Farm Animal Milk Proteomics

J O U RN A L OF P ROT EO M I CS 7 5 ( 2 0 12 ) 42 5 9 4 27 4
Available online at www.sciencedirect.com
www.elsevier.com/locate/jprot
Review
Farm animal milk proteomics

Paola Roncadaa,, Cristian Pirasb , Alessio Soggiuc , Romana Turkd ,
Andrea Urbanie, f , Luigi Bonizzic
a
Istituto Sperimentale Italiano L. Spallanzani, Milano, Italy

Dipartimento di Scienze Zootecniche, Facolt di Agraria, Universit Degli Studi di Sassari, Sassari, Italy
c
Dipartimento di Patologia Animale, Igiene e Sanit Pubblica Veterinaria, Facolt di Medicina Veterinaria, Universit Degli Studi di Milano,
Milano, Italy
d
Department of Pathophysiology, Faculty of Veterinary Medicine, University of Zagreb, Zagreb, Croatia
e
Dipartimento di medicina interna,Universit Tor Vergata, Roma, Italy
f
Fondazione Santa LuciaIRCCS, Rome, Italy
b
AR TIC LE I N FO
ABS TR ACT
Article history:
Milk is one of the most important nutrients for humans during lifetime. Farm animal milk
Received 25 January 2012
in all its products like cheese and other fermentation and transformation products is a
Accepted 16 May 2012
widespread nutrient for the entire life of humans. Proteins are key molecules of the milk
Available online 26 May 2012
functional component repertoire and their investigation represents a major challenge.

Proteins in milk, such as caseins, contribute to the formation of micelles that are different
Keywords:
from species to species in dimension and casein-type composition; they are an integral part
Farm animals
of the MFGM (Milk Fat Globule Membrane) that has being exhaustively studied in recent
Milk
years. Milk proteins can act as enzymes or have an antimicrobial activity; they could act as
Proteomics
hormones and, last but not least, they have a latent physiological activity encoded in their
Safety
primary structure that turns active when the protein is cleaved by fermentation or digestion
Quality
processes. In this review we report the last progress in proteomics, peptidomics and
Dairy products
bioinformatics. These new approaches allow us to better characterize the milk proteome of
farm animal species, to highlight specific PTMs, the peptidomic profile and even to predict
the 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.
Contents
1.
2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . .
Milk proteomics: general strategies and analytical methods
2.1. Prefractionation methods . . . . . . . . . . . . . . .
2.2. Electrophoretic separation . . . . . . . . . . . . . .
2.3. Mass spectrometry . . . . . . . . . . . . . . . . . .
2.4. Bioinformatic tools . . . . . . . . . . . . . . . . . .
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This article is part of a Special Issue entitled: Farm animal proteomics.

Corresponding author.
E-mail address: paola.roncada@guest.unimi.it (P. Roncada).
1874-3919/$ see front matter 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.jprot.2012.05.028
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J O U RN A L OF P ROT EO M IC S 7 5 ( 2 0 12 ) 42 5 9 42 7 4
3.
Milk fractions: an overview in intra and inter specific differences in farm animals
3.1. Caseomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2. Milk fat globules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3. Whey proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.
Proteomic tools in milk safety and quality . . . . . . . . . . . . . . . . . . . . . .
4.1. Milk as a diagnostic fluid . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2. Peptidomics and nutraceutical properties . . . . . . . . . . . . . . . . . . .
4.3. Milk adulteration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.4. Dairy products characterization . . . . . . . . . . . . . . . . . . . . . . . .
5.
Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.
Introduction
In the last two decades, proteomics have become a fundamental research tool for life scientists through its use in
protein characterization and biomarker discovery. Moreover,
diagnostics has emerged as a great promise of medicine. The
greatest challenge of animal production is to better understand the etiology and pathogenesis of disease, to enhance
animal welfare, to improve production and to enhance quality
and safety food. In last decade, great efforts have been
addressed to increase the study of milk proteomics (especially
in human and bovine), which remains a bioactive biological
fluid of great interest. Because of the complexity and
multiplicity of milk components, different research techniques have been combined to explore genetic aspects,
molecular pathways, and cellular functions involved in milk
production, quality, and safety to gain a multifaceted picture
addressing this complexity. The rapid evolution of highthroughput technologies allows generating large-scale data
on the DNA, RNA, and protein levels in milk. Sophisticated
computational tools help to integrate this data set to enhance
information and they are being increasingly used in comparative biology approach wherever (as in case of some farm
animals) complete genome is not completely sequenced. Milk
is one of the most important nutrients for humans during
lifetime. It is consumed since the life beginning to the elderly
age. It could be considered one of the major feeding resources
for humans if considering all the milk products like cheese,
fermentation and transformation products. In contrast to
human milk, that is a nutrient only in the early life, animal
milk and dairy products are nutrients for the entire life of
humans.
Milk is a complex body fluid designed as a useful nutrient
for all newborn mammals. For this reason milk contains
many secreted proteins with different functions: nutrients,
antimicrobials, cytokines and chemokines. All these proteins
contribute to post-partum environmental challenges such as
infections [1,2]. Moreover, for the dairy industry, milk is a high
biological value resource that could be transformed into
cheese and other dairy products. While the major protein
components of both human and bovine milk have been
biochemically characterized two decades ago [3], the analysis
of the less abundant milk proteins have only just recently
been reported for bovine [47] and swine milk [7]. Since 1982,
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when the investigation of milk through 2-DE [8] started,

important progress has been made. For instance, these were
recently performed: the characterization of PTMs as glycosylation and phosphorylation; the identification of variations in
the protein profiles depending on the mammalian species or
on the lactation period; the detection and identification of
new proteins such as the ones present in milk fat globule
membrane (MFGM). However, these last years a significant
increase in the identification of the low-abundant milk
proteins has been observed and these findings are useful for
the characterization of pathways and mechanisms that occur
during lactation and give information on the biological
activity and functionality of these important proteins.
An important task of proteomics is the investigation of major
proteins, including caseins (CNs) (s1-, s2-, - and -casein) and
whey proteins (-lactoglobulin, -lactalbumin, bovine serum
albumin). The polymorphisms of caseins are key characteristics
to be specifically considered in the cheese-manufacturing
industry. Milk proteins are characterized by a great heterogeneity
and the presence of several isoforms different in ruminants.
Proteomics is in particular useful for finding different genetic
variants, changes in the phosphorylation or glycosylation pattern
and other PTMs. Moreover, milk contains a high number of low
abundance proteins, such as lactoferrin, immunoglobulins,
glycoproteins, hormones and enzymes [9]. This review exemplifies the use of proteomics to study milk proteins, from
prefractionation methods to bioinformatic tools with special
highlights in animal pathology, food safety and quality, that are
milestones in animal production. Furthermore, advances in
proteomic analysis of milk from farm animals to investigate the
differences between milk of different species will be described.
This review article focuses on the challenges to overcome when
studying milk from farm animals and it summarizes and
presents new directions, means and a selection of recent
applications useful in livestock production.
2.
Milk proteomics: general strategies and
analytical methods
This section summarizes the general strategies to study
proteomics of milk, starting from raw samples. These
strategies are suitable for every type of milk, either human
or from farm animals. Some strategies are described only for
human and bovine, not for the minor species, however all
methods are applicable on all types of milk (Fig. 1).
2.1.
Prefractionation methods
The main protein fraction of milk comprises of caseins with

the concentration around 25 g/L for bovine milk corresponding to 78% of the total milk proteins; the protein fraction in
whey reaches the concentration of 5.4 g/L in bovine milk: 17%
total milk [10].
Milk also contains a large number of less abundant
proteins which represent 5% of the total milk protein and
are located in whey or in the MFGM. Because of the wide range
in concentration and subcellular location, no unique protocol
yet exists to analyze milk proteome in its entirety.
For proteomics, as in any other biological fluids, it is
important to remove most abundant proteins to enhance
characterization and separation. In the case of milk, there are
different high abundant proteins as caseins. Prefractionation
methods, from centrifugation to the use of hexapeptide
library resins, are fundamental to better understand milk
proteome at different levels.
Milk proteins are present in soluble form in the whey
fraction whereas the caseins are present in micellar form. In
addition, a part of the total protein content is bound to the fat
globule membrane. To obtain different protein fractions from
raw milk, it is necessary to perform the first step of mild
centrifugation (about 3000 rpm). After this passage, the
fraction in the upper layer is composed of lipids and MFGs
and the bottom layer is composed of the skimmed milk
fraction that includes caseins and whey proteins. A more
subtle fractionation of milk proteins could be obtained with
an ultracentrifugation step where it is possible to collect whey
proteins separately from caseins. Fig. 1 shows a scheme of
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milk processing necessary to obtain the different milk

fractions.
In contrast, the analysis of less abundant proteins is
difficult to perform because of the presence of high abundant
milk proteins such as caseins, lactalbumin, lactoferrin and
lactoglobulines.
For the analysis of less abundant proteins it is possible to
use the approach described by Righetti and colleagues who
used combinatorial peptide ligand libraries, containing hexapeptides terminating with a primary amine, or modified with
a terminal carboxyl group [5]. This approach successfully
allowed discovering and identifying a large number of
previously unreported proteins in cow's whey (also for
human) and could be useful for mapping the deep milk
proteome of all species avoiding the problems linked to high
abundant proteins.
2.2.
Electrophoretic separation
For several years, SDS-PAGE and IEF monodimensional

separation of milk proteins were the key tools in casein
analysis, especially for the investigation of the genetic
variants, intra and interspecies. There is a considerable
number of papers that describe alleles using 1D electrophoresis, to enhance species with different milk attitude to make
cheese [1114]. In the last decade, two dimensional electrophoresis has contributed to better understand global milk
proteome providing a direct separation technology of intact
proteins in the light also of post translational modifications.
Two dimensional electrophoresis is useful to optimize separation of proteins of similar molecular weight but different
isoelectric point, which is not resolved using 1-DE. It is
preferable to use 2DE because of its higher resolution but in
some cases 1-DE is the best choice in particular if the
Fig. 1 Milk prefractionation steps and proteomic experimental strategies.
4262
proteomic analysis has to be done on membrane and

