**2. Proteomics approaches**

Goat milk is an excellent source of macro- and micro-nutrients, and proteins that are more easily digested, presumably due to the higher essential fatty acid contents [6]. It is also less allergenic than cow milk, as it was shown to help children at risk of food allergies [7]. While not very popular in the United States, in developing countries where cow milk is not readily available or affordable, goat milk accounts for more than 50% of milk production [8]. In Europe, goat milk is processed mostly for cheese manufacturing [9]. Considering the economic importance in developing countries and the inherent health benefits, goat milk might

Proteins are the key components of milk with many diverse cellular functions. For example, casein micelles provide essential amino acids that are vital for energy, tissue growth, and cellular function. In addition, some proteins can act as hormones, whereas others display antimicrobial properties. Proteomics is the large-scale study of the protein contents of cells and tissues [11]. One of the most promising outcomes of proteome analysis is the discovery of protein biomarkers, which are specific proteins or protein isoforms, whose expression levels change significantly during disease conditions [12]. The identification of these biomarkers in accessible body fluids such as milk could eventually enable farmers and veterinarians to

Mastitis, the focus of several prior veterinary proteomics studies, is defined as inflammation of the breast or udder tissues that is typically caused by invading bacteria. The first proteomic investigation of bovine milk mastitis was conducted by Baeker et al., where they used a comparative proteomics to identify expressed proteins in normal and mastitic bovine milk [13]. This was followed by several other research investigations to identify differentially expressed proteins in cow milk, either following experimentally induced infection or during naturally occurring mastitis [14–16]. Quantification of expressed proteins in clinically healthy cows and cows with experimentally-induced coliform mastitis was also reported using both liquid chromatography tandem-mass spectrometry (LC-MS/MS)-based label-free approach [17] and isobaric peptide tags for relative and absolute quantification (iTRAQ) [18]. As a result, biochemical mechanisms and inflammatory-related biomarkers, especially acute phase proteins

Like other ruminant species that are managed for milk production, goats are also affected by mastitis; however, much less is known about the goat innate immune response to mastitis pathogens or about subsequent changes in goat milk protein expression over the course of a clinical infection. Although some of the apparent clinical signs of mastitis in goats, including udder swelling and redness, increased rectal temperature, and changes in the appearance of the milk are similar to those observed in dairy cattle, limited knowledge of the host response during mastitis in goats exists. Nonetheless, other hallmarks of clinical mastitis in dairy cattle include elevated milk somatic cell counts (SCC), reduced appetite, reduced milk production,

Initial reports of proteomic evaluations of goat milk were limited to the analyses of casein fractions and the determination of the molecular weights of major whey proteins [8, 24]. Later attempts to identify differentially expressed proteins in goat milk employed gel-based assays followed by enzymatic digestion of isolated proteins and matrix-assisted laser desorption/ionization

be a reliable alternative, if not a replacement, for cow milk [10].

monitor diseases and expand treatment options.

increased heart rate, and changes in blood chemistry [22, 23].

(APPs), were identified [19–21].

144 Goat Science

The proteomic field is divided into two main analytical methodologies: the top-down and the bottom-up. The top-down approach relies on the analysis of intact proteins and corresponding fragmentation within the MS. In contrast, the bottom-up approach, which has been increasingly adopted by the proteomics community, proceeds through analysis of peptides that are generated outside the MS. The identified peptide sequences are then reassigned to the proteins they originate from, through database searching. To reduce sample complexities, a protein mixture can also be fractionated by gel-based approaches prior to MS analysis. The spots from the gel are excised and are subjected to an in-gel digestion. In the following sections, gel electrophoresis, MS, and database searching will be briefly discussed.

#### **2.1. Electrophoresis**

Classical proteomic approaches take advantage of protein fractionation by using gel-based assays. 1D-SDS/PAGE and two-dimensional electrophoresis (2-DE) have provided direct separation technologies and contributed to the better understanding of the global milk proteome. 2-DE involves separation by isoelectric focusing in the first dimension, followed by SDS-PAGE in the second dimension. 1D-SDS/PAGE offers a number of important advantages as it produce sharp, molecular weight-separated bands, which increase the dynamic range of the mixture analysis. Fractionation of the complex mixture by spreading it out over 10–20 gel slices dramatically increases the depth of analysis, and hence the number of identified proteins. 2-DE is useful to optimize the separation of proteins of similar molecular weight but with different isoelectric points, which are not resolved using SDS/PAGE. 2-DE provides higher resolution compared to SDS/PAGE and is the best choice for the analysis of phosphorylated or glycosylated proteins as their isoelectric point can shift. In fact, 2-DE is the only technique that provides a visualization of PTM. On the other hand, low abundance, highly charged and hydrophobic proteins such as membrane proteins cannot be well resolved in 2-DE. 2-DE is often time-consuming and requires sample cleanup prior to analysis. In both 1Dand 2-DE analyses, the bands or spot map are visualized by protein staining with Coomassie. The targeted bands from 1D spots or spots from 2D gels are sequenced and identified by mass spectrometry. In a bovine mastitis study, Boehmer et al. performed an optimized protein separation with 2D electrophoresis to analyze whey from control and mastitic bovines. The experiments were conducted with a conventional proteomic approach using 2-DE as a choice of separation method, coupled by MALDI-TOF analysis for the identification [30].

