**Proteomic Analysis of Goat Milk**

**Proteomic Analysis of Goat Milk**

Zohra Olumee-Shabon and Jamie L. Boehmer Boehmer

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

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

Zohra Olumee-Shabon and Jamie L.

#### **Abstract**

The advancement of electrophoresis and chromatography, along with technological developments in mass spectrometry, has widened the potential application of proteomics to study milk from smaller ruminants. The aim of this chapter is to provide an in-depth overview of the development and progress of proteomics applications in goat milk. After examining various proteomic approaches that are currently applied to this field, we narrow our focus on proteomic investigations of mastitis in goat milk. A summary of protein modulation in goat milk during experimentally-induced endotoxin mastitis is discussed. Because the molecular function of proteins is disrupted during disease due to changes in post-translational modifications, we also review the phosphorylation of caseins, which are the predominant phosphoproteins in milk, and discuss the implications of casein modifications during mastitis. These results offer new insights into the changes of protein expression in goat milk during infection.

DOI: 10.5772/intechopen.70082

**Keywords:** goat milk, mastitis, proteomics, casein phosphorylation

#### **1. Introduction**

Milk is an important biological fluid and an essential nutrient for young mammals and humans during their lifetime. It provides macro- and micro-nutrients and is an important source of antimicrobial and immunoregulatory agents [1]. Typically, the U.S. dairy industry relies on cows as the main source of milk and other dairy products. However, cow milk has been implicated in the increasing rates of protein allergies in infants [2, 3], causing gastrointestinal disorders in adults [4], and contains insufficient concentrations of iron [5], thus an interest in finding alternatives to cow milk has emerged.

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

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

mass spectrometry MALDI-MS [25, 26] or further refinements of gel-based assays followed by LC-MS/MS [27]. Until recently, no studies have focused on the proteomic analysis of goat milk protein modulation during clinical mastitis. The first study of goat milk protein modulation over the course of experimentally-induced mastitis using proteomics was recently been reported by our group [28]. Since the molecular function of proteins can be disrupted during the disease due to the changes in post-translational modifications (PTMs), we also evaluated

Proteomic Analysis of Goat Milk

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

This chapter will provide an overview of common proteomic approaches and the application of proteomics to the detection of caprine milk proteins. Likewise, a brief description of the advances in our current understanding of goat mastitis and of associated inflammatory biomarkers detected in goat milk will follow a summary of our proteomic investigations of goat milk during the course of experimentally-induced mastitis. Moreover, the phosphorylation of caprine casein proteins and their potential implications as markers of disease will be discussed.

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 sec-

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

tions, gel electrophoresis, MS, and database searching will be briefly discussed.

the phosphorylation status of caseins [29].

**2. Proteomics approaches**

**2.1. Electrophoresis**

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 monitor diseases and expand treatment options.

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 (APPs), were identified [19–21].

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

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 mass spectrometry MALDI-MS [25, 26] or further refinements of gel-based assays followed by LC-MS/MS [27]. Until recently, no studies have focused on the proteomic analysis of goat milk protein modulation during clinical mastitis. The first study of goat milk protein modulation over the course of experimentally-induced mastitis using proteomics was recently been reported by our group [28]. Since the molecular function of proteins can be disrupted during the disease due to the changes in post-translational modifications (PTMs), we also evaluated the phosphorylation status of caseins [29].

This chapter will provide an overview of common proteomic approaches and the application of proteomics to the detection of caprine milk proteins. Likewise, a brief description of the advances in our current understanding of goat mastitis and of associated inflammatory biomarkers detected in goat milk will follow a summary of our proteomic investigations of goat milk during the course of experimentally-induced mastitis. Moreover, the phosphorylation of caprine casein proteins and their potential implications as markers of disease will be discussed.