hydrophobic proteins. A typical case of a problematic protein
sample to analyze through 2DE is represented by MFGMP, for
this reason some authors prefer to use 1DE [15].
2DE is extremely successful in detecting and to separate
the different phosphorylated or glycosylated proteins because
it can resolve their shift in their isoelectric point. For a good
quality analysis of (whole) milk proteome through 2-DE, the
lipids removal is required and this is achieved by a mild
centrifugation step of raw milk. Before sample loading, total
protein quantification should be performed and only afterwards it is advisable to solubilize the milk protein in a
chaotropic buffer [16].
A study conducted by Claverol and colleagues in 2003 used
a 2-DE approach coupled with MS and revealed how the
sucessfully separated isoforms of a mixture of k-casein had a
different phosphorylation and glycosylation pattern [17].
Other studies performed in 2009 highlight s1-casein isoforms
in donkey milk using 2-DE coupled with MS [17,18]. Caseins
isoform pattern were also investigated in goat milk using a 2DE approach [16].
In 2006 Holland et al. characterized multiple forms of
bovine -casein with 2-DE. Before performing 2-DE for the
identification of casein isoforms, the authors used an enrichment procedure. Authors used a cysteine-tagging enrichment
procedure to identify multiple low abundance isoforms
produced by variable phosphorylation and glycosylation [19].
These isoforms produced by PTMs are present at very low
levels and are difficult to detect or resolve in whole milk
samples, without a specific prefractionation step.
Recently, Alonso-Fauste and colleagues performed an
optimized protein separation with 2D electrophoresis to
analyze whey from control and mastitic animals. The
experiments were conducted with a conventional proteomic
approach using 2D electrophoresis as a choice of separation
method, coupled by MALDI TOF analysis for identification
[20]. Another interesting approach that could be used for
differential analysis of milk proteome is represented by twodimensional difference gel electrophoresis (2D DIGE). 2D DIGE
enables multiple protein extracts from different samples to be
separated on the same 2D gel. This is possible by labeling each
extract using spectrally resolvable, size and charge-matched
fluorescent dyes known as CyDye DIGE fluorophores. This
approach is able to reduce the variability due to electrophoresis experiment because both control and to be investigated
sample can run in the same gel labeled with different
fluorophores. To study milk proteome, this kind of approach
was already being used by Addis and colleagues who used 2D
DIGE to evaluate the Milk fat globules (MFGs) proteome both in
control and in Mycoplasma agalactiae infected sheep [21]. The
same approach has been recently used by Xia and colleagues
to perform a proteomic analysis of plasma from cows affected
with milk fever. Authors detected 23 differentially expressed
protein spots in comparison to control and eight of them were
successfully isolated and identified by MALDI-TOF-MS [22].
This technique, that is very expensive, is particularly suitable
for low abundant samples. Moreover most authors perform
mass spectrometry identifications of protein spots from
preparative 2DE gel stained with Coomassie to enhance
protein amount and to have a better identification.
2.3.
Mass spectrometry
Mass spectrometry investigations in milk samples analysis is

usually coupled with a prefractionation step. Still nowadays,
the most powerful method for the separation of complex
intact protein mixtures is represented, as previously described, by 2D electrophoresis. However several other separation methods are currently used mostly applying LC or
bidimensional (2D) nanoLC coupled with a mass spectrometry
detection for proteolytic fragment analysis of cleaved proteins. This biochemical strategy can be considered orthogonal
to 2-DE in the protein repertoire analysis of milk. In fact,
shotgun proteomics analysis is based on the separation of
proteolytic peptides by nanoHPLC and/or nanoUPLC coupled
to MS analysis. Since the analysis is based on isolated
peptides, the information on the PTMs is leveled out given
the higher number of un-modified peptides which could find
a direct matching in database search. Moreover, the specificity for this kind of acquisition is very high, nonetheless, when
dealing with complex mixtures, co-elution phenomena of
different species commonly happens and the less abundant
are not recorded. Nevertheless the time of data collecting can
be quite fast and the operator is not exposed to carcinogenic
compounds such as acrylamide, thus this experimental setup is increasingly successfully applied especially when
protein PTMs are not of specific interest.
In 2009, Moll and colleagues used in parallel electrospray
(ESI) and matrix-assisted laser desorption (MALDI) ionization
to enhance protein identification. A total of 39 bovine milk
proteins were identified with a high degree of confidence.
More hydrophobic peptides with larger masses were preferentially detected by ESI, whereas smaller and basic peptides
were favored by MALDI. Thus, mass spectrometers with
different ion sources and analyzers may yield complementary
proteome coverage [23]. Affolter and colleagues reported the
qualitative and quantitative profiling of two MFGM-enriched
milk fractions, a whey protein concentrate (WPC) and a
buttermilk protein concentrate (BMP) using different analytical workflows. Authors used an LCMS/MS-based shotgun
approach that revealed 244 protein identities in WPC and 133
in BMP respectively, and provided an extensive characterization of the protein content in those two fractions. Therefore
label-free profiling resulted in rapid and efficient semiquantitative comparison and yielded valuable protein fingerprints. Following these experimental design an absolute
quantification of selected MFGM proteins was achieved by
stable isotope dilution (SID)-MS, in combination with multiple
reaction monitoring (MRM) detection of proteotypic transitions [24].
In 2010, Boehmer and collaborators described the applications of LCMS/MS for the identification of proteins in
complex mixtures, in this case bovine milk under normal
conditions and during experimentally induced mastitis [25].
Recently, MALDI-TOF MS was used in a linear mode to
measure molecular weights of major proteins (-lactalbumin,
-lactoglobulin, and - and -casein) in goat milk in comparison to cow's milk [26]. In this work authors showed that
MALDI TOF MS could be used for rapid determination of MW
of milk proteins without prefractionation steps. Furthermore,
capillary zone electrophoresis [27,28] and capillary isolelectric
focusing [29] coupled to mass spectrometry were used in

characterization of bovine and buffalo milk proteins as a valid
alternative to 2D-PAGE-MS. CE-MS was also used as a rapid
tool for evaluation of ovine and caprine milk adulterations
[30]. In the past years, glycome of human and bovine milk was
investigated in deep using a mass spectrometry experimental
strategy. Wilson and colleagues investigated global N-linked
glycoproteins of human and bovine Milk Fat Globule Membranes. The presence of Lewis b epitope, a target for the
Helicobacter pylori bacteria, was identified only in human milk
fat globule membrane mucins and not in bovine mucins,
supporting the evidence of a protective immune function of
human milk [31]. Using FTICR-MS and IRMPD-MS coupled to
HPLC-Chip with porous graphited carbon Tao [32] and Nwosu
[33] have investigated global glycome in bovine and human
milk evidencing large differences in glycosylation patterns
and a higher concentration of oligosaccharides in human
milk. N-glycomics in early bovine lactation was also performed by chemoselective glycoblotting technique and
MALDI-TOFTOF MS analysis [34].
2.4.
Bioinformatic tools
With the development of fast next-gen DNA sequencing

technology, in the last years several genome projects have
been pursuing farm animal sequencing. The genome sequence was completed for chicken, rabbit, cow, sheep and
pig. The sequencing of a number of other farm animals is still
ongoing or under final annotation [35]. Bovine milk is a major
human food and a valuable farm product. Bovine milk protein
sequences in comparison to the milk of other farm animal
species are important to possibly highlight different molecular functions in nutrition and for health.
The inter-specific variability of milk proteome is a key
topic to be defined at the protein sequence level. In fact, in
caseins, inter-specific sequence homology rapidly decreases
with the phylogenetic distance between species [36,37] (Fig. 2).
A recent work from Khaldi [38] investigated with several
sequence alignment tools (BLASTP, TCOFFEE and CLUSTALX)
the changes of the isoelectric point due to aminoacid variations
in nine major milk proteins in 13 mammals. -casein, lactadherin, and muc1 have undergone the highest change in the
isoelectric point during evolution, probably associated with the
adaptive and functional changes.
Milk contains several types of components that provide
many biological activities. Many of these are proteins that:
protect individuals from exogenous stress, toxins, and pathogens; encourage optimal growth, development, and adaptation to a chosen environment; and promote metabolic
regulation for physical and intellectual performance. Most of
the cited properties are accomplished by milk proteins or by
peptides of milk proteins through the mechanism of proteinprotein interaction. For this reason, it is important to evaluate
and to characterize the whole milk proteome as well as the
protein functions and their interaction network. Omicsoriented approaches are providing a much deeper evaluation
of the proteome of milk and its fractions [39]. Most of bioactive
functions of milk are not carried out by milk proteins in their
whole conformation but by peptides belonging to cleavage
processes of their primary structure. The digestion process
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produces several types of bioactive peptides characteristic of

each protein and animal species. To investigate the putative
nutraceutical properties of milk proteins, several methods
have been proposed. As it will be described afterwards, in the
paragraph about bioactive peptides, Minkiwicz and colleagues
developed a database (BIOPEP) with more than 2000 peptides
classified according to their type of bioactivity useful for
discovering potential bioactive peptides/protein fragments
[40]. New approaches in the investigation of milk protein
molecular functions using functional enrichment of gene
ontologies (GOs) are also reported in literature. In a recent
work D'Alessandro reported FatiGO functional enrichment of
gene ontology (GOs) and a hierarchical clustering analysis of
cow's milk proteins [41]. Another strategy to evaluate the
putative functions of milk proteins is to analyze the protein
protein interactions. It is well known that proteins are the
main actors of most cellular activities in a complex synergic
relationship, and functionally similar proteins are often
related in the same molecular clusters [42]. Such a relationship network can be extracted using specific software based
on a semantic search of database information such as
Ingenuity Pathway Analysis (IPA) or STRING. Using IPA,
D'Alessandro and colleagues have built up a preliminary
map of the human [1] and bovine milk [41] proteins
interactome. This approach provided a preliminary important
network of protein interactions of human and bovine milk.
Recently, Lemay and colleagues used bioinformatic approaches
on high coverage genomic data from Bos taurus and they
showed evolutionary insights into the bovine milk genome
and proteome [43]. Moreover Ibeagha-Awemu showed biological processes, functions, pathways, and molecular networks
that were significantly enriched by proteins that emerged
during E. coli or S. aureus mastitis [44]. Currently generation of
high confidence network is possible only in case of the human
and cattle interactome. Unfortunately, it is still difficult, due to
the lack of data, to obtain the same level of confidence of
physical and functional interactions for mare and sow at the
present time. Currently available information does not allow us
to draw milk interactome of goat, sheep and buffalo. However,
an initial interactome of MFGM proteins for sheep was recently
described [15].
Fig. 2 Phylogenetic tree of average distance based of %

homology of primary structure of kappa-casein in human
and farm animals.
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3.
Milk fractions: an overview in intra and inter
specific differences in farm animals
In this section, a focus on each fraction of milk proteome is
summarized. In particular, caseomics, whey proteins, milk fat
globule and peptides are discussed in separated ways and
they are presented in terms of comparison of data in literature
of farm animals.
3.1.
Caseomics
CNs are organized as macromolecular aggregates with minerals (micelles). Their amount is very variable among species
(80% in bovine milk, about 35% in human [45] and 50% in
equine [46,47]. Caseomics studies are important especially for
the implication in dairy industry. An interesting recent paper
from Larsen and coworkers described the potential difficulties
in cheese making related to casein phosphorylation [48].
Authors investigated the causes of non coagulating milk in
cows with proteomics techniques and demonstrating that it
could be due to a low expression of -casein.
Bramanti and colleagues analyzed the different type of
CNs (Fig. 3) and their concentrations in cows, goats, sheep and
buffalo [49]. Miranda and collaborators in 2004 analyzed the
different types of CNs in equine milk [50] concluding that it is
the most similar to human milk, and could be considered a
good substitute of cow's milk for many children with a severe
IgE-mediated cow milk protein allergy.
Caseins (Fig. 4) could be well resolved using 2D electrophoresis [51]. Holland and colleagues in 2004 used 2-DE with
narrow IPGs to analyze bovine milk proteins. Separation and
detection of 10 different -casein forms ranging from isoelectric point values 4.47 to 5.81 was possible with the creation of
a linear immobilized pH gradient (IPG) 47 that was used for
the first dimension [52].
The same method and IPG was used to compare qualitatively and quantitatively analyzed - and -casein in milk
samples from normal and transgenic cattle. This study
demonstrated the possibility to obtain, by a transgenic
approach, a line of cows that is able to produce milk with
increased casein levels [54].
Fig. 3 Casein composition of cow, goat, sheep, buffalo, mare

