Proteomic Advances in Milk and Dairy Products

molecules Review Proteomic Advances in Milk and Dairy Products Rubén Agregán1, NoemíEchegaray 1, María López-Pedrouso 2, Radwan Kharabsheh 3, Daniel Franco 1 and JoséM. Lorenzo 1,4,*   Citation: Agregán, R.; Echegaray, N.; López-Pedrouso, M.; Kharabsheh, R.; Franco, D.; Lorenzo, J.M. Proteomic Advances in Milk and Dairy Products. Molecules 2021,26, 3832. https:// doi.org/10.3390/molecules26133832 Academic Editor: Antonio-JoséTrujillo Received: 24 May 2021 Accepted: 21 June 2021 Published: 23 June 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1Centro Tecnológico de la Carne de Galicia, Adva. Galicia n◦4, Parque Tecnológico de Galicia, San Cibrao das Viñas, 32900 Ourense, Spain; rubenagr[email protected] (R.A.); [email protected] (N.E.); [email protected] (D.F.) 2Department of Zoology, Genetics and Physical Anthropology, University of Santiago de Compostela, 15872 Santiago de Compostela, Spain; [email protected] 3Business Administration, Faculty of Economics and Administrative Sciences, Applied Science University—Bahrain, Al Hidd 5055, Bahrain; [email protected] 4Área de Tecnología de los Alimentos, Facultad de Ciencias de Ourense, Universidad de Vigo, 32004 Ourense, Spain *Correspondence: jmlor[email protected] Abstract: Proteomics is a new area of study that in recent decades has provided great advances in the field of medicine. However, its enormous potential for the study of proteomes makes it also applicable to other areas of science. Milk is a highly heterogeneous and complex fluid, where there are numerous genetic variants and isoforms with post-translational modifications (PTMs). Due to the vast number of proteins and peptides existing in its matrix, proteomics is presented as a powerful tool for the characterization of milk samples and their products. The technology developed to date for the separation and characterization of the milk proteome, such as two-dimensional gel electrophoresis (2DE) technology and especially mass spectrometry (MS) have allowed an exhaustive characterization of the proteins and peptides present in milk and dairy products with enormous applications in the industry for the control of fundamental parameters, such as microbiological safety, the guarantee of authenticity, or the control of the transformations carried out, aimed to increase the quality of the final product. Keywords: proteomics; proteome; proteins; milk; dairy products; electrophoresis; chromatography; mass spectrometry 1. Introduction Proteomics is an area of knowledge that emerged in the field of biology as a consequence of the discovery of the human genome. The first time this expression was coined was in the mid-1990s as a need to create a specific vocabulary that would describe the work carried out at that time, which was the study of the proteome. This study includes all the proteins of a single cell, together with all the isoforms and modifications, the interactions between them, and their structure and higher-order complexes [ 1 ]. During the early days of proteomics, efforts were focused on the field of medicine, where great advances were made in the diagnosis of diseases. However, the potential of studying the proteome of a biological sample was spread to other fields such as animal production. In particular, great efforts have been carried out to understand the milk proteome, which is defined as complex with a wide variety of compounds, some of which have found to have biological activities of great interest. This study focusses on exploring genetic aspects, molecular pathways, and cellular functions involved in the production of milk, its quality, and safety [2]. Milk is a fluid that contains many low-abundance proteins (5% of the total protein fraction) and they are located in the whey or in the milk fat globule membrane (MGMF) [ 2 ]. To this inconvenience must be added the presence of numerous genetic variants and posttranslational modifications (PTMs), such as glycosylation, phosphorylation, disulphide Molecules 2021,26, 3832. https://doi.org/10.3390/molecules26133832 https://www.mdpi.com/journal/molecules Molecules 2021,26, 3832 2 of 20 bond formation, and proteolysis, which generate a large number of protein variants from a single gene product [ 3 ], which adds even more difficulty to proteomic analysis. Therefore, there is not yet a single protocol that is capable of providing a complete analysis of the milk proteome. In the last few years, analytical chemistry has developed a series of methods that include fractionation and separation of the sample, its concentration and the detection of the proteins present with a resolution and sensitivities enough for a comprehensive determination of the proteome [ 4 ]. The potential represented by the use of proteomics in animals has favored the advancement of technology. Two-dimensional gel electrophoresis (2DE) is generally applied for the separation of proteins or protein subunits according to charge, followed by another separation by molecular mass in SDS-PAGE. After a staining process, the proteins displayed in the form of spots are characterized by mass spectrometry (MS) analysis. In a complementary manner, the sample can be subjected to a digestion process and the peptides obtained analyzed by MS after separation by chromatographic techniques [ 5 ]. These two methodologies are very widespread in the analysis of the milk proteome, with multiple operational variations that are discussed below. This review focused on advances in proteomics for dairy proteome analysis and the applicability of these advances in the safety, authenticity, and quality control of milk and dairy products. 