrophoresis and the determi-nation of the different phosphorylation degrees of the nativeisoforms of αs1-casein was finally achieved by electrosprayionization mass spectrometry. With this experimental strategyauthorswere able to characterize 36 different variants of equineαs1-casein. Phosphorylation level of beta caseins in donkey wasstudied by Cunsolo using MALDI-TOF and nESI-MS/MS [59].Phosphorylation data about alpha s1- and beta-casein wereobtained by MS also for water buffalo by Ferranti [60]. Of all CNsfamily, only κ-CNs are glycosylated. κ-CNs glycosylation wasdescribed in 2005 by Holland at al. [53] who identified thedifferent glycosylation patterns of κ-CNs via 2-DE/MS in bovinemilk. The different isoforms are also visible in Fig. 4.

Caseins show a large inter-specific variability, especially ifconsidering α and β -casein fractions [43,61]. The αs1-casein ofovine milk is very heterogeneous, 10 genetic variants havebeen identified. The differences in sheep's milk caseins arenot due only to the different characteristics of the isoforms,but also the concentration of αs1-casein varies from 0 to 26% oftotal casein and, consequently, the total protein contentvaries considerably. This has major effects on the coagulationproperties of sheep's milk and on the type and quality ofcheese produced [62–64].

Fig. 4 – Bovine master map of 2-DE of bovine milk proteins[51]. It is possible to see the individual glycoforms ofκ-casein. The degree of glycosylation of κ-casein (redarrows) shifts the isoelectric point from the left (more acidic)to the right (more basic) [51–53].

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Human β-casein is phosphorylated [65] as mare's β‐casein[66]. κ-casein plays a key role in micelle formation, it would beexpected that all milk from different species contain thisprotein, but Ochirkhuyag et al. [66] did not identify κ-casein inmare's milk suggesting that an orthologous function could beplayed by β-casein with a low level of phosphorylation. Egitoand collaborators in 2002 using 2-DE-MS showed that theequine CN isoelectric variants were mostly slightly moreacidic than the bovine CN [67].

The properties of casein micelles have been studied incaprine [68,69], ovine [69], buffalo [70], camel [71] and mare[69,72]. Buchheim et al. [73] studied the appearance and thesize of casein micelles discovering that human micelles weresmallest (64 nm) whereas those of goat, camel and donkeywere very large (300–500 nm). In 2010 Chianese and colleaguesanalyzed all the casein isoforms of donkey's milk using aproteomic approach. The methodology used was mainlybased on 1-DE and 2-DE that allowed the contemporaryidentification of donkey CNs and their related heterogeneitydue to phosphorylation, glycosylation and incorrect splicingof RNA in mRNA [74].

3.2. Milk fat globules

Milk fat globules are produced by the mammary gland duringlactation [75]. Their structure is formed by a double phospho-lipid membrane that belongs to lactating cells [75,76]. For thisreason, the proteins present in MFGM could be used formonitoring the pathophysiological state of the mammarygland [4].

It has been demonstrated that, depending on the milksource and its processing, 25–70% of the MFGM is formed byproteins [77]. The composition and function of MFGM proteinsare of high interest because milk fat globule typology andprotein content are different between farm animal species.Most proteins in the MFGM have been identified usingtraditional biochemical approaches [78]. But these methods

Table 1 – % of the sequence identity of major milk and milk fat

Protein name Homosapiens

Bos taurus(Cow)

Bubalus bubalis(Buffalo)

Sus scro(Sow)

as1-casein 100 32 33 35as2-casein NC 100 95 62b-casein 100 56 57 59k-casein 100 53 54 56b-lactoglobulin 44a 100 96 63a-lactalbumin 100 73 73 76lactotransferrin 100 69 70 70Lactoperoxidase 100 83 83 78Osteopontin 100 62 63 69Lactadherinb 100 64 ND 65Lysozyme C 100 80 82 72Bile saltactivated lipase