and human milk samples. From [49,50].
There are several inter-specific differences in the casein

amino acids sequence, the highest similarity has been
observed among ruminants (Table 1).
Caseins family presents a great heterogeneity due to PTMs,
in particular they show a different phosphorylation pattern
on serine/threonine residues. Phosphorylation stoichiometry
of bovine beta-casein and alpha-casein using inductively
coupled plasma mass spectrometry (ICP-MS) was reported by
Ciavardelli [56] and recently Matos and colleagues identified
phosphorylation sites of equine alpha s1 [57] and beta-casein
by nESI-MS/MS [58]. In this case micro-heterogeneity was
evaluated by 2-dimensional electrophoresis and the determination of the different phosphorylation degrees of the native
isoforms of s1-casein was finally achieved by electrospray
ionization mass spectrometry. With this experimental strategy
authors were able to characterize 36 different variants of equine
s1-casein. Phosphorylation level of beta caseins in donkey was
studied by Cunsolo using MALDI-TOF and nESI-MS/MS [59].
Phosphorylation data about alpha s1- and beta-casein were
obtained by MS also for water buffalo by Ferranti [60]. Of all CNs
family, only -CNs are glycosylated. -CNs glycosylation was
described in 2005 by Holland at al. [53] who identified the
different glycosylation patterns of -CNs via 2-DE/MS in bovine
milk. The different isoforms are also visible in Fig. 4.
Caseins show a large inter-specific variability, especially if
considering and -casein fractions [43,61]. The s1-casein of
ovine milk is very heterogeneous, 10 genetic variants have
been identified. The differences in sheep's milk caseins are
not due only to the different characteristics of the isoforms,
but also the concentration of s1-casein varies from 0 to 26% of
total casein and, consequently, the total protein content
varies considerably. This has major effects on the coagulation
properties of sheep's milk and on the type and quality of
cheese produced [6264].
Fig. 4 Bovine master map of 2-DE of bovine milk proteins

[51]. It is possible to see the individual glycoforms of
-casein. The degree of glycosylation of -casein (red
arrows) shifts the isoelectric point from the left (more acidic)
to the right (more basic) [5153].
4265
Human -casein is phosphorylated [65] as mare's casein

[66]. -casein plays a key role in micelle formation, it would be
expected that all milk from different species contain this
protein, but Ochirkhuyag et al. [66] did not identify -casein in
mare's milk suggesting that an orthologous function could be
played by -casein with a low level of phosphorylation. Egito
and collaborators in 2002 using 2-DE-MS showed that the
equine CN isoelectric variants were mostly slightly more
acidic than the bovine CN [67].
The properties of casein micelles have been studied in
caprine [68,69], ovine [69], buffalo [70], camel [71] and mare
[69,72]. Buchheim et al. [73] studied the appearance and the
size of casein micelles discovering that human micelles were
smallest (64 nm) whereas those of goat, camel and donkey
were very large (300500 nm). In 2010 Chianese and colleagues
analyzed all the casein isoforms of donkey's milk using a
proteomic approach. The methodology used was mainly
based on 1-DE and 2-DE that allowed the contemporary
identification of donkey CNs and their related heterogeneity
due to phosphorylation, glycosylation and incorrect splicing
of RNA in mRNA [74].
3.2.
are slow, laborious and are able to analyze only one protein at
time, while proteomic approaches reveal the identifications
and the quantitative analysis of many proteins in one
experiment. Because of hydrophobicity of membrane proteins, it would be better to use SDS-PAGE [79,80]. The loss of
the high resolution provided by 2-DE can be overcome by LCMS/MS. Using this approach Reinhardt and Lippolis identified
up to 120 proteins in cow MFGM. The majority of these
proteins were membrane associated proteins, mainly involved in membrane trafficking or cell signaling [6].
Murgiano and collaborators in 2009 compared the proteome of MFGM from milk samples of individuals belonging to
two different cattle breeds. Authors detected interesting
differences in the amount of proteins linked to mammary
gland development and lipid droplets formation, as well as
host defense mechanisms [81].
A proteomic study on goat MFGM proteome was conducted
by Cebo and collaborators who analyzed the total proteome
and the glycosylation of major proteins [82].
Recently, Pisanu and colleagues mapped the membrane
proteome of sheep's milk fat globule. In this work authors
used a classical SDS-PAGE separation after the MFGM extraction followed by LCMS/MS for protein identification and
characterization [15]. This approach was used to identify in
total 140 unique sheep MFGM proteins. A comparative
analysis of caprine, bovine and human milk fat globules and
their biological activity in a representative model of the
intestinal barrier have been recently obtained by Spertino
and colleagues [83].
Milk fat globules
Milk fat globules are produced by the mammary gland during

lactation [75]. Their structure is formed by a double phospholipid membrane that belongs to lactating cells [75,76]. For this
reason, the proteins present in MFGM could be used for
monitoring the pathophysiological state of the mammary
gland [4].
It has been demonstrated that, depending on the milk
source and its processing, 2570% of the MFGM is formed by
proteins [77]. The composition and function of MFGM proteins
are of high interest because milk fat globule typology and
protein content are different between farm animal species.
Most proteins in the MFGM have been identified using
traditional biochemical approaches [78]. But these methods
3.3.
Whey proteins
Different mammalian species show considerable differences in

protein content. Whey proteins show specific characteristics
which reflect the nutritional or physiological requirements of
the newborn of the different species. Many investigations were
carried out about whey proteins characteristic in human milk,
Table 1 % of the sequence identity of major milk and milk fat globule proteins from human and different farm animals.
Protein name
Homo
sapiens
Bos taurus
(Cow)
Bubalus bubalis
(Buffalo)
Sus scrofa
(Sow)
Capra hircus
(Goat)
Ovis aries
(Sheep)
Equus asinus
(Donkey)
Equus
caballus
(Mare)
as1-casein
as2-casein
b-casein
k-casein
b-lactoglobulin
a-lactalbumin
lactotransferrin
Lactoperoxidase
Osteopontin
Lactadherinb
Lysozyme C
Bile salt
activated lipase
-1-antitrypsin
Serum albumin
100
NC
100
100
44a
100
100
100
100
100
100
100
32
100
56
53
100
73
69
83
62
64
80
NS
33
95
57
54
96
73
70
83
63
ND
82
NC
35
62
59
56
63
76
70
78
69
65
72
NC
33
88
56
52
94
74
70
82
56
64
69
NC
32
89
57
53
93
74
71
83
65
62
70
NC
40
60
57
65
56
75
NC
NC
NC
NC
52
NC
45
57
58
66
59
76
74
86
72
67
50
NC
100
100
68
76
NC
NC
73
76
NC
74
69
75
NC
77
72
76
Protein entries were retrieved from Uniprot (http://www.uniprot.org/) database. All sequences were aligned to human using JalView [55].
Glycodelin (PAEP) in human, b( also Milk fat globule-EGF factor 8). NS: not secreted in milk U: unidentified protein NC: not coded in this
organism.
4266
but few works exist about minor dairy species. The two
principal whey proteins, -Lactalbumin and -Lactoglobulin,
show a high degree of divergence among species. A large
number of papers are published about study of whey proteins
proteomics in human milk, because of implications as hostdefense and immunomodulating factors [84]. Another recent
proteomic study conducted by Tay and colleagues analyzed
simultaneously through 1-DE/MS human, bovine and goat's
milk [14]. Interestingly, authors analyzed the differential
composition of milk through a comparative study design
evaluating the presence of major milk proteins in the three
described samples confirming for example the absence of
lactoferrin in goat's milk in comparison to bovine milk and the
presence of serum albumin, lactoferrin and lysozyme in human
milk in comparison to bovine.
The proteomic whey fraction analysis of milk sample from
donkeys belonging to the Ragusana species of the East of Sicily
was reported in 2007. Authors detected some unknown components, together with the identification of already known whey
proteins, using RP-HPLC/electrospray ionization (ESI)-MS analysis of the whey fraction. Matrix-assisted laser desorption/
ionization (MALDI)-TOF/MS and RP-HPLC/ESI-MS/MS analysis
of the enzymatic digests of the unknown components resulted
in the identification and characterization of two beta-casein
fragments; of the sequence of donkey's serum albumin; and of
the oxidized methionine forms of lysozyme B and alphalactoalbumin [85]. The characterization of donkey's milk protein
fraction was also performed using electrophoretic methods and
mass spectrometric analysis. In this study authors analyzed 51
milk samples demonstrating that donkey's milk proteins
present high phenotypic variability [86].
The content of whey proteins has also been recently analyzed
by reverse-phase high-performance liquid chromatography
coupled with mass spectrometry. Using this approach, the
authors were able to obtain the complete separation of the whey
protein fractions. The adopted RP-HPLC and ESI-MS protocols
provided identification of -lactoglobulin, -lactoalbumin and
serum albumin in Mediterranean water buffalo (Bubalus bubalis)
[87].
Moreover, important inter-species differences in the lessabundant milk proteins have been found: it has been
described that the greatest inter-species differences seem to
occur in the presence/concentration of enzymes [88].
All this evidence shows the usefulness of proteomic
analysis in detection of the inter-intra/specific variability of
whey 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: it

is accessible and it is relatively simple to obtain.
The most used diagnostic strategy for the detection of
bacterial pathologies in bovine milk is the use of PCR [89].
With this method it is possible to detect the sequence of the
genome of a specific bacterial pathogen. PCR-based methods
are currently used for the detection of several pathogens in
animal milk as Mycobacterium avium sub. paratuberculosis [90],
Coxiella burnetii [91], Staphylococcus aureus [92], Mycoplasma