2. Milk Proteins and Their Significance in Human Nutrition Milk may be defined as the liquid excreted by the mammary glands of mammals in order to serve as food for the newborn. However, since ancient times, milk has been considered a very valuable resource by human beings due to its nutritional composition and easy of obtaining. This contextualized vision of milk as an exploitable product to satisfy a need has led to a change in meaning. Hence, from a technological point of view, a more consistent definition of milk would be the product obtained by milking of mammals, which are raised for the only purpose of producing milk [ 6 ]. Different species of animals are used for milk production. Currently, cow’s milk represents around 80% of world production, followed by buffalo (~15%), goat (~2.5%), sheep (~1%), and camel (~0.4%) [ 7 ]. On the other hand, the importance of milk in the human diet goes beyond its consumption as a raw product. The elaboration of fermented dairy products has become a thousand-year-old tradition, which includes a wide variety of foods with different textures, tastes, aromas, shapes, and sizes [8]. Milk is a nutritionally very complete food due to the balance of its different components. These components are not in a fixed proportion, but vary depending on factors, such as the animal species, breed, period of lactation, and diet [ 9 ]. For example, buffalo and sheep milks have a higher fat content (7.5% and 6.4%, respectively) than cow (3.3%) and goat milks (3.9%). These species together with camel are five of the most consumed milks in the world and their composition is presented in Table 1[ 10 ]. Milk is a biologically designed product to provide the neonate with the necessary nutritional requirements, and in particular, the protein fraction is highly valued due to its high nutritional quality. The consumption of milk provides all the essential amino acids and other compounds, such as vitamin-binding and metal-binding proteins and various protein hormones [ 11 ]. The amino acid profile is similar to that of the egg, although limited in sulphur-containing amino acids. However, the variety and quantity of these molecules present in milk is enough to cover the daily needs defined in adults by the FAO/WHO/UNU [ 12 ]. Rafiq et al. [ 13 ] reported a good balance of essential amino acids in cow, sheep, goat, buffalo, and camel milks, especially branched-chain amino acids (leucine, isoleucine, and valine) regarding the daily needs mentioned above. The unique properties of milk proteins and their technological impact by playing a prominent role in the production of dairy products has led to their exhaustive study and characterization [ 14 , 15 ]. This fraction of milk is composed of two types of proteins, caseins and whey proteins. The former is obtained after acid precipitation; at pH 4.6 the casein fraction is insoluble in milk. The remaining liquid is a solution of proteins (whey proteins), which also contains lactose, salts, vitamins, and other compounds at trace levels [ 14 , 16 ]. Molecules 2021,26, 3832 3 of 20 Regarding caseins, up to five types can be distinguish, αs1 -casein, αs2 -casein, β -casein, γ -casein, and κ -casein [ 17 ]. They represent around 80% of the proteins in cow’s milk and other commercial dairy species. They are in the form of colloidal particles of 50–600 nm in diameter, known as micelles. Important and particular features of milk, such as white color, stability to high temperatures or ethanol, and coagulation in the presence of rennet are due to this protein subfraction [ 18 ]. Casein micelles are structurally complex. They are formed by highly phosphorylated caseins, which interact and aggregate with calcium phosphate. The proportion of κ -casein (located on the surface) determines the size of the micelle, as well as many other properties, especially the stability against aggregation [ 19 ], a determining factor in the production of dairy products. Table 1. Nutritional composition of some of the most consumed milks in the world. Composition (g/100 g) Cow Buffalo Goat Sheep Camel Water 87.8 83.2 87.7 82.1 84.8 Protein 3.3 4 3.4 5.6 3.9 Fat 3.3 7.5 3.9 6.4 5 Lactose 4.7 4.4 4.4 5.1 4.2 Ash 0.7 0.8 0.8 0.9 0.9 Data from Muehlhoff et al. [10]. Whey proteins, also called serum proteins, comprise around 20% of total proteins and 0.7% of cow’s milk and, like caseins, they are subdivided into sub-fractions ( β -lactoglobulin, α -lactoalbumin, and several minor proteins) [ 14 ]. The main protein in whey corresponds to β -lactoglobulin. It represents more than half of this protein