**3. Proteomic research in caprine milk**

Milk is composed of three main components: casein, whey proteins, and milk fat globule membrane (MFGM). Using differential centrifugation and ultracentrifugation, it is possible to isolate these three fractions. While the application of proteomics in the milk of larger animals has attracted many research groups, the role of proteomics in the milk of goat remained limited. In recent years, with the advancements of available technologies in proteomics, there has been a growing interest to unravel the dynamic structure of goat milk protein contents. Proteomic research in caprine milk has been applied in the areas of identifying major proteins, comprehensive analysis, MFGM, and identification of PTM (**Table 1**).


**3.1. Identifying major proteins**

assay; MRM, multiple-reaction-monitoring.

**Table 1.** Proteomic investigations of caprine milk.

1

Affinity

chromatography & nLC-Chip-QTOF-MS

protein in many ruminants including goat.

components can be detected.

One of the first proteomic studies in goat milk was reported by Roncada et al., where they used two dimensional electrophoresis (2-DE) combined with MALDI-TOF and ESI-ion trap mass spectrometers to analyze αS1-casein alleles. They determined casein polymorphisms as the key characteristics in the cheese manufacturing industry [24]. Major goat milk proteins including caseins (αS1, αS2, β-, and κ-casein) and some of the whey proteins (albumin, lactoferrin, β-lactoglobulin, and α-lactalbumin) are highly abundant and have been widely studied [8, 25, 37]. Several groups compared the data from proteomics studies of goat milk with those of cow and other species, but their reports involved only the characterization of major proteins [27, 36, 41]. In one of 2-DE analyses [36], highly abundant proteins in each animal species display their own unique pattern [36]. This group further highlighted significant interspecies differences in milk from different ruminants and identified β-lactoglobulin as the major whey

nLC-MS/MS MFGM N-glycosylation comparison in

LC-MS/MS MFGM Phosphoproteome analysis [48]

Literature references of each study: 2-DE, two-dimensional; 1-DE, one-dimensional; nLC, nano liquid chromatography; UPLC, ultra-performance liquid chromatography; SCX, strong cation exchange; CPLL, combinatorial peptide ligand libraries; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; ELISA, enzyme-linked immunosorbent

**Techniques Matrix Proteomic study References1**

Skim milk Lactoferrin N-glycans in

phosphoproteome

human and bovine

mammals

[29]

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[47]

[46]

PTM 1DE & nLC-MS/MS Skim milk Comprehensive caseins

The use of goat milk as a substitute for cow milk for allergic people has been also recently reported [3, 10]. In some parts of the world, milk of donkey or goat is used in newborn and infant feeding because they are less allergenic than cow milk. Accordingly, Di Girolamo et al. used fingerprinting of major milk proteins by MALDI-TOF MS coupled to a robust statistical analysis to determine adulteration and unintended contamination of donkey milk and goat milk [26]. Peptide mass fingerprinting is a simple methodology in proteomics where proteins are cleaved with a protease, such as trypsin. The identification is accomplished by matching the observed peptide masses from MS data to the theoretical masses derived from a sequence database [49]. Comparative proteomic approaches were used to study colostrum and milk of goats, cows, and sheep to determine chemical composition and immunoglobulin concentration [39]. The study revealed that despite the similar immunoglobulin concentrations in colostrum and milk from the three studied species, differences in several immune


1 Literature references of each study: 2-DE, two-dimensional; 1-DE, one-dimensional; nLC, nano liquid chromatography; UPLC, ultra-performance liquid chromatography; SCX, strong cation exchange; CPLL, combinatorial peptide ligand libraries; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; ELISA, enzyme-linked immunosorbent assay; MRM, multiple-reaction-monitoring.