bovis [93] and many others. However, this method is useful
only if the genome of the pathogen has already been
sequenced.
Another method for indirect detection of an etiologic agent
is the research of the specific immunoglobulin in milk, as well
as in serum. Immunoglobulins could be used for the diagnosis
of several animal pathologies i.e. paratuberculosis [94,95] or
the infection with other bacteria [96]. The election method for
the discovery of immunoreactive epitopes is the use of 2D
electrophoresis of the proteins of the etiologic agent immunoblotted against whey or serum of an infected animal. The
discovered immunoreactive proteins could be studied for the
development of an ELISA kit.
One of the most investigated animal pathologies through
milk proteomics is mastitis. Different proteomics approaches
were applied to study mastitis in milk; but aready to use
proteome biomarker in milk is yet far to obtain. Several works
on milk proteomics of cows with mastitis gave a contribute to
the comprehension of biochemical mechanisms of the basis
of inflammation especially for the Acute Phase Proteins (APP)
[97100].
Bovine mastitis is a major disease that causes economic
losses to dairy industry going from decreased milk production
to reproductive disorders in dairy cows. Detection of clinical
mastitis is relatively easy, but subclinical mastitis is difficult
to detect due to the absence of any visible clinical sign. Clear
understanding of the pathogenesis of mastitis is crucial for
the development of adequate tools for mastitis diagnosis.
Currently, mastitis can be monitored by measuring milk
electrical conductivity [101], somatic cell counts (SCCs) [102] or
the enzyme activity of lactate dehydrogenase [103,104].
However, biomarkers which could predict mastitis at earlier
stages are required because all other milk biomarkers are able to
detect mastitis only when it is in the clinical phase and it is too
late to treat animals with antibiotics. There are several
proteomic studies conducted on milk that highlight putative
proteins useful as possible biomarkers for early stage or
subclinical mastitis. In 2004 Hogarth and collaborators analyzed
with a classical proteomic approach both bovine normal and
mastitic whey, reporting an increased concentration of proteins
of blood serum origin as serotransferrin and albumin, while
concentrations of the major whey proteins -lactalbumin and
-lactoglobulin were reduced in mastitic whey [105].
In 2010 Danielsen and colleagues analyzed with a proteomic approach the differential proteome of milk collected after
a lipopolysaccharide-mediated inflammation. Forty-nine differentially expressed proteins were identified including some
interesting proteins like several apolipoproteins and other
anti-inflammatory proteins in milk, which are important for
the cow's ability to balance the immune response. Moreover
authors found an up-regulation of both complement C3 and
C4, which indicates that more than one complement pathway
could be activated during LPS-induced mastitis [106].
Smolenski and colleagues in 2007 used a 2-DE/MS approach to study the milk proteome (both whey and MFGM
proteins) in order to find the proteins involved in host defense
mechanisms [107]. Recently Alonso-Fauste and colleagues
used a proteomic approach for the diagnosis of mastitis. A
proteomic approach was used for the analysis of both serum
and whey proteins and authors obtained encouraging results

analyzing the differential proteomic profile of normal whey in
comparison to mastitic whey. In particular, authors found
several proteins from the somatic cells whose number is
strongly increased in milk in case of an acute phase situation
[20].
Prostaglandin D synthase was described as a putative
biomarker for bovine mastitis diagnosis. In particular, one
isoform was found with a defined cysteine residue that was
oxidized to a sulfonic acid [108].
All the described experimental evidence could be used as
tools to help the diagnosis of bovine mastitis. The presence of
high amounts of somatic cell proteins in the mastitic whey
could be used as an index to evaluate the severity or to detect
mastitis in its subclinical form. Moreover, the described
upregulation of C3 and C4 indicates the immune reaction
against lipopolysaccharide that leads to bacterial opsonization. Upregulation of C3 and C4 is a proof that demonstrates
the presence of bacterial growth in the organism [109].
4.2.
Peptidomics and nutraceutical properties
Milk naturally contains a considerable number of bioactive

compounds as lysozyme, lactoferrin, growth factors, and
hormones, which are directly secreted in their active form by
the mammary gland. Colostrum is rich in nutrients and
provides protection against pathogens thanks to its high
concentration of antimicrobial proteins and, in particular,
immunoglobulins [110,111]. Many milk proteins are precursors of bioactive peptides that are generated by digestive
enzymes and during milk fermentation [112]. Biological
activity can be interpreted as a beneficial or negative
influence on an organism [113,114]. Peptides are considered
bioactive when they possess a hormone, or drug-like, activity
which modulates physiological functions through binding
interaction to specific receptors. Peptides with various bioactivities have been identified in several dairy-products, such
as milk protein hydrolysates, fermented milk and many
cheese varieties [115]. The peptidome is represented by the
entire number of peptides present in food products or raw
materials, or obtained during processing and storage. Peptides
present in several kinds of cheese have already being studied:
4267
Cheddar [116], Parmigiano Reggiano [117,118], Grana Padano

[119], and Emmental [119].
Recently, Panchaud and colleagues described in a review
how novel proteomic techniques together with bioinformatics
could be helpful in finding hidden bioactive peptides in
analyzed proteome [120]. On the same topic, Minkiwicz and
colleagues developed a database (BIOPEP) with more than
2000 peptides classified according to their type of bioactivity
useful to discover potential bioactive peptides/protein fragments [40]. The same database was used by Iwaniak and
Dziuba who used a bioinformatics approach and reported an
interesting study on the bioactivity and the protein structure.
Authors evidenced the structural requirements for peptide(s)
to be regarded as biologically active (bioactive). In particular
the structure and bioactivity analysis revealed that if peptides
encode for a bio-action, it is essential that they assume the
structure of a coil (or combination of coil and a-helix) in the
sequence of their protein precursors [121].
A good description of bioactive proteins and peptides has
been recently done by Nagpal and colleagues [122] who, as
shown in Table 2, described exhaustively all known bioactive
peptides and their original protein.
Table 2 shows how milk protein derived peptides could
have several biological functions such as: opioid activity,
antihypertensive properties, antithrombotic properties, mineral binding properties, immunomodulating activities, antimicrobial properties.
Opioid activity is due to the affinity of these peptides for an
opiate receptor that produces opiate-like effects. Specific receptors are responsible for physiological effects, e.g., the receptor for emotional behavior and a suppression of intestinal
motility, the -receptor for emotional behavior, and the receptor for sedation and food intake. One of the first bioactive
peptides with opioid properties that has been studied is casomorphin. There are several types of casomorphins and
most of them present a high affinity for receptor [135]. Many
other peptides from milk peptides have an opioid agonist
activity as -Lactorphin and -Casein exorphins that respectively belong to -Lactalbumin and s1-Casein. -casoxin
belongs to -casein and has an opioid antagonist function.
Antihypertensive properties are achieved by peptides that
act as Angiotensin-converting enzyme (ACE) inhibitors. There
Table 2 Bioactive peptides derived from milk proteins of several farm animals.
Precursor
-Lactalbumin
-Lactoglobulin
Lactoferrin
-Casein
s1-Casein
k-Casein
Bovine serum albumin
Bioactive peptide
Function
-Lactorphin
-Lactorphin
Lactoferricin
-Casomorphins
-Casokinins
Casein phosphopeptide
-Casein exorphins
-Casokinin
Casoxins
Casoplatelin
Serorphin
Albutensin A
Opioid agonist, ACE-inhibition

Non-opioid stimulatory effect on ileum, ACE-inhibition
Antimicrobial
Opioid agonist, ACE-inhibition, immunomodulation
ACE-inhibition, immunomodulation
Stimulation of mineral absorption
Opioid agonist
ACE inhibition, immunomodulation
Opioid antagonist
Antithrombotic
Opioid
Ileum contraction, ACE-inhibition
References
[123]
[123]
[124]
[125127]
[125,128]
[129]
[129]
[125,130]
[131]
[132]
[133]
[134]
4268
is a lot of experimental evidence that demonstrates how

several tripeptides belonging to milk proteins could carry out
an anti-hypertensive function [136139]. As resumed in
Table 2, almost all milk proteins have bioactive peptides
with ACE inhibitory function in their sequence.
Other functions carried out by milk bioactive peptides are
the immunomodulating and the antimicrobial function.
The immunomodulating function is carried out by
-Casomorphins, -Casokinins and -Casokinin. Even if the
physiological mode of action is not yet known, it has been
demonstrated that they may stimulate the proliferation and
maturation of immune system cells. Synthetic peptides corresponding to fragments of bovine k-casein and -lactalbumin
have been shown to enhance proliferation of human peripheral
blood lymphocytes. These peptides were Tyr-Gly and Tyr-Gly-Gly
belonging from -Casomorphins, -Casokinins showed suppression and stimulation of lymphocyte proliferation depending on
the peptide concentration [140].
The antimicrobial function is mainly carried out by Lactoferricin peptide that pertains to lactoferrin. Bellamy and
colleagues demonstrated how lactoferricin B, a peptide produced by gastric pepsin digestion of bovine lactoferrin, is able to
inhibit growth of several microorganisms as Escherichia coli,
Salmonella enteritidis, Klebsiella pneumoniae, Proteus vulgaris,
Yersinia enterocolitica, Pseudomonas aeruginosa, Campylobacter
jejuni, S. aureus, Streptococcus mutans, Corynebacterium diphtheriae,
Listeria monocytogenes and Clostridium perfringens.
4.3.
Milk adulteration
Milk and dairy products economically driven adulteration

represents a major problem in food production. Usually the
most common adulteration arises from a mixing of high
quality food products with cheaper ingredients. For example,
one of the most common milk adulterations is characterized
by the mixing goat's milk with bovine milk to be directly sold
as entire goat's milk or for goat cheese production. This kind
of adulteration is difficult to detect and usually it is performed
through DNA-based methods like PCR [141143].
Only in the past decade proteomics expanded the objectives to the study of food products in order to detect milk
adulteration.
As suggested by D'Ambrosio and colleagues [144], proteomics could be used for detection of milk adulteration. The
detection of adulteration in buffalo's milk is still based on
1-DE (Italian Gazzetta Ufficiale n.160, 11/07/1994), but it could
be replaced by 2-DE to have a more exhaustive adulteration
analysis. In that article authors analyzed the Italian buffalo
(B. bubalis) whole milk proteome with 2-DE/MS. They analyzed almost all proteins present in the 2D map and their
isoforms through MS giving an exhaustive characterization
of PTM [144].
The collected evidence is useful to prevent adulteration of
buffalo's milk and derived products. This kind of approach
could be applied to milk of all other species in order to better
characterize the inter-specific differences of whole milk
proteome. Using a mass spectrometric approach Cuollo and
colleagues [145] showed the possibility of detecting extraneous milk in single species cheese-milk through the monitoring of casein proteotypic peptides [145].
A proteomic approach has also been applied by Arena and

colleagues to study protein modifications in milk during and
after processing [146,147]. Pinto in a recent work showed that
casein lactosylation is a function of the heating intensity
[148]. Holland described temperature dependent molecular
changes in milk proteins (non-disulfide cross-linking, deamidation and lactosylation) during storage of UHT-treated milk
using 2-DE coupled to MALDI-TOF MS [149].
In conclusion, through a proteomic approach, it is possible
to evaluate the provenience of specific milk and whether it
has been mixed or not with milk from other species. This kind
of adulteration is particularly common for buffalo's milk. A
further potential application carried out by proteomics
analysis consists in the evaluation of proper milk storage
and processing. As previously described, it is possible to
determine, analyzing protein post-translational modifications, the thermal process and the pasteurization process
which has been applied.
4.4.
Dairy products characterization
Raw milk can be processed to obtain a huge variety of related

dairy products. Quality and production in diary industry are
assessed using probiotics and starter bacteria. In particular
lactic acid bacteria (LAB) are used in the dairy industry as starter
cultures for the production of fermented milk products. LAB
produce lactic acid from lactose resulting in acidification of the
substrate, which inhibits pathogen growth [9]. Several microorganisms typical of dairy fermented products have been studied
through proteomics [150,151] such as: Lactococcus lactis, Streptococcus thermophilus, Lactobacillus delbrueckii ssp. lactis, Lactobacillus
acidophilus, and Propionibacterium freudenreichii. Proteomic approaches have also been used to investigate the adaptation of
probiotic lactobacilli, bifidobacteria, and propionibacteria to
digestive stress. In particular, Gagnaire et al. identified bacterial
proteins released after lysis of the microflora in Emmental
cheese, a complex dairy matrix [152].
Cheese manufacturing is often performed through the aid
of chymosin. Traditionally, chymosin is the major enzyme
responsible for the coagulation of milk proteins and it is one
of the main enzymes present in rennet. Chymosin is the
principal protease used for cheese making because it has
highly specific milk-clotting activity relative to its proteolytic
activity and it is specific for the cleavage of -CN. Recently,
Hsieh and colleagues analyzed the coagulation of milk
proteins induced by chymosin through the proteomic profiling. Authors reported an interesting time-course where they
documented the chymosin-related -CN hydrolysis through
1DE and 2DE [48].
The components of cheese are proteins and fat derived
from milk. Different types of cheese are produced from
different milk. They are produced by the coagulation of casein
fraction using specific enzymes. Many chemical and biochemical reactions occur during cheese ripening, and the
proteolytic mechanism is the most important. Proteolysis
contributes to give cheese a typical texture and flavor,
through generation of large polypeptides, and with the
formation of a wide range of intermediate-sized and small
peptides, including free amino acids and their degradation
products [153]. The first proteomics-like studies about
casein proteolysis in cheese date back to the nineties