fraction and, in turn, is subdivided into two genetic variants, differentiated by the substitution of a glycine residue (variant A) for another of aspartic acid (variant B). The β -lactoglobulin molecule is characterized by the absence of phosphorous and by the presence of two disulfide and one free sulfhydryl groups. Other abundant protein in whey is α -lactoalbumin, which account for approximately 13% of the total serum proteins. The molecule of this protein contains four disulfide linkages and, like β -lactoglobulin, does not have phosphorus. There are other proteins in much lower amounts in whey, such as inmunoglobulins, which comprise 2% of serum proteins, and are divided into four classes, IgG1, IgG2, IgA, and IgM, or bovine serum albumin (BSA) [ 20 ]. The major milk proteins of several highly consumed species, including human, are represented in Table 2. Recently, the study of the milk proteins and related compounds has revealed multitude of beneficial properties for health. It is suggested that the consumption of milk and dairy products might help prevent and delay the appearance of certain types of cancer [ 21 – 23 ]. In this line, Kim et al. [ 24 ] reported that α -caseins, β -caseins, and β -lactoglobulin can protect cells from oxidative stress, inhibiting cellular senescence. These compounds might be used as supplements for the prevention of aging-associated diseases, especially in the prevention of skeletal muscle loss. Milk is a very nutritious medium, a natural source of fatty acids, essential amino acids, and the sugar lactose, which makes it an optimal medium for microbial growth. However, a number of compounds with antimicrobial activity, such as immunoglubulins, lactoferrin, lactoperoxidase, and lysozyme, can be found naturally in milk. In addition, antimicrobial activity has been also observed in peptides found in fermented dairy products [ 25 ]. These compounds help keep the raw product stable for longer and they might be used as nutraceuticals in novel food products. Molecules 2021,26, 3832 4 of 20 Table 2. Major milk proteins of several highly consumed animal species. Milk Compound (g/L of Milk) Cow aGoat aSheep bBuffalo cCamel d Crude protein 29.4 35.84 55.83 60.10 26.5 Casein 24 29.64 41.39 50.38 17.34 αs-casein 11.12 9.83 17.69 24.14 2.89 β-casein 12.88 19.80 13.95 18.45 12.78 k-casein 9.74 7.79 1.67 Whey protein 5.04 6.20 - - - α-lactoalbumin - - 6.57 4.30 2.01 β-lactoglobulin - - 7.86 5.42 - aCeballos et al. [26]; bPelmus et al., [27]; cBonfatti et al., [28]; dOmar et al., [29]. Overall, milk proteins, mainly those derived from whey, have been found to exhibit a wide variety of biological activities, acting as health promoters. Research in the field suggests that their use in the treatment of chronic diseases, such as obesity, hypertension, or type 2 diabetes could improve the prognosis of patients [30]. 3. Milk Proteomics 3.1. Concept of Proteomics and Applied Strategies Proteomics is a term that describes an area of knowledge framed within the field of biology formed by the combination of the words “protein” and “genomics”, that was first coined in mid-1990s, although the concept was proposed in 1979 by Anderson and Leigh when in the presentation entitled “Human Proteins Index Project” they hoped to unlock the genome by identifying the respective proteins using the recently developed technique of 2DE [ 31 ]. Wilkins et al. [ 32 ] was the first to stablish the concept of proteomics or proteome analysis, as the separation, identification, and quantification of the set of proteins expressed by a genome, cell, or tissue. Another definition could be that given by Anderson et al. [ 33 ], who determined the concept of proteomics as “the use of quantitative protein-level measurements of gene expression to characterize biological processes and decipher the mechanisms of gene expression control”. Basically, by the term of proteomics we refer to the set of proteins encoded by the genome, and by proteomics to the study of these proteins. However, this is a simplistic conception of the term, since proteomics not only includes all the proteins in a given cell, but also the set of all isoforms and modifications of the proteins, the interactions between them, and the description of their structures and higher-order complexes [1]. The characterization of proteomes is an arduous and complex task given the vast variability of the forms in which proteins can appear. Unfortunately, at present there are no methods available that allow the complete analysis of proteomes in a single and simple event. However, technologies developed in recent decades have made it possible to exponentially improve speed, accuracy, and level of detail [34]. 