Detection of Casein Phosphopeptides in Goat Milk - American

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Detection of Casein Phosphopeptides in Goat Milk Zohra Olumee-Shabon, and Jamie L. Boehmer J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/pr3010666 • Publication Date (Web): 16 Apr 2013 Downloaded from http://pubs.acs.org on April 29, 2013

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Journal of Proteome Research

Detection of Casein Phosphopeptides in Goat Milk

Zohra Olumee-Shabon§* and Jamie L. Boehmer§ § U.S. Food and Drug Administration Center for Veterinary Medicine, Laurel, MD 20708

*Corresponding author: Zohra Olumee-Shabon U.S. Food and Drug Administration Center for Veterinary Medicine 8401 Muirkirk Road, Laurel, MD 20708 Phone: 301-210-4253 Fax: 301-210-4685 E-mail: [email protected]

Keywords: casein, phosphorylation, phosphoproteome, post-translational modification, milk, mastitis

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ABSTRACT The aims of this study were to profile casein phosphopeptides in goat milk, to accurately determine the site of phosphorylation, and to evaluate whether or not any of the casein phosphorylation patterns were specific to a given physiological condition. Goat milk, collected before and after experimental induction of endotoxin mastitis, was separated by SDS-PAGE. Casein bands were digested with trypsin and the resulting peptides were analyzed by nLCMS/MS. Eight out of nine predicted tryptic phosphopeptides corresponding to eighteen different phosphorylation sites were detected in αS1-, αS2-, and β-casein.

Characterization of the

phosphorylation sites illustrated the capability of tandem MS to accurately localize phosphorylated residues among a number of other putative sites. Despite apparent lower abundance, almost all the phosphopeptides were also detected in milk samples obtained from the goats following experimental-induction of endotoxin mastitis. However, a tetra-phosphopeptide in αS2-casein was only observed in the milk samples obtained from healthy animals. The absence of this multi-phosphopeptide in the mastitic goat milk samples could indicate changes in phosphorylation as a result of disease, and potentially be used as a marker for milk quality. This study represents the first comprehensive analysis of casein phosphoproteome and reveals a much higher level of phosphorylation than previously demonstrated in goat milk.

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INTRODUCTION Among post-translational modifications (PTMs) of proteins, phosphorylation is of particular interest due to its involvement in critical cellular functions such as intracellular signaling, apoptosis, metabolism, gene repression, cell cycling, and gene transcription.1-2 Phosphorylation occurs primarily on serine (S), followed by threonine (T), and then tyrosine (Y) residues. Compared to unmodified peptides, analysis of phosphopeptides can be challenging because of the inherent lower ionization efficiency, relatively low stoichiometry, and variation of phosphorylation site(s).3-4 In the current analysis collision-induced dissociation (CID) was used, a process that is usually accompanied by existence of a strong internal fragmentation due to the loss of phosphoric acid or a phosphate group and water, also called neutral loss. While presence of neutral loss is useful in phosphopeptide identification, it usually diminishes y- and b- ions. This is especially true for p-S and p-T which are more labile, and thus readily undergo gas-phase elimination of the phosphate moiety from both precursor and fragment ions during MS/MS.5 The milk caseins have been used as a classical example of phosphorylation in many studies, due to the fact that caseins are the most abundant phosphoproteins in milk, and are utilized as sources of amino acids, carbohydrates, calcium, and phosphorus.6 Caseins exist in four different variants including αS1-, αS2, β-, and κ-casein. Despite little homology, there is a rare conserved sequence of (SSSEE) present in αS1-, αS2, and β-casein that serves as a multi-phosphorylation site.7

Although κ-casein does not contain this conserved sequence motif, it does possess two

phosphorylation sites embedded in a very large tryptic peptide. These two phosphorylation sites of κ-casein were recently characterized using the truncated form of the protein because both phosphorylation and glycosylation are found exclusively in the C-terminus.8



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Analyses of the multi-phosphorylation sites of bovine β-casein by matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) has been recently reported.9 Similarly phosphorylation status of αS1- and β-casein in infant formula is profiled using LCMS/MS.10 The latter study only reported the MS/MS spectrum of tetra-phosphopeptide in βcasein, but no tandem mass spectra were provided for αS1-casein multi-phosphopeptides. In a separate study, multi-phosphopeptides of both standard β-casein and β-casein from bovine milk samples

were

characterized

using

μHPLC–ESI-MS/MS

following

enrichment

and

dephosphorylation by alkaline phosphatase strategies.11 However, the sites of phosphorylation were only estimated by analyzing the partially dephosphorylated peptides which were generated in the incomplete dephosphorylation process.