[117,118,154156]. Peptides derived from casein proteolysis as
a marker of ripening were analyzed in cheeses like Grana
Padano [119,157] and Parmigiano Reggiano [158160] and also
phosphopeptides are well described [156,161].
The complexity of cheese is due to the concomitant
presence of different proteins which are a part of the milk
microbial ecosystem [162]. These different pools of proteins
are drawn from the cheese proteome that is typical and
specific for each kind of cheese [151]. This process completely
depends on the fermentation and transformation processes of
lactic acid bacteria [163,164]. Thus it is important to characterize proteins secreted by bacterial starters in cheese as a
marker of the ripening or fermentation process [165,166].
Moreover, the mandatory evaluation of the safety and
traceability [167,168] of these important dairy products can
be achieved monitoring specific protein or peptide products
[169].
5.
Concluding remarks
A large body of evidence has been collected in recent years in

the development of different proteomics strategies principally
for the analysis of human and bovine milk. The progress
achieved in milk proteomics represents only an initial goal in
the field of biomarker discovery and quality-safety food
related research. Many new opportunities and challenges
remain to be explored in the coming years, especially for new
insights in milk of small ruminants and in general of other
farm animals. Although, it is also true that small dairy species
will never be able to compete with cattle in terms of quantity
and quality of the milk production. The contribution that milk
from other secondary (domesticated) dairy species can give to
the survival and well-being of mankind around the world is
immense and fundamental. Especially when it concerns the
developing countries, the secondary dairy species play a
critical role in supplying the food and nutritional needs of
populations that live in those areas. Furthermore, in developing countries, unavailability of cow's milk, linked to a very low
consumption of meat, represents a huge problem. For this
reason, milk of small dairy species such as goat, buffalo, sheep
and possibly donkey, could well replace daily food sources of
protein, phosphate and calcium for large number of people in
the world. Last, but not least, because of its important and
related hypoallergenic properties, milk from small farm
species such as goat or donkey, but also mare, have been
often recommended as substitutes in diets in cases where of
cow's milk allergies and immunocompromised patients. For
all these aspects, this review describes how proteomics is
central for a better understanding and characterization of
farm animal milk proteins and dairy products. As discussed in
this paper, high throughput and advanced separation
methods now available are able to deeply characterize milk
proteins 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 is
necessary to implement knowledge on milk proteins. As
previously described high resolution 2-DE is useful to obtain
an optimal separation of the milk analysis of protein isoforms.
4269
Such an approach could be useful as a tool to investigate the

differences between milk from different species or lactation
period, as well as for the diagnosis of animal pathologies or for
assessing milk quality. Moreover, a proteomic and peptidomic
investigation could be extremely important to counteract food
adulteration, to develop novel traceability methods and also
to find putative nutraceutical properties of different farm
animal milk and milk products. In particular, bioinformatics
methods could provide the necessary tools to discover
nutraceutical properties of identified proteins and it is able
to predict the possible production of bioactive peptides after
proteolytic cleavages.
In conclusion, milk contains a wide array of proteins that
provide a number of biological activities; the deep knowledge
of farm animal milk proteomics could be useful to answer the
increasing interest of industry in the application of functional
food proteins. Furthermore, the study of milk proteome can
contribute to human and animal welfare thus providing
important elements for improving the milk formula in
nutrition. Finally, the research in milk proteomics of small
dairy species as well, will continue to expand in various
directions in the near future, and many new fascinating
applications and properties will be investigated, in compliance with the sustainable progress era in which we all live.
Acknowledgment
Authors are grateful to the COST ACTION FA1002 Farm
Animal proteomics for the network provided.
REFERENCES
[1] D'Alessandro A, Scaloni A, Zolla L. Human milk proteins: an

interactomics and updated functional overview. J Proteome
Res 2010;9:333973.
[2] Lnnerdal B. Bioactive proteins in human milk: mechanisms
of action. J Pediatr 2010;156:S2630.
[3] O'Donnell R, Holland J, Deeth H, Alewood P. Milk proteomics.
Int Dairy J 2004;14:101323.
[4] Bianchi L, Puglia M, Landi C, Matteoni S, Perini D, Armini A,
et al. Solubilization methods and reference 2-DE map of cow
milk fat globules. J Proteomics 2009;72:85364.
[5] D'Amato A, Bachi A, Fasoli E, Boschetti E, Peltre G, Snchal
H, et al. In-depth exploration of cow's whey proteome via
combinatorial peptide ligand libraries. J Proteome Res
2009;8:392536.
[6] Reinhardt TA, Lippolis J. Bovine milk fat globule membrane
proteome. J Dairy Res 2006;73:406.
[7] Wu W, Wang X, Wu G, Kim S, Chen F, Wang J. Differential
composition of proteomes in sow colostrum and milk from
anterior and posterior mammary glands. J Anim Sci 2010;88:
265764.
[8] Anderson N, Powers M, Tollaksen S. Proteins of human milk.
I. Identification of major components. Clin Chem 1982;28:
104555.
[9] Fox P, Kelly A. Developments in the chemistry and
technology of milk proteins. Food Aust 2003;55:231.
[10] Gagnaire V, Jardin J, Jan G, Lortal S. Proteomics of milk and
bacteria used in fermented dairy products: from qualitative
to quantitative advances. J Dairy Sci 2009;92:81125.
4270
[11] Chianese L, Calabrese MG, Ferranti P, Mauriello R, Garro G,

De Simone C, et al. Proteomic characterization of donkey
milk caseome. J Chromatogr A 2010;1217:483440.
[12] Jovanovic S, Barac M, Macej O, Vucic T, Lacnjevac C.
SDS-PAGE analysis of soluble proteins in reconstituted milk
exposed to different heat treatments. Sensors 2007;7:37183.
[13] Hayes K, Nielsen S. Plasmin levels in fresh milk whey and
commercial whey protein products. J Dairy Sci 2000;83:
38794.
[14] Tay EP, Gam LH. Proteomics of human and the domestic
bovine and caprine milk. Asia-Pac J Mol Biol Biotechnol
2011;19:4553.
[15] Pisanu S, Ghisaura S, Pagnozzi D, Biosa G, Tanca A, Roggio T,
et al. The sheep milk fat globule membrane proteome.
J Proteomics 2011;74:3508.
[16] Roncada P, Gaviraghi A, Liberatori S, Canas B, Bini L, Greppi
GF. Identification of caseins in goat milk. Proteomics 2002;2:
7236.
[17] Claverol S, Burlet-Schiltz O, Gairin JE, Monsarrat B.
Characterization of protein variants and post-translational
modifications: ESI-MSn analyses of intact proteins eluted
from polyacrylamide gels. Mol Cell Proteomics 2003;2:
48393.
[18] Cunsolo V, Cairone E, Fontanini D, Criscione A, Muccilli V,
Saletti R, et al. Sequence determination of s1casein
isoforms from donkey by mass spectrometric methods.
J Mass Spectrom 2009;44:174253.
[19] Holland JW, Deeth HC, Alewood PF. Resolution and
characterisation of multiple isoforms of bovine casein by
2DE following a reversible cysteinetagging enrichment
strategy. Proteomics 2006;6:308795.
[20] Alonso-Fauste I, Andrs M, Iturralde M, Lampreave F, Gallart
J, lava MA. Proteomic characterization by 2-DE in bovine
serum and whey from healthy and mastitis affected farm
animals. J Proteomics 2011 http://dx.doi.org/10.1016/j.jprot.
2011.11.035.
[21] Addis MF, Pisanu S, Ghisaura S, Pagnozzi D, Marogna G,
Tanca A, et al. Proteomics and pathway analyses of the milk
fat globule in sheep naturally infected by Mycoplasma
agalactiae provide indications of the in vivo response of the
mammary epithelium to bacterial infection. Infect Immun
2011;79:383345.
[22] Xia C, Zhang H, Wu L, Xu C, Zheng J, Yan Y, et al. Proteomic
analysis of plasma from cows affected with milk fever using
two-dimensional differential in-gel electrophoresis and
mass spectrometry. Res Vet Sci 2011 http://dx.doi.org/10.
1016/j.rvsc.2011.10.025.
[23] Moll D, Jardin J, Piot M, Pasco M, Lonil J, Gagnaire V.
Comparison of electrospray and matrix-assisted laser
desorption ionization on the same hybrid quadrupole
time-of-flight tandem mass spectrometer: application to
bidimensional liquid chromatography of proteins from
bovine milk fraction. J Chromatogr A 2009;1216:242432.
[24] Affolter M, Grass L, Vanrobaeys F, Casado B, Kussmann M.
Qualitative and quantitative profiling of the bovine milk
fat globule membrane proteome. J Proteomics 2010;73:
107988.
[25] Boehmer JL, DeGrasse JA, McFarland MA, Tall EA, Shefcheck
KJ, Ward JL, et al. The proteomic advantage: label-free
quantification of proteins expressed in bovine milk during
experimentally induced coliform mastitis. Vet Immunol
Immunopathol 2010;138:25266.
[26] Ham JS, Han GS, Jeong SG, Seol KH, Jang AR, Oh MH, et al.
Determination of molecular weights of caprine milk
proteins by matrix-assisted laser desorption/ionization
mass spectrometry. J Dairy Sci 2012;95:159.
[27] Catal-Clariana S, Benavente F, Gimnez E, Barbosa J,
Sanz-Nebot V. Identification of bioactive peptides in
hypoallergenic infant milk formulas by capillary
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
[45]
[46]
electrophoresismass spectrometry. Anal Chim Acta