mass analyzers for ESI are single or triple quadrupoles and quadrupole ion traps. Because it is a liquid-based method, ESI is more compatible with the LC separations and has consequently became the standard ionization method in LC-MS/MS experiments. With the advent of ultraperformance liquid chromatography (UPLC), chromatographic separations have improved in terms of both the resolving power and the speed of the separation. Nanoflow LC (nLC) is now also widely used in proteomics because it is amenable to LC columns with a smaller internal diameter, which increases the column backpressure and results in greater sensitivity [32].

Proteomic Analysis of Goat Milk

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http://dx.doi.org/10.5772/intechopen.70082

The accurate identification of proteins and peptides in a complex mixture using tandem mass spectrometry, data can be achieved by database searching strategies. The databases are normally protein sequences translated from genomic data. Considering a database containing potential proteins that could be present in the sample, these proteins are digested in silico by a search engine. For example, for tryptic peptides, the search engine would calculate the masses of all peptides that could be produced by cleavages after lysine and arginine residues and would create a virtual peptide database [33]. Similarly, for the identification of peptides in the sample, the search engine first filters all potential peptides with the same mass, then performs an in silico fragmentation of each of these peptides. After matching peptide sequences with the list of fragment masses observed in the MS/MS spectrum, the search

The success of MS-based proteomics analyses depends on the availability of complete and accurately annotated databases containing the gene and protein sequence information for the animal species of interest. Unfortunately, as of yet, only a limited number of annotated genome sequences are available for the goat. In recent years, despite an increase in the number of proteomic investigations of various body fluids from the goat, identifying these proteins is recognized as a greater technical challenge [34]. In the absence of annotated protein databases, researchers are forced to extrapolate their data from the bovine genome or other mammalian protein databases [28]. If a given database does not contain the amino acid sequences of all of the proteins in the sample, suitable matches cannot be made; thus the proteins will

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 pro-

teins, comprehensive analysis, MFGM, and identification of PTM (**Table 1**).

**2.3. Bioinformatics**

engine assigns a score [33].

remain unidentified.

**3. Proteomic research in caprine milk**

#### **2.2. Mass spectrometry**

Although gel-based assays are useful for fractionating complex mixtures and for removing contaminants from a sample that might interfere with the MS analysis, gel-free approaches are simple and straightforward. Using a gel-free approach, a complex protein mixture is directly digested in solution prior to MS analysis. Reduction and alkylation steps, though not required prior to enzymatic cleavage, provide greater peptide yields and can enhance peptide sequence coverage. Prior to MS analysis, peptides can be fractionated using different forms of chromatography including reverse-phase, strong and weak ion exchange, size exclusion, and affinity capture chromatography. Although reverse-phase chromatography is the most widely used method of LC separation in proteomics, affinity capture chromatography is commonly used to enrich or isolate intact proteins, or to investigate PTMs prior to MS analysis.

There are different types of MS instruments for the analysis of a particular type of sample, each with different scanning speed and resolution capabilities. Nonetheless, all MS instrument systems have three common and distinct components: an ionization source for the generation of ions, a mass analyzer for the separation of ions, and a mass detector for the detection of ions.

In regards to ionization, it is important to note that soft ionization techniques such as MALDI and electrospray ionization (ESI) have revolutionized the analysis of large biomolecules. In MALDI, a sample is mixed with a UV-absorbing crystalline matrix, most commonly derivatives of cinnamic acid, and spotted onto a metal target plate. The plate is then inserted into the MS instrument, where it is placed in a vacuum and irradiated with a UV laser. The matrix absorbs the irradiation, which simultaneously heats, volatilizes, and ionizes the sample [16]. Once ionized, the proteins or peptides are analyzed in a mass analyzer, which is typically time-of-flight (TOF) in a MALDI instrument. The TOF mass analyzer is ideal for MALDI because it has a virtually unlimited mass range, which is advantageous because MALDI yields singly charged molecular ions that can have a high mass-to-charge (*m/z*) ratio [30]. TOF instruments can also operate in tandem mode, using a second mass analyzer to monitor fragment ions (i.e., TOF/TOF).