**Table 1.** Proteomic investigations of caprine milk.

#### **3.1. Identifying major proteins**

**Techniques Matrix Proteomic study References1**

adulteration

proteins

LPS induced

Brazilian breeds

allergic properties

proteins/adulteration

quantification

quantification

abundance proteins

(MRM) Whey powder

Milk Comprehensive of low

Milk Comparison study sheep and goat

composition

biological activity

proteins

[35]

[36]

[28]

[37]

[3]

[26]

[38]

[39]

[40]

[41]

[6]

[42]

[8]

[44]

Major proteins 2-DE & MALDI-TOF Milk Casein profile [24]

nLC-MS/MS Cheese Comparative proteomics/

2-DE & MALDI-TOF Skim milk Farm animals principle

2-DE & nLC-MS/MS Skim milk Comparison of healthy and

1DE/2-DE/HPLC Skim milk Comparative proteomics

MALDI-TOF Skim milk Anti-inflammatory/anti-

MALDI-TOF-MS Skim milk Fingerprinting of principle

MALDI-TOF-MS Skim milk Fingerprinting of major

UPLC-XevoTQS Milk powder Absolute quantification

Comprehensive SCX & nLC-MS/MS Whey Farm animals comparative &

MGFM 1D SDS/PAGE MFGM Protein composition of MFGM [43]

1DE &MALD-TOF MFGM Assessment of protein

LC-MS/MS MFGM Proteome profile and

nLC-MS/MS MFGM Colostrum & mature milk [45]

2-DE/nLC-MS/MS MFGM Quantification of mammalian [46]

CPLL, 1DE & nLC-MS/MS

148 Goat Science

2-DE & MALDI-TOF & LC-MS/MS

2-DE/ELISA Colostrum/skim milk IgG/IgM bindings profile &

1-DE & LC-MS/MS Skim milk Comparative proteomics [27]

MALDI-TOF Raw milk Comparative proteomics [25]

One of the first proteomic studies in goat milk was reported by Roncada et al., where they used two dimensional electrophoresis (2-DE) combined with MALDI-TOF and ESI-ion trap mass spectrometers to analyze αS1-casein alleles. They determined casein polymorphisms as the key characteristics in the cheese manufacturing industry [24]. Major goat milk proteins including caseins (αS1, αS2, β-, and κ-casein) and some of the whey proteins (albumin, lactoferrin, β-lactoglobulin, and α-lactalbumin) are highly abundant and have been widely studied [8, 25, 37]. Several groups compared the data from proteomics studies of goat milk with those of cow and other species, but their reports involved only the characterization of major proteins [27, 36, 41]. In one of 2-DE analyses [36], highly abundant proteins in each animal species display their own unique pattern [36]. This group further highlighted significant interspecies differences in milk from different ruminants and identified β-lactoglobulin as the major whey protein in many ruminants including goat.

The use of goat milk as a substitute for cow milk for allergic people has been also recently reported [3, 10]. In some parts of the world, milk of donkey or goat is used in newborn and infant feeding because they are less allergenic than cow milk. Accordingly, Di Girolamo et al. used fingerprinting of major milk proteins by MALDI-TOF MS coupled to a robust statistical analysis to determine adulteration and unintended contamination of donkey milk and goat milk [26]. Peptide mass fingerprinting is a simple methodology in proteomics where proteins are cleaved with a protease, such as trypsin. The identification is accomplished by matching the observed peptide masses from MS data to the theoretical masses derived from a sequence database [49]. Comparative proteomic approaches were used to study colostrum and milk of goats, cows, and sheep to determine chemical composition and immunoglobulin concentration [39]. The study revealed that despite the similar immunoglobulin concentrations in colostrum and milk from the three studied species, differences in several immune components can be detected.