2010;683:11925.
Somma A, Ferranti P, Addeo F, Mauriello R, Chianese L.
Peptidomic approach based on combined capillary
isoelectric focusing and mass spectrometry for the
characterization of the plasmin primary products from
bovine and water buffalo -casein. J Chromatogr A
2008;1192:294300.
Lecoeur M, Gareil P, Varenne A. Separation and quantitation
of milk whey proteins of close isoelectric points by on-line
capillary isoelectric focusingelectrospray ionization mass
spectrometry in glycerolwater media. J Chromatogr A
2010;1217:7293301.
Mller L, Bartak P, Bedn P, Fryov I, evk J, Lemr K.
Capillary electrophoresismass spectrometrya fast and
reliable tool for the monitoring of milk adulteration.
Electrophoresis 2008;29:208893.
Wilson NL, Robinson LJ, Donnet A, Bovetto L, Packer NH,
Karlsson NG. Glycoproteomics of milk: differences in sugar
epitopes on human and bovine milk fat globule membranes.
J Proteome Res 2008;7:368796.
Tao N, DePeters E, Freeman S, German J, Grimm R, Lebrilla C.
Bovine milk glycome. J Dairy Sci 2008;91:376878.
Nwosu CC, Aldredge DL, Lee H, Lerno LA, Zivkovic AM,
German JB, et al. Comparison of the human and bovine milk
N-glycome via high-performance microfluidic chip liquid
chromatography and tandem mass spectrometry. J Proteome
Res 2012, doi:10.1021/pr300008u.
Takimori S, Shimaoka H, Furukawa JI, Yamashita T, Amano M,
Fujitani N, et al. Alteration of the Nglycome of bovine milk
glycoproteins during early lactation. FEBS J 2011, doi:10.1111/
j.1742-4658.2011.08299.x.
Fadiel A, Anidi I, Eichenbaum KD. Farm animal genomics
and informatics: an update. Nucleic Acids Res 2005;33:
630818.
Cunsolo V, Muccilli V, Saletti R, Foti S. Applications of mass
spectrometry techniques in the investigation of milk
proteome. Eur J Mass Spectrom (Chichester, Eng) 2011;17(4):
30520.
Ginger MR, Grigor MR. Comparative aspects of milk caseins.
Comp Biochem Physiol B Biochem Mol Biol 1999;124:13345.
Nora K, Denis S. Shift in the isoelectric-point of milk
proteins as a consequence of adaptive divergence between
the milks of mammalian species. Biol Direct Jul. 29 2011;6:40.
Casado B, Affolter M, Kussmann M. OMICS-rooted studies of
milk proteins, oligosaccharides and lipids. J Proteomics
2009;73:196208.
Minkiewicz P, Dziuba J, Iwaniak A, Dziuba M, Darewicz M.
Biopep database and other programs for processing
bioactive peptide sequences. J AOAC Int 2008;91:96580.
D'Alessandro A, Zolla L, Scaloni A. The bovine milk
proteome: cherishing, nourishing and fostering molecular
complexity. An interactomics and functional overview.
Mol Biosyst 2011;7:57997.
Barabsi AL, Oltvai ZN. Network biology: understanding the
cell's functional organization. Nat Rev Genet 2004;5:10113.
Lemay DG, Lynn DJ, Martin WF, Neville MC, Casey TM,
Rincon G, et al. The bovine lactation genome: insights
into the evolution of mammalian milk. Genome Biol
2009;10:R43.
Ibeagha-Awemu EM, Ibeagha AE, Messier S, Zhao X.
Proteomics, genomics and pathway analyses of E. coli and S.
aureus infected milk whey reveal molecular pathways and
networks involved in mastitis. J Proteome Res Sep. 3
2010;9(9):460419.
Lnnerdal B. Nutritional and physiologic significance of
human milk proteins. Am J Clin Nutr 2003;77:1537S43S.
Malacarne M, Martuzzi F, Summer A, Mariani P. Protein and
fat composition of mare's milk: some nutritional remarks
[47]
[48]
[49]
[50]
[51]
[52]
[53]
[54]
[55]
[56]
[57]
[58]
[59]
[60]
[61]
[62]
[63]
[64]
[65]
with reference to human and cow's milk. Int Dairy J 2002;12:

86977.
Guo H, Pang K, Zhang X, Zhao L, Chen S, Dong M, et al.
Composition, physiochemical properties, nitrogen fraction
distribution, and amino acid profile of donkey milk. J Dairy
Sci 2007;90:163543.
Frederiksen P, Andersen K, Hammershj M, Poulsen H,
Srensen J, Bakman M, et al. Composition and effect of
blending of noncoagulating, poorly coagulating, and
well-coagulating bovine milk from individual Danish
Holstein cows. J Dairy Sci 2011;94:478799.
Bramanti E, Sortino C, Onor M, Beni F, Raspi G. Separation
and determination of denatured s1-, s2-, - and -caseins
by hydrophobic interaction chromatography in cows', ewes'
and goats' milk, milk mixtures and cheeses. J Chromatogr A
2003;994:5974.
Miranda G, Mah MF, Leroux C, Martin P. Proteomic tools to
characterize the protein fraction of Equidae milk.
Proteomics 2004;4:2496509.
D'auria E, Agostoni C, Giovannini M, Riva E, Zetterstrm R,
Fortin R, et al. Proteomic evaluation of milk from different
mammalian species as a substitute for breast milk. Acta
Paediatr 2005;94:170813.
Holland JW, Deeth HC, Alewood PF. Proteomic analysis of
casein microheterogeneity. Proteomics 2004;4:74352.
Holland JW, Deeth HC, Alewood PF. Analysis of
Oglycosylation site occupancy in bovine casein
glycoforms separated by twodimensional gel
electrophoresis. Proteomics 2005;5:9901002.
Brophy B, Smolenski G, Wheeler T, Wells D, L'Huillier P,
Laible G. Cloned transgenic cattle produce milk with higher
levels of bold beta-casein and kappa-casein. Nat Biotechnol
2003;21:15762.
Waterhouse AM, Procter JB, Martin DMA, Clamp M, Barton
GJ. Jalview Version 2a multiple sequence alignment editor
and analysis workbench. Bioinformatics 2009;25:118991.
Ciavardelli D, Sacchetta P, Federici G, Di Ilio C, Urbani A.
Protein phosphorylation stoichiometry by simultaneous
ICP-QMS determination of phosphorus and sulfur oxide
ions: a multivariate optimization of plasma operating
conditions. Talanta 2010;80:151325.
Matos A, Miclo L, Moll D, Dary A, Girardet JM, Gaillard JL.
Equine [alpha] S1-casein: Characterization of alternative
splicing isoforms and determination of phosphorylation
levels. J Dairy Sci 2009;92:360415.
Matos A, Girardet JM, Moll D, Corbier C, Gaillard JL, Miclo L.
Identification of phosphorylation sites of equine casein
isoforms. Rapid Commun Mass Spectrom 2010;24:153342.
Cunsolo V, Cairone E, Saletti R, Muccilli V, Foti S. Sequence
and phosphorylation level determination of two donkey
caseins by mass spectrometry. Rapid Commun Mass
Spectrom 2009;23:190716.
Ferranti P, Scaloni A, Caira S, Chianese L, Malorni A, Addeo
F. The primary structure of water buffalo s1-and -casein:
identification of phosphorylation sites and characterization
of a novel -casein variant. J Protein Chem 1998;17:
83544.
Fox PF. Advanced dairy chemistry: proteins. Elsevier; 2003.
Amigo L, Recio I, Ramos M. Genetic polymorphism of ovine
milk proteins: its influence on technological properties of
milka review. Int Dairy J 2000;10:13549.
Clark S, Sherbon J. Genetic variants of alphas1-CN in goat
milk: breed distribution and associations with milk
composition and coagulation properties. Small Rumin Res
2000;38:13543.
Greppi G, Roncada P, Fortin R. Protein components of goat's
milk. Dairy goats feeding and nutrition, 2; 2008. p. 7194.
Atkinson SA, Lnnerdal B. Protein and non-protein nitrogen
in human milk. CRC Press; 1989.
4271
[66] Ochirkhuyag B, Chobert JM, Dalgalarrondo M, Haertl T.

Characterization of mare caseins. Identification
of-and-caseins. Lait 2000;80:22335.
[67] Egito A, Miclo L, Lopez C, Adam A, Girardet JM, Gaillard JL.
Separation and characterization of mares' milk s1-, -,
-caseins, -casein-like, and proteose peptone component
5-like peptides. J Dairy Sci 2002;85:697706.
[68] Ono T, Creamer L. Structure of goat casein micelles. N Z J
Dairy Sci Technol 1986;21:5764.
[69] Ono T, Kohno H, Odagiri S, Takagi T. Subunit components of
casein micelles from bovine, ovine, caprine and equine
milks. J Dairy Res 1989;56:618.
[70] Patel R, Mistry V. Physicochemical and structural properties
of ultrafiltered buffalo milk and milk powder1. J Dairy Sci
1997;80:8127.
[71] Attia H, Kherouatou N, Nasri M, Khorchani T.
Characterization of the dromedary milk casein micelle and
study of its changes during acidification. Lait 2000;80:
50315.
[72] Welsch U, Schumacher U, Buchheim W, Schinko I, Jenness
P, Patton S. Histochemical and biochemical observations on
milk-fat-globule membranes from several mammalian
species. Acta Histochem Suppl 1990;40:59.
[73] Buchheim W, Lund S, Scholtissek J. Vergleichende
Untersuchungen zur Struktur und Grsse von
Caseinmicellen in der Milch verschiedener Species. Kieler
Milchw Forsch 1989;41:25365.
[74] Chianese L, Calabrese MG, Ferranti P, Mauriello R, Garro G,
De Simone C, et al. Proteomic characterization of donkey
milk caseome. J Chromatogr A 2010;1217:483440.
[75] Heid HW, Keenan TW. Intracellular origin and secretion of
milk fat globules. Eur J Cell Biol 2005;84:24558.
[76] Mather IH, Keenan TW. Origin and secretion of milk lipids.
J Mammary Gland Biol Neoplasia 1998;3:25973.
[77] Fortunato D, Giuffrida MG, Cavaletto M, Garoffo LP,
Dellavalle G, Napolitano L, et al. Structural proteome of
human colostral fat globule membrane proteins. Proteomics
2003;3:897905.
[78] Mather IH. A review and proposed nomenclature for major
proteins of the milk-fat globule membrane. J Dairy Sci
2000;83:20347.
[79] Gu S, Chen J, Dobos KM, Bradbury EM, Belisle JT, Chen X.
Comprehensive proteomic profiling of the membrane
constituents of a Mycobacterium tuberculosis strain. Mol Cell
Proteomics 2003;2:128496.
[80] Peirce MJ, Wait R, Begum S, Saklatvala J, Cope AP. Expression
profiling of lymphocyte plasma membrane proteins. Mol
Cell Proteomics 2004;3:5665.
[81] Murgiano L, Timperio AM, Zolla L, Bongiorni S, Valentini A,
Pariset L. Comparison of milk fat globule membrane (MFGM)
proteins of Chianina and Holstein cattle breed milk samples
through proteomics methods. Nutrients 2009;1:30215.
[82] Cebo C, Caillat H, Bouvier F, Martin P. Major proteins of the
goat milk fat globule membrane. J Dairy Sci 2010;93:
86876.
[83] Spertino S, Cipriani V, De Angelis C, Giuffrida MG, Marsano
F, Cavaletto M. Proteome profile and biological activity of
caprine, bovine and human milk fat globules. Mol Biosyst
2012;8(4):96774.
[84] Senda A, Fukuda K, Ishii T, Urashima T. Changes in the
bovine whey proteome during the early lactation period.
Anim Sci J 2011;82(5):698706.
[85] Cunsolo V, Saletti R, Muccilli V, Foti S. Characterization of
the protein profile of donkey's milk whey fraction. J Mass
Spectrom 2007;42:116274.
[86] Criscione A, Cunsolo V, Bordonaro S, Guastella AM, Saletti R,
Zuccaro A, et al. Donkeys' milk protein fraction investigated
by electrophoretic methods and mass spectrometric
analysis. Int Dairy J 2009;19:1907.
4272
[87] Buffoni JN, Bonizzi I, Paciullo A, Ramunno L, Feligini M.