100 NS NC NC

α-1-antitrypsin 100 68 NC 73Serum albumin 100 76 NC 76

Protein entries were retrieved from Uniprot (http://www.uniprot.org/) daGlycodelin (PAEP) in human, b( also Milk fat globule-EGF factor 8). NS:organism.

are slow, laborious and are able to analyze only one protein attime, while proteomic approaches reveal the identificationsand the quantitative analysis of many proteins in oneexperiment. Because of hydrophobicity of membrane pro-teins, it would be better to use SDS-PAGE [79,80]. The loss ofthe high resolution provided by 2-DE can be overcome by LC-MS/MS. Using this approach Reinhardt and Lippolis identifiedup to 120 proteins in cow MFGM. The majority of theseproteins were membrane associated proteins, mainly in-volved in membrane trafficking or cell signaling [6].

Murgiano and collaborators in 2009 compared the prote-ome of MFGM from milk samples of individuals belonging totwo different cattle breeds. Authors detected interestingdifferences in the amount of proteins linked to mammarygland development and lipid droplets formation, as well ashost defense mechanisms [81].

A proteomic study on goat MFGM proteomewas conductedby Cebo and collaborators who analyzed the total proteomeand the glycosylation of major proteins [82].

Recently, Pisanu and colleagues mapped the membraneproteome of sheep's milk fat globule. In this work authorsused a classical SDS-PAGE separation after the MFGM extrac-tion followed by LC–MS/MS for protein identification andcharacterization [15]. This approach was used to identify intotal 140 unique sheep MFGM proteins. A comparativeanalysis of caprine, bovine and human milk fat globules andtheir biological activity in a representative model of theintestinal barrier have been recently obtained by Spertinoand colleagues [83].

3.3. Whey proteins

Different mammalian species show considerable differences inprotein content. Whey proteins show specific characteristicswhich reflect the nutritional or physiological requirements ofthe newborn of the different species. Many investigations werecarried out about whey proteins characteristic in human milk,

globule proteins from human and different farm animals.

fa Capra hircus(Goat)

Ovis aries(Sheep)

Equus asinus(Donkey)

Equuscaballus(Mare)

33 32 40 4588 89 60 5756 57 57 5852 53 65 6694 93 56 5974 74 75 7670 71 NC 7482 83 NC 8656 65 NC 7264 62 NC 6769 70 52 50NC NC NC NC

NC 69 NC 7274 75 77 76

atabase. All sequences were aligned to human using JalView [55].not secreted in milk U: unidentified protein NC: not coded in this

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but few works exist about minor dairy species. The twoprincipal whey proteins, α-Lactalbumin and β-Lactoglobulin,show a high degree of divergence among species. A largenumber of papers are published about study of whey proteinsproteomics in human milk, because of implications as host-defense and immunomodulating factors [84]. Another recentproteomic study conducted by Tay and colleagues analyzedsimultaneously through 1-DE/MS human, bovine and goat'smilk [14]. Interestingly, authors analyzed the differentialcomposition of milk through a comparative study designevaluating the presence of major milk proteins in the threedescribed samples confirming for example the absence oflactoferrin in goat's milk in comparison to bovine milk and thepresence of serumalbumin, lactoferrin and lysozyme in humanmilk in comparison to bovine.

The proteomic whey fraction analysis of milk sample fromdonkeys belonging to the ‘Ragusana’ species of the East of Sicilywas reported in 2007. Authors detected some unknown compo-nents, together with the identification of already known wheyproteins, using RP-HPLC/electrospray ionization (ESI)-MS analy-sis of the whey fraction. Matrix-assisted laser desorption/ionization (MALDI)-TOF/MS and RP-HPLC/ESI-MS/MS analysisof the enzymatic digests of the unknown components resultedin the identification and characterization of two beta-caseinfragments; of the sequence of donkey's serum albumin; and ofthe oxidized methionine forms of lysozyme B and alpha-lactoalbumin [85]. The characterization of donkey'smilk proteinfraction was also performed using electrophoreticmethods andmass spectrometric analysis. In this study authors analyzed 51milk samples demonstrating that donkey's milk proteinspresent high phenotypic variability [86].

The content of whey proteins has also been recently analyzedby reverse-phase high-performance liquid chromatographycoupled with mass spectrometry. Using this approach, theauthors were able to obtain the complete separation of the wheyprotein fractions. The adopted RP-HPLC and ESI-MS protocolsprovided identification of β-lactoglobulin, α-lactoalbumin andserum albumin in Mediterranean water buffalo (Bubalus bubalis)[87].

Moreover, important inter-species differences in the less-abundant milk proteins have been found: it has beendescribed that the greatest inter-species differences seem tooccur in the presence/concentration of enzymes [88].

All this evidence shows the usefulness of proteomicanalysis in detection of the inter-intra/specific variability ofwhey protein composition.

4. Proteomic tools in milk safety and quality

4.1. Milk as a diagnostic fluid

Milk represents a basic biological fluid useful for diagnosis: itis accessible and it is relatively simple to obtain.

The most used diagnostic strategy for the detection ofbacterial pathologies in bovine milk is the use of PCR [89].With this method it is possible to detect the sequence of thegenome of a specific bacterial pathogen. PCR-based methodsare currently used for the detection of several pathogens inanimal milk as Mycobacterium avium sub. paratuberculosis [90],

Coxiella burnetii [91], Staphylococcus aureus [92], Mycoplasmabovis [93] and many others. However, this method is usefulonly if the genome of the pathogen has already beensequenced.

Another method for indirect detection of an etiologic agentis the research of the specific immunoglobulin in milk, as wellas in serum. Immunoglobulins could be used for the diagnosisof several animal pathologies i.e. paratuberculosis [94,95] orthe infection with other bacteria [96]. The election method forthe discovery of immunoreactive epitopes is the use of 2Delectrophoresis of the proteins of the etiologic agent immu-noblotted against whey or serum of an infected animal. Thediscovered immunoreactive proteins could be studied for thedevelopment of an ELISA kit.

One of the most investigated animal pathologies throughmilk proteomics is mastitis. Different proteomics approacheswere applied to study mastitis in milk; but a—ready to use—proteome biomarker in milk is yet far to obtain. Several workson milk proteomics of cows with mastitis gave a contribute tothe comprehension of biochemical mechanisms of the basisof inflammation especially for the Acute Phase Proteins (APP)[97–100].

Bovine mastitis is a major disease that causes economiclosses to dairy industry going from decreasedmilk productionto reproductive disorders in dairy cows. Detection of clinicalmastitis is relatively easy, but subclinical mastitis is difficultto detect due to the absence of any visible clinical sign. Clearunderstanding of the pathogenesis of mastitis is crucial forthe development of adequate tools for mastitis diagnosis.

Currently, mastitis can be monitored by measuring milkelectrical conductivity [101], somatic cell counts (SCCs) [102] orthe enzyme activity of lactate dehydrogenase [103,104].

However, biomarkers which could predict mastitis at earlierstages are requiredbecause all othermilk biomarkers are able todetect mastitis only when it is in the clinical phase and it is toolate to treat animals with antibiotics. There are severalproteomic studies conducted on milk that highlight putativeproteins useful as possible biomarkers for early stage orsubclinicalmastitis. In 2004Hogarth and collaborators analyzedwith a classical proteomic approach both bovine normal andmastitic whey, reporting an increased concentration of proteinsof blood serum origin as serotransferrin and albumin, whileconcentrations of the major whey proteins α-lactalbumin andβ-lactoglobulin were reduced in mastitic whey [105].