Proteomics is instrumental in detecting milk adulteration as adulteration has been a significant problem in the dairy industry. In a comparative proteomic study, different cheese samples obtained from milk of cow, sheep, and goat were analyzed using HPLC-chip-MS/ MS [35]. The authors found κ-casein to have a unique primary structure and suggested that it could be used to determine the origin of milk in different cheese samples. In another study, a MALDI-TOF-MS platform was used to profile milk samples for the rapid detection of illegal adulterations caused by the addition of either nondeclared cow milk, milk of other species, or the addition of powdered milk to the fresh counterpart [38]. For this purpose, peptide and protein markers of cow, water buffalo, goat, and sheep milk were identified and the effects of thermal treatment-associated adulterated milk samples were evaluated. This study introduced an independent, complementary peptide profiling measurement and extended proteomic approaches to the analysis of thermal treatment. Yang et al. analyzed milk whey samples obtained from a number of species including goat, cow, buffalo, yak, and camel. They detected certain proteins as the characterizing traits for a given species that could be used to evaluate adulteration [41].

when cow milk was added to sheep or goat milk [40]. In this study, two peptides derived from β-lactoglobulin were chosen as the protein markers. Similar isotopically labeled peptides as internal standards were designed and synthesized to minimize the matrix effect, which led to more accurate quantification results. MRM has been used for many years to measure and quantify small molecules, drugs, and metabolites. However, the application of MRM to obtain absolute quantitation of proteins is relatively new and offers great potential

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Goat milk is a great source of essential fatty acids, which are concentrated in MFGM along with a complex mixture of proteins, glycoproteins, and enzymes [52]. Although MFGM proteins account for a small fraction of the total milk protein, they have been extensively studied in human and bovine milk. In previous bovine MFGM studies, many health benefits including anticancer, antimicrobial, and antiviral effects have been attributed to the glycoproteins [53].

The protein composition of goat MFGM has been described by several groups [8, 43, 44, 53]. Cebo et al. used 1DE and MALDI-TOF-MS and reported butyrophilin, lactadherin, mucin, and lectin as major MFGM proteins in goat milk. Interestingly, lactadherin from goat milk consisted of a single polypeptide chain whereas 2 polypeptide chains were detected in bovine milk [8]. In addition, the MFGM of colostrum and mature milk was also investigated [45]. As expected, the acute phase proteins were higher in colostrum MFGM, signifying the impor-

In a more recent 2016 study by Yang et al., the N-glycoproteome of MFGMs, obtained from a group of mammals' milk including human, Holstein, Jersey, buffalo, yak, camel, horse, and goat was investigated. They found that protein components of MFGM fractions from ruminants were more similar to each other when compared to nonruminants [46]. In a comprehensive analysis of goat milk MFGM, Henry et al. reported the use of high-resolution LC-MS/MS to expand the MFGM proteome in goat milk to 442 functional groups [48]. The main focus of their study was to probe the phosphoproteome of goat MFGM that will be

PTMs are chemical modifications that play a key role in functional proteomics. The characterization of PTMs, although challenging, is very important as they regulate protein function and control numerous important biological processes. A large number of different PTMs have been reported, but by far, phosphorylation and glycosylation are the most important and well-studied [54]. Since phosphorylation has a low stoichiometry, there is a need for enrichment of phosphopeptides in an analysis of a complex mixture. The current method to enrich phosphopeptides is based on affinity purification using phosphospecific antibodies immobilized-metal affinity chromatography (IMAC). Zhong et al. optimized the selective isolation of mono- and multi-phosphorylated peptides by using different forms of iron ions [55].

Therefore, there is a need to characterize this important component in goat milk.

tance of colostrum intake to the immune system of newborns.

to the field.

**3.3. Analysis of MFGM**

covered in the next section.