In contrast to MALDI, ESI produces multiply charged species for each molecule. This is due to the mechanism of ion formation in ESI in which an electrical field leads to the transfer of ions from a solution into the gaseous phase. The transfer of an ionic species from solution into the gas phase by ESI involves three steps that include the dispersion of charged droplets, followed by solvent evaporation, and finally the ejection of the ion from the highly charged droplets [31]. Although the generation of multiply charged ions complicates the mass analysis, it greatly enhances fragmentation potential, which is necessary for structure elucidation. Ideal mass analyzers for ESI are single or triple quadrupoles and quadrupole ion traps. Because it is a liquid-based method, ESI is more compatible with the LC separations and has consequently became the standard ionization method in LC-MS/MS experiments. With the advent of ultraperformance liquid chromatography (UPLC), chromatographic separations have improved in terms of both the resolving power and the speed of the separation. Nanoflow LC (nLC) is now also widely used in proteomics because it is amenable to LC columns with a smaller internal diameter, which increases the column backpressure and results in greater sensitivity [32].

#### **2.3. Bioinformatics**

2-DE. 2-DE is often time-consuming and requires sample cleanup prior to analysis. In both 1Dand 2-DE analyses, the bands or spot map are visualized by protein staining with Coomassie. The targeted bands from 1D spots or spots from 2D gels are sequenced and identified by mass spectrometry. In a bovine mastitis study, Boehmer et al. performed an optimized protein separation with 2D electrophoresis to analyze whey from control and mastitic bovines. The experiments were conducted with a conventional proteomic approach using 2-DE as a choice

Although gel-based assays are useful for fractionating complex mixtures and for removing contaminants from a sample that might interfere with the MS analysis, gel-free approaches are simple and straightforward. Using a gel-free approach, a complex protein mixture is directly digested in solution prior to MS analysis. Reduction and alkylation steps, though not required prior to enzymatic cleavage, provide greater peptide yields and can enhance peptide sequence coverage. Prior to MS analysis, peptides can be fractionated using different forms of chromatography including reverse-phase, strong and weak ion exchange, size exclusion, and affinity capture chromatography. Although reverse-phase chromatography is the most widely used method of LC separation in proteomics, affinity capture chromatography is commonly used to enrich or isolate intact proteins, or to investigate PTMs prior to MS analysis.

There are different types of MS instruments for the analysis of a particular type of sample, each with different scanning speed and resolution capabilities. Nonetheless, all MS instrument systems have three common and distinct components: an ionization source for the generation of ions, a mass analyzer for the separation of ions, and a mass detector for the detection of ions.

In regards to ionization, it is important to note that soft ionization techniques such as MALDI and electrospray ionization (ESI) have revolutionized the analysis of large biomolecules. In MALDI, a sample is mixed with a UV-absorbing crystalline matrix, most commonly derivatives of cinnamic acid, and spotted onto a metal target plate. The plate is then inserted into the MS instrument, where it is placed in a vacuum and irradiated with a UV laser. The matrix absorbs the irradiation, which simultaneously heats, volatilizes, and ionizes the sample [16]. Once ionized, the proteins or peptides are analyzed in a mass analyzer, which is typically time-of-flight (TOF) in a MALDI instrument. The TOF mass analyzer is ideal for MALDI because it has a virtually unlimited mass range, which is advantageous because MALDI yields singly charged molecular ions that can have a high mass-to-charge (*m/z*) ratio [30]. TOF instruments can also operate in tandem mode, using a second mass analyzer to monitor fragment ions (i.e., TOF/TOF).

In contrast to MALDI, ESI produces multiply charged species for each molecule. This is due to the mechanism of ion formation in ESI in which an electrical field leads to the transfer of ions from a solution into the gaseous phase. The transfer of an ionic species from solution into the gas phase by ESI involves three steps that include the dispersion of charged droplets, followed by solvent evaporation, and finally the ejection of the ion from the highly charged droplets [31]. Although the generation of multiply charged ions complicates the mass analysis, it greatly enhances fragmentation potential, which is necessary for structure elucidation. Ideal

of separation method, coupled by MALDI-TOF analysis for the identification [30].

**2.2. Mass spectrometry**

146 Goat Science

The accurate identification of proteins and peptides in a complex mixture using tandem mass spectrometry, data can be achieved by database searching strategies. The databases are normally protein sequences translated from genomic data. Considering a database containing potential proteins that could be present in the sample, these proteins are digested in silico by a search engine. For example, for tryptic peptides, the search engine would calculate the masses of all peptides that could be produced by cleavages after lysine and arginine residues and would create a virtual peptide database [33]. Similarly, for the identification of peptides in the sample, the search engine first filters all potential peptides with the same mass, then performs an in silico fragmentation of each of these peptides. After matching peptide sequences with the list of fragment masses observed in the MS/MS spectrum, the search engine assigns a score [33].

The success of MS-based proteomics analyses depends on the availability of complete and accurately annotated databases containing the gene and protein sequence information for the animal species of interest. Unfortunately, as of yet, only a limited number of annotated genome sequences are available for the goat. In recent years, despite an increase in the number of proteomic investigations of various body fluids from the goat, identifying these proteins is recognized as a greater technical challenge [34]. In the absence of annotated protein databases, researchers are forced to extrapolate their data from the bovine genome or other mammalian protein databases [28]. If a given database does not contain the amino acid sequences of all of the proteins in the sample, suitable matches cannot be made; thus the proteins will remain unidentified.