In 2010 Danielsen and colleagues analyzed with a proteo-mic approach the differential proteome of milk collected aftera lipopolysaccharide-mediated inflammation. Forty-nine dif-ferentially expressed proteins were identified including someinteresting proteins like several apolipoproteins and otheranti-inflammatory proteins in milk, which are important forthe cow's ability to balance the immune response. Moreoverauthors found an up-regulation of both complement C3 andC4, which indicates that more than one complement pathwaycould be activated during LPS-induced mastitis [106].

Smolenski and colleagues in 2007 used a 2-DE/MS ap-proach to study the milk proteome (both whey and MFGMproteins) in order to find the proteins involved in host defensemechanisms [107]. Recently Alonso-Fauste and colleaguesused a proteomic approach for the diagnosis of mastitis. Aproteomic approach was used for the analysis of both serum

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and whey proteins and authors obtained encouraging resultsanalyzing the differential proteomic profile of normal whey incomparison to mastitic whey. In particular, authors foundseveral proteins from the somatic cells whose number isstrongly increased in milk in case of an acute phase situation[20].

Prostaglandin D synthase was described as a putativebiomarker for bovine mastitis diagnosis. In particular, oneisoform was found with a defined cysteine residue that wasoxidized to a sulfonic acid [108].

All the described experimental evidence could be used astools to help the diagnosis of bovine mastitis. The presence ofhigh amounts of somatic cell proteins in the mastitic wheycould be used as an index to evaluate the severity or to detectmastitis in its subclinical form. Moreover, the describedupregulation of C3 and C4 indicates the immune reactionagainst lipopolysaccharide that leads to bacterial opsoniza-tion. Upregulation of C3 and C4 is a proof that demonstratesthe presence of bacterial growth in the organism [109].

4.2. Peptidomics and nutraceutical properties

Milk naturally contains a considerable number of bioactivecompounds as lysozyme, lactoferrin, growth factors, andhormones, which are directly secreted in their active form bythe mammary gland. Colostrum is rich in nutrients andprovides protection against pathogens thanks to its highconcentration of antimicrobial proteins and, in particular,immunoglobulins [110,111]. Many milk proteins are precur-sors of bioactive peptides that are generated by digestiveenzymes and during milk fermentation [112]. Biologicalactivity can be interpreted as a beneficial or negativeinfluence on an organism [113,114]. Peptides are consideredbioactive when they possess a hormone, or drug-like, activitywhich modulates physiological functions through bindinginteraction to specific receptors. Peptides with various bio-activities have been identified in several dairy-products, suchas milk protein hydrolysates, fermented milk and manycheese varieties [115]. The peptidome is represented by theentire number of peptides present in food products or rawmaterials, or obtained during processing and storage. Peptidespresent in several kinds of cheese have already being studied:

Table 2 – Bioactive peptides derived from milk proteins of seve

Precursor Bioactive peptide

α-Lactalbumin α-Lactorphin Opioid aβ-Lactoglobulin β-Lactorphin Non-opiLactoferrin Lactoferricin Antimicβ-Casein β-Casomorphins Opioid a

β-Casokinins ACE-inhCasein phosphopeptide Stimula

αs1-Casein α-Casein exorphins Opioid aα-Casokinin ACE inh

k-Casein Casoxins Opioid aCasoplatelin Antithro

Bovine serum albumin Serorphin OpioidAlbutensin A Ileum co

Cheddar [116], Parmigiano Reggiano [117,118], Grana Padano[119], and Emmental [119].

Recently, Panchaud and colleagues described in a reviewhow novel proteomic techniques together with bioinformaticscould be helpful in finding hidden bioactive peptides inanalyzed proteome [120]. On the same topic, Minkiwicz andcolleagues developed a database (BIOPEP) with more than2000 peptides classified according to their type of bioactivityuseful to discover potential bioactive peptides/protein frag-ments [40]. The same database was used by Iwaniak andDziuba who used a bioinformatics approach and reported aninteresting study on the bioactivity and the protein structure.Authors evidenced the structural requirements for peptide(s)to be regarded as biologically active (bioactive). In particularthe structure and bioactivity analysis revealed that if peptidesencode for a ‘bio-action’, it is essential that they assume thestructure of a coil (or combination of coil and a-helix) in thesequence of their protein precursors [121].