**3.4. Post-translational modifications (PTM)**

#### **3.2. Comprehensive analysis**

Proteomic investigation is challenging due to the wide dynamic range of protein expression where the presence of high abundance protein masks or prevents the detection of low abundance proteins. Recently, the most comprehensive proteomic dataset of goat milk has been reported by Cunsolo et al. [6]. This group fractionated the total milk samples using combinatorial hexapeptide ligand libraries (CPLL; such as ProteoMiner) at different pH levels to reduce the dynamic range of protein concentrations. They identified 452 unique gene products including many low abundance proteins in goat milk. Their success was also related to the use of further fractionation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), high resolution, nLC-MS/MS, and an advanced bioinformatics platform [6]. New strategies employing multi-enzyme digestion coupled with CID and ETD for protein sequencing and characterization was also used to increase sequence coverage and localization of PTMs [50]. In a separate study, using two complementary proteomic approaches, Anagnostopoulos et al. investigated the milk whey of three Greek sheep and goat breeds. They identified about 600 protein groups, most of which were involved in nutrient transport and immune system responses [42]. These findings provide the most comprehensive description of the goat milk proteome that can be utilized to build a goat protein sequence database. As it was pointed out by Soares et al. the success of proteomic-based investigations largely depend on the availability of complete and annotated databases containing the gene and protein sequence information for different animal species [51]. Hence, the data generated from these recent, more comprehensive proteomics analysis of the goat milk proteome could facilitate the detection and characterization of additional milk proteins in future proteomics analyses of goat milk.

While proteomic analyses have been used to qualitatively identify adulterants in milk, no reliable, selective, and sensitive method existed to obtain absolute quantification. In a clever analysis, Chen et al. used multiple reaction monitoring (MRM) to quantify milk adulteration when cow milk was added to sheep or goat milk [40]. In this study, two peptides derived from β-lactoglobulin were chosen as the protein markers. Similar isotopically labeled peptides as internal standards were designed and synthesized to minimize the matrix effect, which led to more accurate quantification results. MRM has been used for many years to measure and quantify small molecules, drugs, and metabolites. However, the application of MRM to obtain absolute quantitation of proteins is relatively new and offers great potential to the field.

#### **3.3. Analysis of MFGM**

Proteomics is instrumental in detecting milk adulteration as adulteration has been a significant problem in the dairy industry. In a comparative proteomic study, different cheese samples obtained from milk of cow, sheep, and goat were analyzed using HPLC-chip-MS/ MS [35]. The authors found κ-casein to have a unique primary structure and suggested that it could be used to determine the origin of milk in different cheese samples. In another study, a MALDI-TOF-MS platform was used to profile milk samples for the rapid detection of illegal adulterations caused by the addition of either nondeclared cow milk, milk of other species, or the addition of powdered milk to the fresh counterpart [38]. For this purpose, peptide and protein markers of cow, water buffalo, goat, and sheep milk were identified and the effects of thermal treatment-associated adulterated milk samples were evaluated. This study introduced an independent, complementary peptide profiling measurement and extended proteomic approaches to the analysis of thermal treatment. Yang et al. analyzed milk whey samples obtained from a number of species including goat, cow, buffalo, yak, and camel. They detected certain proteins as the characterizing traits for a given species that could be

Proteomic investigation is challenging due to the wide dynamic range of protein expression where the presence of high abundance protein masks or prevents the detection of low abundance proteins. Recently, the most comprehensive proteomic dataset of goat milk has been reported by Cunsolo et al. [6]. This group fractionated the total milk samples using combinatorial hexapeptide ligand libraries (CPLL; such as ProteoMiner) at different pH levels to reduce the dynamic range of protein concentrations. They identified 452 unique gene products including many low abundance proteins in goat milk. Their success was also related to the use of further fractionation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), high resolution, nLC-MS/MS, and an advanced bioinformatics platform [6]. New strategies employing multi-enzyme digestion coupled with CID and ETD for protein sequencing and characterization was also used to increase sequence coverage and localization of PTMs [50]. In a separate study, using two complementary proteomic approaches, Anagnostopoulos et al. investigated the milk whey of three Greek sheep and goat breeds. They identified about 600 protein groups, most of which were involved in nutrient transport and immune system responses [42]. These findings provide the most comprehensive description of the goat milk proteome that can be utilized to build a goat protein sequence database. As it was pointed out by Soares et al. the success of proteomic-based investigations largely depend on the availability of complete and annotated databases containing the gene and protein sequence information for different animal species [51]. Hence, the data generated from these recent, more comprehensive proteomics analysis of the goat milk proteome could facilitate the detection and characterization of additional milk proteins in future proteomics