3.1.1. Separation Techniques The proteome is a complex matrix that contains thousands of different protein molecules, the characterization of which requires analyzing each protein, its forms, and abundance within that matrix [ 34 ]. Hence, in order to correctly identify each protein, a previous separation step is necessary. The separation of all proteins contained in cells, tissues, and biofluids has been and continues to be a challenge for science, which for many years has tried to develop methods of fractionation, separation, concentration, and detection with sufficient resolution to separate large amounts of proteins, and with an adequate sensibility and dynamic range to detect those proteins present in low abundance [4]. To achieve protein separation, scientists have relied on electrophoretic and chromatographic technologies, separately and in combination, both offline and online [ 35 ]. Regarding the first of the aforementioned technologies, electrophoresis, one of the most popular and effective methods is the two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) based on two-dimensional gel electrophoresis (2DE) technology (Figure 1). Molecules 2021,26, 3832 5 of 20 This method is based on the combination of two orthogonal separation techniques, isoelectric focusing (IEF) and sodium dodecyl sulphate (SDS) PAGE. First, with IEF technique, proteins are separated according to their different isoelectric point (pI) (first dimension). Then, proteins are separated again, but this time based on their electrophoretic mobility using SDS-PAGE (second dimension). Finally, the proteins are visualized and quantified by different procedures, including Coomassie, silver, or fluorescence stains [ 36 ]. The two-dimensional SDS-PAGE technique is usually not very effective in resolving proteins with similar molecular mass. Similarly, proteins with a similar pI are difficult to resolve using IEF gels, particularly when trying to analyze samples with high concentrations of individual proteins, such as milk. However, using a two-dimensional approach, these limitations are largely resolved, greatly improving resolution [ 3 ]. Despite the high strength showed by 2D-PAGE in the separation of proteomes, a significant number of disadvantages have been found, such as the low ability to detect proteins with low abundance [ 37 ], the scarce efficiency to separate polypeptide chains with molecular masses outside the 150-8 kDa range, or the difficulty of resolution of proteins with an extremely acidic (pI < 3) or basic (pI > 12) pH [3]. Molecules 2021, 26, x FOR PEER REVIEW 5 of 20 popular and effective methods is the two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) based on two-dimensional gel electrophoresis (2DE) technology (Figure 1). This method is based on the combination of two orthogonal separation techniques, isoelectric focusing (IEF) and sodium dodecyl sulphate (SDS) PAGE. First, with IEF technique, proteins are separated according to their different isoelectric point (pI) (first dimension). Then, proteins are separated again, but this time based on their electrophoretic mobility using SDS-PAGE (second dimension). Finally, the proteins are visualized and quantified by different procedures, including Coomassie, silver, or fluorescence stains [36]. The twodimensional SDS-PAGE technique is usually not very effective in resolving proteins with similar molecular mass. Similarly, proteins with a similar pI are difficult to resolve using IEF gels, particularly when trying to analyze samples with high concentrations of individual proteins, such as milk. However, using a two-dimensional approach, these limitations are largely resolved, greatly improving resolution [3]. Despite the high strength showed by 2D-PAGE in the separation of proteomes, a significant number of disadvantages have been found, such as the low ability to detect proteins with low abundance [37], the scarce efficiency to separate polypeptide chains with molecular masses outside the 150-8 kDa range, or the difficulty of resolution of proteins with an extremely acidic (pI < 3) or basic (pI > 12) pH [3]. . Figure 1. Advantages and disadvantages of two-dimensional gel electrophoresis (2DE) technology. Data from Rabilloud et al. [38]. Gel image from Zagorchev et al. [39] The utility and protein resolution capacity of 2D-PAGE has been reported in analysis of milk proteomes. Hsieh et al. [40] identified up to 15 proteins in cow’s milk, three αs1- caseins, three αs2-caseins, three β-caseins, three κ-caseins, two β-lactoglobulins, and one serum albumin, in a study that intended the proteomic analysis of the effects of chymosin on the coagulation of individual milk proteins. By using 2D-PAGE, D’Auria et al. [41] managed to detect the presence of four main protein spots, corresponding to albumin, caseins, β-lactoglobulin and α-lactalbumin, in milks from different animal species, included human. Up to eight and nine different proteins were separated and identified in human and cow’s milk, respectively. Liquid phase separation is another separation method increasingly used that includes techniques, such as capillary electrophoresis (CE) or liquid chromatography (LC) [42–46]. The increase in its use in proteomic analysis is due to its numerous advantages over 2D-PAGE, such as its great sensibility, superior dynamic range, easy automatization, and speed. On the other hand, this method is very versatile, since it can work with different separation mechanisms (e.g., size exclusion, reverse phase, and ion exchange) and in Figure 