A good description of bioactive proteins and peptides hasbeen recently done by Nagpal and colleagues [122] who, asshown in Table 2, described exhaustively all known bioactivepeptides and their original protein.

Table 2 shows how milk protein derived peptides couldhave several biological functions such as: opioid activity,antihypertensive properties, antithrombotic properties, min-eral binding properties, immunomodulating activities, anti-microbial properties.

Opioid activity is due to the affinity of these peptides for anopiate receptor that produces opiate-like effects. Specific re-ceptors are responsible for physiological effects, e.g., the μ-receptor for emotional behavior and a suppression of intestinalmotility, the σ-receptor for emotional behavior, and the κ-receptor for sedation and food intake. One of the first bioactivepeptides with opioid properties that has been studied is β-casomorphin. There are several types of casomorphins andmost of them present a high affinity for μ receptor [135]. Manyother peptides from milk peptides have an opioid agonistactivity as α-Lactorphin and α-Casein exorphins that respec-tively belong to α-Lactalbumin and αs1-Casein. Κ-casoxinbelongs to κ-casein and has an opioid antagonist function.

Antihypertensive properties are achieved by peptides thatact as Angiotensin-converting enzyme (ACE) inhibitors. There

ral farm animals.

Function References

gonist, ACE-inhibition [123]oid stimulatory effect on ileum, ACE-inhibition [123]robial [124]gonist, ACE-inhibition, immunomodulation [125–127]ibition, immunomodulation [125,128]tion of mineral absorption [129]gonist [129]ibition, immunomodulation [125,130]ntagonist [131]mbotic [132]

[133]ntraction, ACE-inhibition [134]

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is a lot of experimental evidence that demonstrates howseveral tripeptides belonging to milk proteins could carry outan anti-hypertensive function [136–139]. As resumed inTable 2, almost all milk proteins have bioactive peptideswith ACE inhibitory function in their sequence.

Other functions carried out by milk bioactive peptides arethe immunomodulating and the antimicrobial function.

The immunomodulating function is carried out byβ-Casomorphins, β-Casokinins and α-Casokinin. Even if thephysiological mode of action is not yet known, it has beendemonstrated that they may stimulate the proliferation andmaturation of immune system cells. Synthetic peptides corre-sponding to fragments of bovine k-casein and α-lactalbuminhave been shown to enhance proliferation of human peripheralblood lymphocytes. These peptideswere Tyr-Gly andTyr-Gly-Glybelonging from β-Casomorphins, β-Casokinins showed suppres-sion and stimulation of lymphocyte proliferation depending onthe peptide concentration [140].

The antimicrobial function is mainly carried out by Lacto-ferricin peptide that pertains to lactoferrin. Bellamy andcolleagues demonstrated how lactoferricin B, a peptide pro-duced by gastric pepsin digestion of bovine lactoferrin, is able toinhibit growth of several microorganisms as Escherichia coli,Salmonella enteritidis, Klebsiella pneumoniae, Proteus vulgaris,Yersinia enterocolitica, Pseudomonas aeruginosa, Campylobacterjejuni, S. aureus, Streptococcus mutans, Corynebacterium diphtheriae,Listeria monocytogenes and Clostridium perfringens.

4.3. Milk adulteration

Milk and dairy products economically driven adulterationrepresents a major problem in food production. Usually themost common adulteration arises from a mixing of highquality food products with cheaper ingredients. For example,one of the most common milk adulterations is characterizedby the mixing goat's milk with bovine milk to be directly soldas entire goat's milk or for goat cheese production. This kindof adulteration is difficult to detect and usually it is performedthrough DNA-based methods like PCR [141–143].