Characterization of the major whey proteins from milk of
Mediterranean water buffalo (Bubalus bubalis). Food Chem
2011;127:151520.
[88] Fox P, Kelly A. Indigenous enzymes in milk: overview and
historical aspectsPart 1. Int Dairy J 2006;16:50016.
[89] Lorenz H, Jger C, Willems H, Baljer G. PCR detection of
Coxiella burnetii from different clinical specimens, especially
bovine milk, on the basis of DNA preparation with a silica
matrix. Appl Environ Microbiol 1998;64:42347.
[90] Millar D, Ford J, Sanderson J, Withey S, Tizard M, Doran T,
et al. IS900 PCR to detect Mycobacterium paratuberculosis in
retail supplies of whole pasteurized cows' milk in England
and Wales. Appl Environ Microbiol 1996;62:344652.
[91] Muramatsu Y, Yanase T, Okabayashi T, Ueno H, Morita C.
Detection of Coxiella burnetii in cow's milk by
PCR-enzyme-linked immunosorbent assay combined with a
novel sample preparation method. Appl Environ Microbiol
1997;63:21426.
[92] Kim CH, Khan M, Morin D, Hurley W, Tripathy D, Kehrli M,
et al. Optimization of the PCR for detection of Staphylococcus
aureus nuc gene in bovine milk. J Dairy Sci 2001;84:7483.
[93] Cai HY, Bell-Rogers P, Parker L, Prescott JF. Development of a
real-time PCR for detection of Mycoplasma bovis in bovine
milk and lung samples. J Vet Diagn Invest 2005;17:537.
[94] Salgado M, Kruze J, Collins MT. Diagnosis of
paratuberculosis by fecal culture and ELISA on milk and
serum samples in two types of Chilean dairy goat herds.
J Vet Diagn Invest 2007;19:99.
[95] Cho D, Sung N, Collins MT. Identification of proteins of
potential diagnostic value for bovine paratuberculosis.
Proteomics 2006;6:578594.
[96] Matsushita T, Dinsmore RP, Eberhart RJ, Jones GM,
McDonald JS, Sears PM, et al. Performance studies of an
enzyme-linked immunosorbent assay for detecting
Staphylococcus aureus antibody in bovine milk. J Vet Diagn
Invest 1990;2:163.
[97] Eckersall P, Young F, McComb C, Hogarth C, Safi S,
Fitzpatrick J, et al. Acute phase proteins in serum and milk
from dairy cows with clinical mastitis. Vet Rec 2001;148:
3541.
[98] Grnlund U, Hultn C, Eckersall PD, Hogarth C, Waller KP.
Haptoglobin and serum amyloid A in milk and serum during
acute and chronic experimentally induced Staphylococcus
aureus mastitis. J Dairy Res 2003;70:37986.
[99] Zeng R, Bequette B, Vinyard B, Bannerman D. Determination
of milk and blood concentrations of
lipopolysaccharide-binding protein in cows with naturally
acquired subclinical and clinical mastitis. J Dairy Sci 2009;92:
9809.
[100] Tabrizi AD, Batavani R, Rezaei SA, Ahmadi M. Fibrinogen
and ceruloplasmin in plasma and milk from dairy cows
with subclinical and clinical mastitis. Pak J Biol Sci 2008;11:
571.
[101] De Mol R, Ouweltjes W. Detection model for mastitis in cows
milked in an automatic milking system. Prev Vet Med
2001;49:7182.
[102] Kamphuis C, Sherlock R, Jago J, Mein G, Hogeveen H.
Automatic detection of clinical mastitis is improved by in-line
monitoring of somatic cell count. J Dairy Sci 2008;91:456070.
[103] Chagunda M, Friggens N, Rasmussen MD, Larsen T. A model
for detection of individual cow mastitis based on an
indicator measured in milk. J Dairy Sci 2006;89:298098.
[104] Friggens N, Chagunda M, Bjerring M, Ridder C, Hojsgaard S,
Larsen T. Estimating degree of mastitis from time-series
measurements in milk: a test of a model based on lactate
dehydrogenase measurements. J Dairy Sci 2007;90:
541527.
[105] Hogarth CJ, Fitzpatrick JL, Nolan AM, Young FJ, Pitt A,
Eckersall PD. Differential protein composition of bovine
whey: a comparison of whey from healthy animals and from
those with clinical mastitis. Proteomics 2004;4:2094100.
[106] Danielsen M, Codrea MC, Ingvartsen KL, Friggens NC,
Bendixen E, Rntved CM. Quantitative milk
proteomicshost responses to
lipopolysaccharidemediated inflammation of bovine
mammary gland. Proteomics 2010;10:22409.
[107] Smolenski G, Haines S, Fiona YSK, Bond J, Farr V, Davis SR,
et al. Characterisation of host defence proteins in milk using
a proteomic approach. J Proteome Res 2007;6:20715.
[108] Baeker R, Haebel S, Schlatterer K, Schlatterer B.
Lipocalin-type prostaglandin D synthase in milk: a new
biomarker for bovine mastitis. Prostaglandins Other Lipid
Mediat 2002;67:7588.
[109] Barrio MB, Rainard P, Poutrel B. Milk complement and the
opsonophagocytosis and killing of Staphylococcus aureus
mastitis isolates by bovine neutrophils. Microb Pathog
2003;34:19.
[110] Pakkanen R, Aalto J. Growth factors and antimicrobial
factors of bovine colostrum. Int Dairy J 1997;7:28597.
[111] Chianese L, Caira S, Ferranti P, Laezza P, Malorni A,
Mucchetti G, et al. The oligopeptides of sweet and acid
cheese whey. Lait 1997;77:699715.
[112] Korhonen H, Pihlanto A. Bioactive peptides: production and
functionality. Int Dairy J 2006;16:94560.
[113] Kilara A, Panyam D. Peptides from milk proteins and their
properties. Crit Rev Food Sci Nutr 2003;43:60733.
[114] Fitzgerald RJ, Murray BA. Bioactive peptides and lactic
fermentations. Int J Dairy Technol 2006;59:11825.
[115] Gobbetti M, Stepaniak L, De Angelis M, Corsetti A, Di Cagno
R. Latent bioactive peptides in milk proteins: proteolytic
activation and significance in dairy processing. Crit Rev
Food Sci Nutr 2002;42:22339.
[116] Singh T, Fox P, Hojrup P, Healy A. A scheme for the
fractionation of cheese nitrogen and identification of
principal peptides. Int Dairy J 1994;4:11122.
[117] Addeo F, Chianese L, Sacchi R, Musso SS, Ferranti P, Malorni
A. Characterization of the oligopeptides of
Parmigiano-Reggiano cheese soluble in 120 g trichloroacetic
acid/l. J Dairy Res 1994;61:36574.
[118] Addeo F, Chianese L, Salzano A, Sacchi R, Cappuccio U,
Ferranti P, et al. Characterization of the 12% trichloroacetic
acid-insoluble oligopeptides of Parmigiano-Reggiano
cheese. J Dairy Res 1992;59:40111.
[119] Ferranti P, Itolli E, Barone F, Malorni A, Garro G, Laezza P,
et al. Combined high resolution chromatographic
techniques (FPLC and HPLC) and mass spectrometry-based
identification of peptides and proteins in Grana Padano
cheese. Lait 1997;77:68397.
[120] Panchaud A, Affolter M, Kussmann M. Mass spectrometry
for nutritional peptidomics: how to analyze food bioactives
and their health effects. J Proteomics 2011. http://dx.doi.org/
10.1016/j.jprot.2011.12.022.
[121] Iwaniak A, Dziuba J. BIOPEP-PBIL tool for the analysis of the
structure of biologically active motifs derived from food
proteins. Food Technol Biotechnol 2011;49:11827.
[122] Nagpal R, Behare P, Rana R, Kumar A, Kumar M, Arora S,
et al. Bioactive peptides derived from milk proteins and their
health beneficial potentials: an update. Food Funct 2011;2:
1827.
[123] Antila P, Paakkari I, Jrvinen A, Mattila M, Laukkanen M,
Pihlanto-Leppl A, et al. Opioid peptides derived from
in-vitro proteolysis of bovine whey proteins. Int Dairy J
1991;1:21529.
[124] Bellamy W, Takase M, Wakabayashi H, Kawase K, Tomita M.
Antibacterial spectrum of lactoferricin B, a potent
[125]
[126]
[127]
[128]
[129]
[130]
[131]
[132]
[133]
[134]
[135]
[136]
[137]
[138]
[139]
[140]
[141]
[142]
[143]
[144]
[145]
bactericidal peptide derived from the Nterminal region of