1. Advantages and disadvantages of two-dimensional gel electrophoresis (2DE) technology. Data from Rabilloud et al. [38]. Gel image from Zagorchev et al. [39]. The utility and protein resolution capacity of 2D-PAGE has been reported in analysis of milk proteomes. Hsieh et al. [ 40 ] identified up to 15 proteins in cow’s milk, three αs1 - caseins, three αs2 -caseins, three β -caseins, three κ -caseins, two β -lactoglobulins, and one serum albumin, in a study that intended the proteomic analysis of the effects of chymosin on the coagulation of individual milk proteins. By using 2D-PAGE, D’Auria et al. [ 41 ] managed to detect the presence of four main protein spots, corresponding to albumin, caseins, β -lactoglobulin and α -lactalbumin, in milks from different animal species, included human. Up to eight and nine different proteins were separated and identified in human and cow’s milk, respectively. Liquid phase separation is another separation method increasingly used that includes techniques, such as capillary electrophoresis (CE) or liquid chromatography (LC) [ 42 – 46 ]. The increase in its use in proteomic analysis is due to its numerous advantages over 2D- PAGE, such as its great sensibility, superior dynamic range, easy automatization, and speed. On the other hand, this method is very versatile, since it can work with different separation mechanisms (e.g., size exclusion, reverse phase, and ion exchange) and in this way, it Molecules 2021,26, 3832 6 of 20 can analyze basic or acid proteins and of any molecular mass. However, liquid phase separation suffers in term of resolution, being lower than using 2D-PAGE [ 47 ]. However, Omar et al. [ 29 ] highlighted the excellent resolution of the CE technique, greater than that of 2D-PAGE, in the separation of camel milk proteins. Using this technique, these authors managed to identify and quantify the major caseins ( α -casein, β -casein, and κ -casein) and whey proteins (α-lactoalbumin, serum albumin, and lactoferrin). 3.1.2. Characterization Techniques Once the proteins have been selected, their identification or comprehensive characterization is carried out preferably using MS. Recent advances in this determination technology have allowed proteins to be characterized in a rapid and exhaustive manner [ 47 ], establishing itself as a powerful technology for protein characterization [ 48 ]. A mass spectrometer basically ionizes analytes using an ion source and provides information about the mass-charge ratio (m/z) of these and the number of ions at each value of m/z, using a mass analyzer and a detector, respectively. Moreover, in the case of peptides, a previous breakdown step is necessary through a process called dissociation, for which there are different techniques, such as collision-induced dissociation (CID), electron-capture dissociation (ECD), or electron-transfer dissociation (ETD) [ 49 ]. Electrospray Ionization (ESI) and Matrix Assisted Laser Desorption/Ionization (MALDI) MS techniques have greatly improved the biomolecule analysis, including proteins. The MALDI technique is frequently used for the characterization of simple proteomes. It has the advantage that it is partially resistant to interferences caused by buffers commonly used in protein analysis, such as phosphate, tris, or urea. In this way, the formation of singly charged ions is promoted, thus facilitating the interpretation of the generated mass spectra. These advantages have led to the use of MALDI-MS in many analytical areas. However, some important drawbacks persist, such as the variability of the signal intensities and the resolution between points of the same sample [ 50 ]. At this point, a delayed extraction technique could increase resolution, allowing peaks with a similar mass range to be differentiated. Cozzolino et al. [ 51 ] successfully applied this technology along with MALDI-MS, using a time-of-flight (TOF) detector as a rapid and easy strategy to identify possible adulteration in raw milk samples. The limited resolution of MALDI was partially resolved by the delayed extraction technique, which allowed to separate peaks with a mass range of up to 20-30 kDa, using the main whey proteins α -lactalbumin and β -lactoglobulin as molecular markers. The authors proposed this protocol for the detection of cow milk in ewe and water buffalo milks, which proved to be practical and fast enough, managing to analyze around 100 samples in less than an hour. In contrast, the ESI technique produces the generation of multiply charged ions. An important advantage of ESI is its easy in line coupling with high-performance separation techniques such the aforementioned CE and HPLC [ 52 ]. Vincent et al. [ 53 ] greatly optimized the separation of intact cow milk proteins by HPLC and their analysis by MS using ESI as ionization source. The quantification of proteins was successfully applied to Holstein-Friesian and Jersey milks to assess differences in proteins variant levels. In recent years, the evolution in MS, with the appearance of new instruments, has led to the development of new ion activation techniques and reduced the detection limit. In addition, the latest evolutions of this technology have achieved to improve the understanding of structures of peptides and proteins [ 54 ]. However, the identification of these compounds remains particularly difficult since the data libraries do not always offer reliable results. Furthermore, negatives, false positives, and unassigned spectra can be produced [ 49 ]. Along with these drawbacks, it should be noted the unaffordable price for a fragile technology that requires continuous maintenance. 