Only in the past decade proteomics expanded the objec-tives to the study of food products in order to detect milkadulteration.

As suggested by D'Ambrosio and colleagues [144], prote-omics could be used for detection of milk adulteration. Thedetection of adulteration in buffalo's milk is still based on1-DE (Italian Gazzetta Ufficiale n.160, 11/07/1994), but it couldbe replaced by 2-DE to have a more exhaustive adulterationanalysis. In that article authors analyzed the Italian buffalo(B. bubalis) whole milk proteome with 2-DE/MS. They ana-lyzed almost all proteins present in the 2D map and theirisoforms through MS giving an exhaustive characterizationof PTM [144].

The collected evidence is useful to prevent adulteration ofbuffalo's milk and derived products. This kind of approachcould be applied to milk of all other species in order to bettercharacterize the inter-specific differences of whole milkproteome. Using a mass spectrometric approach Cuollo andcolleagues [145] showed the possibility of detecting extrane-ous milk in single species cheese-milk through the monitor-ing of casein proteotypic peptides [145].

A proteomic approach has also been applied by Arena andcolleagues to study protein modifications in milk during andafter processing [146,147]. Pinto in a recent work showed thatcasein lactosylation is a function of the heating intensity[148]. Holland described temperature dependent molecularchanges in milk proteins (non-disulfide cross-linking, deami-dation and lactosylation) during storage of UHT-treated milkusing 2-DE coupled to MALDI-TOF MS [149].

In conclusion, through a proteomic approach, it is possibleto evaluate the provenience of specific milk and whether ithas beenmixed or not with milk from other species. This kindof adulteration is particularly common for buffalo's milk. Afurther potential application carried out by proteomicsanalysis consists in the evaluation of proper milk storageand processing. As previously described, it is possible todetermine, analyzing protein post-translational modifica-tions, the thermal process and the pasteurization processwhich has been applied.

4.4. Dairy products characterization

Raw milk can be processed to obtain a huge variety of relateddairy products. Quality and production in diary industry areassessed using probiotics and starter bacteria. In particularlactic acid bacteria (LAB) are used in the dairy industry as startercultures for the production of fermented milk products. LABproduce lactic acid from lactose resulting in acidification of thesubstrate, which inhibits pathogen growth [9]. Several microor-ganisms typical of dairy fermented products have been studiedthrough proteomics [150,151] such as: Lactococcus lactis, Strepto-coccus thermophilus, Lactobacillus delbrueckii ssp. lactis, Lactobacillusacidophilus, and Propionibacterium freudenreichii. Proteomic ap-proaches have also been used to investigate the adaptation ofprobiotic lactobacilli, bifidobacteria, and propionibacteria todigestive stress. In particular, Gagnaire et al. identified bacterialproteins released after lysis of the microflora in Emmentalcheese, a complex dairy matrix [152].

Cheese manufacturing is often performed through the aidof chymosin. Traditionally, chymosin is the major enzymeresponsible for the coagulation of milk proteins and it is oneof the main enzymes present in rennet. Chymosin is theprincipal protease used for cheese making because it hashighly specific milk-clotting activity relative to its proteolyticactivity and it is specific for the cleavage of κ-CN. Recently,Hsieh and colleagues analyzed the coagulation of milkproteins induced by chymosin through the proteomic profil-ing. Authors reported an interesting time-course where theydocumented the chymosin-related κ-CN hydrolysis through1‐DE and 2‐DE [48].

The components of cheese are proteins and fat derivedfrom milk. Different types of cheese are produced fromdifferent milk. They are produced by the coagulation of caseinfraction using specific enzymes. Many chemical and bio-chemical reactions occur during cheese ripening, and theproteolytic mechanism is the most important. Proteolysiscontributes to give cheese a typical texture and flavor,through generation of large polypeptides, and with theformation of a wide range of intermediate-sized and smallpeptides, including free amino acids and their degradationproducts [153]. The first “proteomics-like” studies about

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casein proteolysis in cheese date back to the nineties[117,118,154–156]. Peptides derived from casein proteolysis asa marker of ripening were analyzed in cheeses like GranaPadano [119,157] and Parmigiano Reggiano [158–160] and alsophosphopeptides are well described [156,161].