While proteomic analyses have been used to qualitatively identify adulterants in milk, no reliable, selective, and sensitive method existed to obtain absolute quantification. In a clever analysis, Chen et al. used multiple reaction monitoring (MRM) to quantify milk adulteration

used to evaluate adulteration [41].

**3.2. Comprehensive analysis**

150 Goat Science

analyses of goat milk.

Goat milk is a great source of essential fatty acids, which are concentrated in MFGM along with a complex mixture of proteins, glycoproteins, and enzymes [52]. Although MFGM proteins account for a small fraction of the total milk protein, they have been extensively studied in human and bovine milk. In previous bovine MFGM studies, many health benefits including anticancer, antimicrobial, and antiviral effects have been attributed to the glycoproteins [53]. Therefore, there is a need to characterize this important component in goat milk.

The protein composition of goat MFGM has been described by several groups [8, 43, 44, 53]. Cebo et al. used 1DE and MALDI-TOF-MS and reported butyrophilin, lactadherin, mucin, and lectin as major MFGM proteins in goat milk. Interestingly, lactadherin from goat milk consisted of a single polypeptide chain whereas 2 polypeptide chains were detected in bovine milk [8]. In addition, the MFGM of colostrum and mature milk was also investigated [45]. As expected, the acute phase proteins were higher in colostrum MFGM, signifying the importance of colostrum intake to the immune system of newborns.

In a more recent 2016 study by Yang et al., the N-glycoproteome of MFGMs, obtained from a group of mammals' milk including human, Holstein, Jersey, buffalo, yak, camel, horse, and goat was investigated. They found that protein components of MFGM fractions from ruminants were more similar to each other when compared to nonruminants [46]. In a comprehensive analysis of goat milk MFGM, Henry et al. reported the use of high-resolution LC-MS/MS to expand the MFGM proteome in goat milk to 442 functional groups [48]. The main focus of their study was to probe the phosphoproteome of goat MFGM that will be covered in the next section.

#### **3.4. Post-translational modifications (PTM)**

PTMs are chemical modifications that play a key role in functional proteomics. The characterization of PTMs, although challenging, is very important as they regulate protein function and control numerous important biological processes. A large number of different PTMs have been reported, but by far, phosphorylation and glycosylation are the most important and well-studied [54]. Since phosphorylation has a low stoichiometry, there is a need for enrichment of phosphopeptides in an analysis of a complex mixture. The current method to enrich phosphopeptides is based on affinity purification using phosphospecific antibodies immobilized-metal affinity chromatography (IMAC). Zhong et al. optimized the selective isolation of mono- and multi-phosphorylated peptides by using different forms of iron ions [55]. In this study, they selected α-casein and two synthesized mono- and di-phosphopeptides as a model system to demonstrate that NiZnFe<sup>2</sup> O4 was highly selective for multi-phosphopeptides whereas Fe3 O4 , NiFe2 O4 , and ZnFe<sup>2</sup> O4 had a higher affinity for mono-phosphopeptides. Along with the improvements of IMAC for phosphoproteomic experiments, instrument enhancements including improved acquisition speed allowed the identification of many more phosphopeptides per analysis. As mentioned before, in a comprehensive analysis of goat milk MFGM phosphoproteome, Henry et al. used TiO2 for enrichment of the MFGM samples. Using nLC-MS/MS and high resolution mass spectrometer, they characterized the phosphorylation of several key mammary gland proteins in goat MFGM. This group, leveraging the strengths of high resolution and faster acquisition time, reported the detection of 271 sites of phosphorylation on 124 unique goat MFGM proteins [48].