3.2. Advances in Proteomics in the Characterization of Milk and Dairy Products During the 1980s, 2DE procedures were used extensively for the mapping of milk proteomes, including that of some dairy products. Thus, the protein patterns of different milks were obtained by using the aforementioned IEF and SDS-PAGE techniques, which Molecules 2021,26, 3832 7 of 20 has made it possible to compare their most abundant proteins. The quantification of protein expression in images enables this comparison among experiments, popularizing the use of 2DE gels [ 55 ]. Nevertheless, low abundant proteins remained undetectable. With the emergence of immobilized pH gradients (IPGs) this problem was partially solved, increasing the resolution for a better visualization of the protein profile [ 56 ]. In this manner, Conti et al. [ 57 ] were able to carry out the isolation and subsequent characterization of a genetic variant of bovine β -lactoglobulin. By means of a preparative HPLC gel filtration and preparative IEF-IPG they reached a highly purified protein. Although the use of 2DE technology was a milestone in the early days of proteomics and provided great results in the past, the reality is that its use as the sole protein characterization technique allows only a part to be identified from the analytical sample. The combined use of 2DE technology with specific detection systems has increased sensitivity when characterizing protein profiles. The coupling of 2DE to MS was one of the most important advances in milk proteomics. Protein analysis using this combination of technologies in which identification is achieved by MS after proteins have been separated by 2DE and digested with trypsin represents an interesting methodology with a powerful application in the study of dairy products [ 58 ]. Up to 30 proteins were identified successfully through this strategy in the aqueous extract of Swiss-type of cheese during the ripening stage, including those proteins expressed by Streptococcus thermophilus ITG ST20 and Lactobacillus helveticus ITG LH1 microorganisms used as starters [ 59 ]. Similarly, Pourjoula et al. [ 60 ] characterized the proteome and peptidome of kashz, a typical Iranian dairy product made from sour milk, combining technologies such as SDS-PAGE and HPLC separation techniques coupled to MS. The results showed a protein profile similar to yogurt and exposure to in vitro plasmin hydrolysis reveled that it is actually a product resulting from acid coagulation in the absence of γ-caseins. Milk proteome has been found to be highly heterogeneous due to the existence of numerous protein isoforms [ 61 ]. These modifications may arise from alternative mRNA splicing, single point mutations, and PTMs. These changes in protein structure often cause variations in the molecular mass and net charge [ 62 ]. Some of these isoforms, the so-called PTMs are very important when characterizing the proteome as they influence some structural or functional aspect of the protein involved. These modifications generally originate after protein synthesis on cellular ribosomes [ 63 , 64 ]. PTM is the second pathway after genetic polymorphism by which the milk proteome expands, notably increasing its complexity. Many proteins undergo a multitude of PTMs, including phosphorylation, acetylation, glycosylation, disulfide crosslinking, lipid conjugation, and proteolytic cleavages, critical for important functions of milk proteins, such as micellar stability [ 64 ], vital for making cheese. Many of these particular modifications have been found to be affected by different factors. Thus, Fang et al. [ 65 ] reported variations in the relative concentrations of αs -casein phosphorylation isoforms in cow milk depending of intrinsic and extrinsic factors. The use of LC-ESI MS for the proteome characterization showed that parity, lactation stage, and genetic variation among animals positive contributed to phenotypic variation in the relative concentrations of individual αs -casein phosphorylation isoforms and in the phosphorylation degree of αs -caseins. Variations in the phosphorylation degree of this kind of caseins play a major role in the milk industrial properties. When investigation the causes that differentiate good coagulation from poor coagulation, even its absence, Frederiksen et al. [ 66 ] found that the presence of a lower proportion of two less phosphorylated variants of α -casein ( αs1 -casein-8P and αs2 -casein-11P) in Danish Holstein milk is related to worse coagulation compared to other milk samples were its presence was higher. Less significant appears to be the level of phosphorylation in κ -casein for milk coagulation. In a study carried out with the Holstein and Jersey cow breeds, six isoforms of κ -casein were identified that varied in the levels of phosphorylation and glycosylation (95 to 96% of the total κ -casein was phosphorylated, while 34 to 35% of the κ -casein isoforms were glycosylated). However, despite this, the authors of the study reported a very consistent κ-casein PTM patterns independent of milk coagulation ability [67]. Molecules 2021,26, 3832 8 of 20 These modifications in protein molecules have technological consequences for the dairy industry. Thus, protein functions such as micellar stabilization, among others, are governed by protein phosphorylation-dephosphorylation reactions. Advances in proteomics have improved the identification of these compounds. Through an approach using the connection of several mass analyzers (MS/MS), it has been possible to decipher proteins of the membrane of milk fat globules of different species [ 68 ]. Pisanu et al. [ 69 ] managed to identify 140 proteins belonging to this membrane in sheep’s milk using LC tandem mass spectrometry (LC-MS/MS). The connection of several mass analyzers in series allows more detailed information regarding the molecular structure of the compound. Equipment with ESI-MS or MALDI-MS are really effective for the determination of molecular ions and are sometimes even capable of providing valuable structural information. However, to obtain more detailed information regarding the molecular structure of the compound it is necessary to connect several serial mass analyzers. In this way, the first analyzer isolates the ions and then their fragmentation occurs. Subsequently, the final analyzer separates these fragments based on their m/z values [ 70 ]. Recent original research studies have shown the great utility of this evolution of MS. Milkovska-Stamenova and Hoffmann [ 71 ] identified 14 types of protein-derived advanced glycation end products (AGEs) produced during dairy processing and storage using nanoRP- ultraperformance liquid chromatography (UPLC)-ESI-MS/MS. These AGEs can alter protein functions, decrease nutritional value, and produce other potentially harmful health effects. For at least one identified AGE, 132 modified peptides were found, with AGE-amide being the main group of compounds. The identification of low abundance proteins in milk and dairy products has recently gained importance due to the proven beneficial health effects of a large quantity of peptides and proteins. Tacoma et al. [ 72 ] found a total of 935 low abundance proteins in the skimmed milks of Holstein and Jersey cows using LC-MS/MS. In the study, it was observed that a total of 43 of these proteins were expressed differentially between the two breeds. Similarly, Verma et al. [ 73 ] used nano-scale liquid chromatography tandem mass spectrometry (nLC-MS/MS) for the identification of 1307 functional proteins comprising casein and other low abundance proteins. The authors reported that the vast majority of these proteins were involved in binding function and catalytic and structural activities. 3.3. Benefits of Recent Proteomic Advances in Milk and Dairy Production In the dairy industry, the protection of consumer health and the guarantee of a nutritionally similar milk to the original has become an essential task and a large part of its efforts lies in guaranteeing these quality parameters. The origin of the milk is fundamental, since for example, that from a sick cow with mastitis can present a high number of somatic cells, which leads to an altered concentration of caseins, salts, free fatty acids, or lactose. Thus, a poor-quality milk will affect the dairy products made from it. In order to identify this type of anomaly, it is necessary to analyze protein patterns, as well as PTMs and even the presence of allergens [ 74 ], in order to provide the pertinent information aimed at protecting the consumer. 3.3.1. Ensuring the Safety of Milk and Dairy Products Proteomics has been a revolutionary technology for the analysis of samples and the early detection of diseases that can affect the entire production chain in the dairy industry, such as mastitis. MALDI-TOF MS has helped to overcome this problem by improving the detection of bacteria in sample by identifying biomarkers that represent ribosomal proteins from different bacteria. This methodology has improved to microbiological methods where there is an inherent error in the sample preparation and conditions of the culture medium, which can now be solved [ 74 ]. With regard to the aforementioned mastitis disease, it usually causes the discard of numerous liters of milk in the farms if it is not prevented or controlled properly, in addition to representing an industrial problem if it goes unnoticed. It is defined as an inflammatory disease due to infection of the udder tissue, caused by numerous pathogenic microorganisms, but usually staphylococci, streptoccci, and enter- Molecules 2021,26, 3832 9 of 20 obacteria are involved, with Staphylococcus aureus and Streptococcus agalactiae being the most contagious and responsible for the vast majority