The complexity of cheese is due to the concomitantpresence of different proteins which are a part of the milkmicrobial ecosystem [162]. These different pools of proteinsare drawn from the cheese proteome that is typical andspecific for each kind of cheese [151]. This process completelydepends on the fermentation and transformation processes oflactic acid bacteria [163,164]. Thus it is important to charac-terize proteins secreted by bacterial starters in cheese as amarker of the ripening or fermentation process [165,166].Moreover, the mandatory evaluation of the safety andtraceability [167,168] of these important dairy products canbe achieved monitoring specific protein or peptide products[169].

5. Concluding remarks

A large body of evidence has been collected in recent years inthe development of different proteomics strategies principallyfor the analysis of human and bovine milk. The progressachieved in milk proteomics represents only an initial goal inthe field of biomarker discovery and quality-safety food –related research. Many new opportunities and challengesremain to be explored in the coming years, especially for newinsights in milk of small ruminants and in general of otherfarm animals. Although, it is also true that small dairy specieswill never be able to compete with cattle in terms of quantityand quality of the milk production. The contribution that milkfrom other secondary (domesticated) dairy species can give tothe survival and well-being of mankind around the world isimmense and fundamental. Especially when it concerns thedeveloping countries, the secondary dairy species play acritical role in supplying the food and nutritional needs ofpopulations that live in those areas. Furthermore, in develop-ing countries, unavailability of cow's milk, linked to a very lowconsumption of meat, represents a huge problem. For thisreason,milk of small dairy species such as goat, buffalo, sheepand possibly donkey, could well replace daily food sources ofprotein, phosphate and calcium for large number of people inthe world. Last, but not least, because of its important andrelated hypoallergenic properties, milk from small farmspecies such as goat or donkey, but also mare, have beenoften recommended as substitutes in diets in cases where ofcow's milk allergies and immunocompromised patients. Forall these aspects, this review describes how proteomics iscentral for a better understanding and characterization offarm animalmilk proteins and dairy products. As discussed inthis paper, high throughput and advanced separationmethods now available are able to deeply characterize milkproteins of different species including the analysis of PTMs.Most of the literature is focused on human and bovine milk;about other animal species, in particular farm animals, it isnecessary to implement knowledge on milk proteins. Aspreviously described high resolution 2-DE is useful to obtainan optimal separation of themilk analysis of protein isoforms.

Such an approach could be useful as a tool to investigate thedifferences between milk from different species or lactationperiod, as well as for the diagnosis of animal pathologies or forassessingmilk quality. Moreover, a proteomic and peptidomicinvestigation could be extremely important to counteract foodadulteration, to develop novel traceability methods and alsoto find putative nutraceutical properties of different farmanimal milk and milk products. In particular, bioinformaticsmethods could provide the necessary tools to discovernutraceutical properties of identified proteins and it is ableto predict the possible production of bioactive peptides afterproteolytic cleavages.

In conclusion, milk contains a wide array of proteins thatprovide a number of biological activities; the deep knowledgeof farm animal milk proteomics could be useful to answer theincreasing interest of industry in the application of functionalfood proteins. Furthermore, the study of milk proteome cancontribute to human and animal welfare thus providingimportant elements for improving the milk formula innutrition. Finally, the research in milk proteomics of smalldairy species as well, will continue to expand in variousdirections in the near future, and many new fascinatingapplications and properties will be investigated, in compli-ance with the sustainable progress era in which we all live.

Acknowledgment

Authors are grateful to the COST ACTION FA1002 FarmAnimal proteomics for the network provided.

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