bovine milk proteome, only a limited understanding of the goat innate immune response to mastitis pathogens or the subsequent changes in goat milk protein expression over the course of a clinical infection exists. Nonetheless, like other ruminants that are managed for milk pro-

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Soluble mediators of inflammation in bovine milk and plasma during clinical mastitis have been studied extensively using antibody-based strategies [59]. Although antibody-based methodologies are both quantitative and accurate, they have limited detection capabilities. Conversely, mass spectrometric-based proteomic technologies allow for the simultaneous analysis of a larger number of proteins without the reliance on antibodies. Using MS-based proteomics, a number of biomarkers including APPs were identified in bovine serum and milk, which were correlated with pain and disease status [19, 60]. The concentration of most APPs typically increased during infection or inflammation, and the increased levels were relatively stable and persisted for a number of days, or even weeks, after the original insult or stimulus [19]. Despite the fact that our knowledge of the modulation of the bovine milk proteome during mastitis continues to expand, very little comparative data exists on lactat-

In regards to the study of the goat milk proteome, our group detected increases in haptoglobin (Hp), serum amyloid A (SAA), and lactoferrin in the milk of goats following an intramammary infusion of lipopolysaccharide (LPS) to induce coliform mastitis [28]. Other studies also documented significantly increased blood levels of Hp and SAA in an experimentally induced subacute ruminal acidosis in goats [61]. The majority of APPs are known to be glycosylated. Due to the high extent of its carbohydrate moiety, the APP alpha-1-acid glycoprotein (AGP) has been established as a biomarker of inflammation in goats [62]. AGP was also reported to potentially inhibit neutrophil migration to the site of infection, leading to inadequate bacterial clearance and resulting in increased risk of mortality [63]. Further, Heller et al. determined the species-specific reference intervals for four APPs including Hp, SAA, AGP, and lipopolysaccharide-binding protein (LBP), which is a soluble polypeptide that binds to bacterial LPS and increases its proinflammatory activity up to 1000 fold, in goat

Modulations in the expression of goat milk proteins have been examined following an experimental induction of endotoxin mastitis by intra-mammary infusion with LPS. For details of challenge study and sample preparation, see materials and methods section in Ref. [28]. Crude milk samples were separated by 2DE prior to nLC-MS/MS analysis. The unique proteins identified following the 2DE analysis of skim milk from healthy goats and skim milk collected from the same goats 18 h post infusion with LPS are summarized in **Table 2**. In the absence of goat specific database, we used the Swiss-Prot other mammalia taxonomy, which includes only a limited number of goat sequences. Though some goat specific proteins were identified, the majority of the protein identifications were assigned to other species. As shown in this table, caseins constitute the most abundant proteins in milk; thus, a marked number of casein variants, specifically β- and αS2-caseins, which were detected in 13 and 6 separate

**4.1. Effects of experimentally-induced mastitis on the goat proteome**

duction, goats are also susceptible to and affected by mastitis.

ing dairy goats.

milk [64, 65].

The identification of PTMs is especially useful for the detection and characterization of acute phase proteins (APPs) during disease because APPs are subjected to modification. The N-glycan profiles of goat milk lactoferrin were compared with human and bovine milk using advanced mass spectrometry techniques [47]. The characterization of glycan composition established high mannose, hybrid, and complex N-glycans. Among the N-glycan compositions, 37% were sialylated and 34% were fucosylated. This group highlighted the existence of similar glycan composition between human and goat milk and discovered a novel glycan in goat milk that was not detected in human milk. A recent 2016 study investigated N-glycoproteome analysis of MFGMs from a number of mammals' milk [46]. They observed different glycosylation patterns in certain proteins that were previously reported with varying molecular weights based on the analysis by SDS-PAGE. They concluded that these discrepancies were the result of the differences in carbohydrate content of these proteins.