of infections [ 75 ]. When the disease occurs, milk is not adequate for transformation and changes in the nutritional profile are produced. Ogola et al. [ 76 ] observed an increase in the levels of non-casein fractions, sodium, chloride, and free fatty acids, while the casein content, lactose, caseins, potassium, and calcium decreased respect the normal levels present in the milk from a healthy cow. This disease is characterized by the presence of a high number of somatic cells that in turn are related to a variable proteome [ 77 ]. Therefore, proteomics represents an important advance in the field of early detection of this anomaly in milk. Pisanu et al. [ 78 ] found the increase of 119 proteins related to innate immune defense or structural functions in buffalo milk with mastitis after using SDS-PAGE and LC-MS/MS, which included vimentin, cathelicidins, histones, S100 and neutrophil granule proteins, haptoglobin, and lysozyme. In the same experiment, the authors also identified up to 33 lowering proteins that were found to be involved in lipid metabolism, including butyrophilin, xanthine dehydrogenase/oxidase, and lipid biosynthetic enzymes. In the same line, Abdelmegid et al. [ 79 ] used proteomics to find changes in the protein profiles in the whey of cows suffering from mastitis in order to identify biomarkers that help diagnose the disease. Using two-dimensional differential gel electrophoresis (2D-DIGE) coupled with LC-MS/MS, 28 highly abundant proteins were identified in milk affected by S. aureus (Figure 2). Nine of these proteins were involved in host defense functions, such as acute phase proteins, which play a role in the innate immunity and antimicrobial functions (e.g., serotransferrin, complement C3, fibrinogen γ -B chain, and cathepsin B), and proteins associated with the immune response to pathogens, such as polymeric immunoglobulin receptor-line protein, MHC class I antigen, and β-2-microglobulin. Molecules 2021, 26, x FOR PEER REVIEW 9 of 20 where there is an inherent error in the sample preparation and conditions of the culture medium, which can now be solved [74]. With regard to the aforementioned mastitis disease, it usually causes the discard of numerous liters of milk in the farms if it is not prevented or controlled properly, in addition to representing an industrial problem if it goes unnoticed. It is defined as an inflammatory disease due to infection of the udder tissue, caused by numerous pathogenic microorganisms, but usually staphylococci, streptoccci, and enterobacteria are involved, with Staphylococcus aureus and Streptococcus agalactiae being the most contagious and responsible for the vast majority of infections [75]. When the disease occurs, milk is not adequate for transformation and changes in the nutritional profile are produced. Ogola et al. [76] observed an increase in the levels of non-casein fractions, sodium, chloride, and free fatty acids, while the casein content, lactose, caseins, potassium, and calcium decreased respect the normal levels present in the milk from a healthy cow. This disease is characterized by the presence of a high number of somatic cells that in turn are related to a variable proteome [77]. Therefore, proteomics represents an important advance in the field of early detection of this anomaly in milk. Pisanu et al. [78] found the increase of 119 proteins related to innate immune defense or structural functions in buffalo milk with mastitis after using SDS-PAGE and LC-MS/MS, which included vimentin, cathelicidins, histones, S100 and neutrophil granule proteins, haptoglobin, and lysozyme. In the same experiment, the authors also identified up to 33 lowering proteins that were found to be involved in lipid metabolism, including butyrophilin, xanthine dehydrogenase/oxidase, and lipid biosynthetic enzymes. In the same line, Abdelmegid et al. [79] used proteomics to find changes in the protein profiles in the whey of cows suffering from mastitis in order to identify biomarkers that help diagnose the disease. Using two-dimensional differential gel electrophoresis (2D-DIGE) coupled with LCMS/MS, 28 highly abundant proteins were identified in milk affected by S. aureus (Figure 2). Nine of these proteins were involved in host defense functions, such as acute phase proteins, which play a role in the innate immunity and antimicrobial functions (e.g., serotransferrin, complement C3, fibrinogen γ-B chain, and cathepsin B), and proteins associated with the immune response to pathogens, such as polymeric immunoglobulin receptor-line protein, MHC class I antigen, and β-2-microglobulin. Figure 2. Proteins in mastitic milk (red spots) vs proteins from healthy milk (green spots) in twodimensional differential gel electrophoresis (2D-DIGE) image. Proteins with similar levels in both groups are indicated by white spots [80.] Figure 2. Proteins in mastitic milk (red spots) vs proteins from healthy milk (green spots) in twodimensional differential gel electrophoresis (2D-DIGE) image. Proteins with similar levels in both groups are indicated by white spots [80]. 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