bovine lactoferrin. J Appl Microbiol 1992;73:4729.
FitzGerald RJ, Meisel H. Milk protein-derived peptide
inhibitors of angiotensin-I-converting enzyme. Br J Nutr
2000;84:337.
Pihlanto-Leppl A. Bioactive peptides derived from bovine
whey proteins: opioid and ace-inhibitory peptides. Trends
Food Sci Technol 2000;11:34756.
Gill HS, Doull F, Rutherfurd K, Cross M. Immunoregulatory
peptides in bovine milk. Br J Nutr 2000;84:1117.
Park YW. Overview of bioactive components in milk and
dairy products. Wiley Online Library; 2009.
Hansen M, Sandstrm B, Jensen M, Srensen SS. Casein
phosphopeptides improve zinc and calcium absorption
from rice-based but not from whole-grain infant cereal.
J Pediatr Gastroenterol Nutr 1997;24:56.
Meisel H. Multifunctional peptides encrypted in milk
proteins. Biofactors 2004;21:5561.
Patten GS, Head RJ, Abeywardena MY. Effects of casoxin 4 on
morphine inhibition of small animal intestinal contractility
and gut transit in the mouse. Clin Exp Gastroenterol 2011;4:23.
Phelan M, Kerins D. The potential role of milk-derived
peptides in cardiovascular disease. Food Funct 2011;2:15367.
Nagpal R, Behare P, Rana R, Kumar A, Kumar M, Arora S,
et al. Bioactive peptides derived from milk proteins and their
health beneficial potentials: an update. Food Funct 2011;2:
1827.
Marshall SH, Arenas G. Antimicrobial peptides: a natural
alternative to chemical antibiotics and a potential
for applied biotechnology. Electron J Biotechnol 2003;6:
27184.
Chiba H, Tani F, Yoshikawa M. Opioid antagonist peptides
derived from b-casein. J Dairy Res 1989;56:3636.
Nakamura Y, Yamamoto N, Sakai K, Okubo A, Yamazaki S,
Takano T. Purification and characterization of angiotensin
I-converting enzyme inhibitors from sour milk. J Dairy Sci
1995;78:77783.
Jkl P, Vapaatalo H. Antihypertensive peptides from milk
proteins. Pharmaceuticals 2010;3:25172.
Muro Urista C, lvarez Fernndez R, Riera Rodriguez F,
Arana Cuenca A, Tllez Jurado A. Review: production and
functionality of active peptides from milk. Food Sci Technol
Int 2011;17:293317.
Jauhiainen T, Pilvi T, Cheng ZJ, Kautiainen H, Vapaatalo H,
Korpela R, et al. Milk products containing bioactive
tripeptides have an antihypertensive effect in double
transgenic rats (dTGR) harbouring human renin and human
angiotensinogen genes. J Nutr Metab 2009;2010.
Kayser H, Meisel H. Stimulation of human peripheral blood
lymphocytes by bioactive peptides derived from bovine milk
proteins. FEBS Lett 1996;383:1820.
Adamczyk E. Application of polymerase chain reaction for
detection of goats' milk adulteration by milk of cow. J Dairy
Res 2001;68:3336.
Maudet C, Taberlet P. Detection of cows' milk in goats'
cheeses inferred from mitochondrial DNA polymorphism.
J Dairy Res 2001;68:22935.
Lopez-Calleja I, Gonzalez I, Fajardo V, Rodriguez M,
Hernandez P, Garcia T, et al. Rapid detection of cows' milk in
sheeps' and goats' milk by a species-specific polymerase
chain reaction technique. J Dairy Sci 2004;87:283945.
D'Ambrosio C, Arena S, Salzano AM, Renzone G, Ledda L,
Scaloni A. A proteomic characterization of water buffalo milk
fractions describing PTM of major species and the identification
of minor components involved in nutrient delivery and defense
against pathogens. Proteomics 2008;8:365766.
Cuollo M, Caira S, Fierro O, Pinto G, Picariello G, Addeo F.
Toward milk speciation through the monitoring of casein
[146]
[147]
[148]
[149]
[150]
[151]
[152]
[153]
[154]
[155]
[156]
[157]
[158]
[159]
[160]
[161]
[162]
[163]
4273
proteotypic peptides. Rapid Commun Mass Spectrom

2010;24:168796.
Arena S, Renzone G, Novi G, Paffetti A, Bernardini G,
Santucci A, et al. Modern proteomic methodologies for the
characterization of lactosylation protein targets in milk.
Proteomics 2010;10:341434.
Arena S, Renzone G, Novi G, Scaloni A. Redox proteomics of
fat globules unveils broad protein lactosylation and
compositional changes in milk samples subjected to various
technological procedures. J Proteomics 2011;74:245375.
Pinto G, Caira S, Cuollo M, Fierro O, Nicolai MA, Chianese L,
et al. Lactosylated casein phosphopeptides as specific
indicators of heated milks. Anal Bioanal Chem 2012;402:
196172.
Holland JW, Gupta R, Deeth HC, Alewood PF. Proteomic
analysis of temperature-dependent changes in stored UHT
milk. J Agric Food Chem 2011;59:183746.
Champomier-Verges MC, Maguin E, Mistou MY, Anglade P,
Chich JF. Lactic acid bacteria and proteomics: current
knowledge and perspectives. J Chromatogr B Analyt Technol
Biomed Life Sci 2002;771:32942.
Manso M, Leonil J, Jan G, Gagnaire V. Application of
proteomics to the characterisation of milk and dairy
products. Int Dairy J 2005;15:84555.
Gagnaire V, Piot M, Camier B, Vissers JPC, Jan G, Leonil J.
Survey of bacterial proteins released in cheese: a proteomic
approach. Int J Food Microbiol 2004;94:185201.
Fox PF, McSweeney PLH. Proteolysis in cheese during
ripening. Food Rev Int 1996;12:457509.
Belitz HD, Kaiser KP. Monitoring cheddar cheese ripening by
chemical indices of proteolysis. 3. Identification of several
high-molecular mass peptides. Z Lebensm Unters Forsch
1993;197:11822.
Addeo F, Garro G, Intorcia N, Pellegrino L, Resmini P,
Chianese L. Gel electrophoresis and immunoblotting for the
detection of casein proteolysis in cheese. J Dairy Res 1995;62:
297309.
Ferranti P, Barone F, Chianese L, Addeo F, Scaloni A,
Pellegrino L, et al. Phosphopeptides from Grana Padano
cheese: nature, origin and changes during ripening. J Dairy
Res 1997;64:60115.
Sforza S, Ferroni L, Galaverna G, Dossena A, Marchelli R.
Extraction, semi-quantification, and fast on-line
identification of oligopeptides in Grana Padano cheese by
HPLC-MS. J Agric Food Chem 2003;51:21305.
Gaiaschi A, Beretta B, Poiesi C, Conti A, Giuffrida MG,
Galli CL, et al. Proteolysis of alphas-casein as a
marker of Grana Padano cheese ripening. J Dairy Sci 2000;83:
27339.
Gaiaschi A, Beretta B, Poiesi C, Conti A, Giuffrida MG, Galli
CL, et al. Proteolysis of beta-casein as a marker of Grana
Padano cheese ripening. J Dairy Sci 2001;84:605.
Sforza S, Galaverna G, Dossena A, Marchelli R, Neviani E,
Pinelli C. Study of the oligopeptide fraction in Grana Padano
and Parmigiano-Reggiano cheeses by liquid
chromatography electrospray ionization mass
spectrometry. Eur J Mass Spectrom (Chichester, Eng)
2004;10:4217.
Lund M, Ardo Y. Purification and identification of
water-soluble phosphopeptides from cheese using Fe(III)
affinity chromatography and mass spectrometry. J Agric
Food Chem 2004;52:661622.
Gagnaire V, Piot M, Camier B, Vissers JP, Jan G, Leonil J.
Survey of bacterial proteins released in cheese: a proteomic
approach. Int J Food Microbiol 2004;94:185201.
Pessione A, Lamberti C, Pessione E. Proteomics as a tool for
studying energy metabolism in lactic acid bacteria.
Mol Biosyst 2010;6:141930.
4274
[164] Champomier-Verges MC, Maguin E, Mistou MY, Anglade P,

Chich JF. Lactic acid bacteria and proteomics: current
knowledge and perspectives. J Chromatogr B Analyt Technol
Biomed Life Sci 2002;771:32942.
[165] Herve-Jimenez L, Guillouard I, Guedon E, Gautier C,
Boudebbouze S, Hols P, et al. Physiology of Streptococcus
thermophilus during the late stage of milk fermentation with
special regard to sulfur amino-acid metabolism. Proteomics
2008;8:427386.
[166] Streit F, Delettre J, Corrieu G, Beal C. Acid adaptation of
Lactobacillus delbrueckii subsp. bulgaricus induces
physiological responses at membrane and cytosolic levels
that improves cryotolerance. J Appl Microbiol 2008;105:
107180.
[167] Guarino C, Fuselli F, La Mantia A, Longo L, Faberi A,

Marianella RM. Peptidomic approach, based on liquid
chromatography/electrospray ionization tandem mass
spectrometry, for detecting sheep's milk in goat's and cow's
cheeses. Rapid Commun Mass Spectrom 2010;24:70513.
[168] Saz JM, Marina ML. High performance liquid
chromatography and capillary electrophoresis in the
analysis of soybean proteins and peptides in foodstuffs.
J Sep Sci 2007;30:43151.
[169] Meyer B, AlDiab D, Vollmer G, Pischetsrieder M. Mapping
the glycoxidation product Ncarboxymethyllysine in the
milk proteome. Proteomics 2011;11:4208.

Farm Animal Milk Proteomics

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J O U RN A L OF P ROT EO M I CS 7 5 ( 2 0 12 ) 42 5 9 4 27 4

Available online at www.sciencedirect.com

Farm animal milk proteomics

Istituto Sperimentale Italiano L. Spallanzani, Milano, Italy

Received 25 January 2012

Accepted 16 May 2012

Available online 26 May 2012

functional component repertoire and their investigation represents a major challenge.

This article is part of a Special Issue entitled: Farm animal proteomics.

when the investigation of milk through 2-DE [8] started,

The main protein fraction of milk comprises of caseins with

milk processing necessary to obtain the different milk

For several years, SDS-PAGE and IEF monodimensional

Fig. 1 Milk prefractionation steps and proteomic experimental strategies.

proteomic analysis has to be done on membrane and

Mass spectrometry investigations in milk samples analysis is

focusing [29] coupled to mass spectrometry were used in

With the development of fast next-gen DNA sequencing

produces several types of bioactive peptides characteristic of

Fig. 2 Phylogenetic tree of average distance based of %

Fig. 3 Casein composition of cow, goat, sheep, buffalo, mare

There are several inter-specific differences in the casein

Fig. 4 Bovine master map of 2-DE of bovine milk proteins

Human -casein is phosphorylated [65] as mare's casein

Milk fat globules

Milk fat globules are produced by the mammary gland during

Different mammalian species show considerable differences in

Proteomic tools in milk safety and quality

Milk as a diagnostic fluid

Milk represents a basic biological fluid useful for diagnosis: it

Coxiella burnetii [91], Staphylococcus aureus [92], Mycoplasma

and whey proteins and authors obtained encouraging results

Peptidomics and nutraceutical properties

Milk naturally contains a considerable number of bioactive

Cheddar [116], Parmigiano Reggiano [117,118], Grana Padano

Opioid agonist, ACE-inhibition

is a lot of experimental evidence that demonstrates how

Milk and dairy products economically driven adulteration

A proteomic approach has also been applied by Arena and

Dairy products characterization

Raw milk can be processed to obtain a huge variety of related

casein proteolysis in cheese date back to the nineties

A large body of evidence has been collected in recent years in

Such an approach could be useful as a tool to investigate the

[1] D'Alessandro A, Scaloni A, Zolla L. Human milk proteins: an

[11] Chianese L, Calabrese MG, Ferranti P, Mauriello R, Garro G,

electrophoresismass spectrometry. Anal Chim Acta

with reference to human and cow's milk. Int Dairy J 2002;12:

[66] Ochirkhuyag B, Chobert JM, Dalgalarrondo M, Haertl T.

[87] Buffoni JN, Bonizzi I, Paciullo A, Ramunno L, Feligini M.

bactericidal peptide derived from the Nterminal region of

proteotypic peptides. Rapid Commun Mass Spectrom

[164] Champomier-Verges MC, Maguin E, Mistou MY, Anglade P,

[167] Guarino C, Fuselli F, La Mantia A, Longo L, Faberi A,

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