Recently, αS1-, αS2, and β-casein standards and

native bovine αS1-, αS2, and β-casein phosphopeptides were identified in bovine milk using LCMS/MS.12 Though the published report claimed identifying 20 phosphopeptides, most of them were variations of the same mono- and di-phosphopeptides. Sporadic proteomic reports of casein phosphorylation in ovine milk 13-14 and caprine milk15 also exist in the literature. Despite the fact that many phosphopeptides have been reported, only a limited number of phosphorylation sites are determined. The proteomic evaluation of milk and milk protein modulation during clinical mastitis has also been the focus of several studies.16-20 Although knowledge of host response to pathogens during mastitis in bovine milk has grown goats.

21-22

, little comparative data exists on lactating dairy

Due to inherent nutritional factors of goat milk, and intolerance, allergies, and

gastrointestinal disorders that have been related to bovine milk, the consumption of goat milk has risen steadily in developing countries.23-25 The first analysis of goat milk protein modulation over the course of experimentally induced mastitis using proteomics has recently been reported by our


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group.26 We observed various degree of PTM in several milk proteins including the caseins. Accordingly, the aims of the current study were to generate a comprehensive profile of goat milk casein phosphopeptides and to accurately determine the phosphorylation sites. Because the molecular functions of proteins can be disrupted during disease due to changes in PTM profiles, an additional goal was to evaluate whether any of the phosphorylation patterns evidenced in the goat milk were altered in milk collected from goats with experimentally-induced endotoxin mastitis.

MATERIALS AND METHODS Goats: In these experiments, six clinically healthy mixed breed dairy goats in their first lactation, approximately 100-150 days in milk, and ranging from 49 to 63 kg in weight were used. All goats were free of major mastitis pathogens as determined by negative bacteriological examinations (Quality Milk Production Services, Cornell University, Ithaca, NY), and had no prior incidence or treatments for mastitis. The care and use of all animals in the current study were approved by the Center for Veterinary Medicine Office of Research Institute Animal Care and Use Committee.

Intra-mammary challenge with LPS: Intra-mammary challenge with LPS was performed as previously described.26 Briefly, on the day of challenge, the right udder half on each of the 6 does was infused with 4 µg/kg LPS in 2 mL of pyrogen-free saline using a sterile disposable teat cannula. The left half of each udder was infused with 2 mL of sterile phosphate buffered saline (PBS) in order to serve as a control to the challenged half.



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Sample Collection and Handling: Approximately 30 mL of milk was collected aseptically from the challenged half of the goat mammary gland as outlined by the National Mastitis Council. Briefly, a few streams of milk were discarded and the teat end scrubbed with 70% ethanol until clean. Milk samples were collected 3 days prior to challenge for baseline milk samples, and at 24 hour following LPS challenge. After collection, the milk was centrifuged at 4000×g at 4oC for 15 min, and the resulting fat layer removed. The skim milk was transferred to sterile 1.5 mL microcentrifuge tubes and kept frozen at -20oC until further analysis.

SDS-PAGE: Approximately 100 μg of each sample was re-suspended in 45 μL loading buffer (0.5 M Tris, pH 6.8, 10% SDS, 38% glycerol, 0.1% bromophenol blue) and boiled for 7 min, allowed to cool to room temperature, and centrifuged at 14,000×g for 5 min prior to loading. Each sample was loaded into a well of an one-dimensional 4–20% SDS-PAGE precast gel (BioRad, Hercules, CA) and electrophoresed at 150 V for 55 min. Gels were washed with deionized water (dH2O) and stained with Coomassie Blue stain (Bio-Rad Laboratories). The gels were destained in dH2O for 1 hr and imaged using a laser densitometer (Model PDSI, Molecular Dynamics Inc, Sunnyvale, CA). Casein variants were isolated by excision of gel bands at MW 20-30 kD and subjected to in-gel digestion as previously described 27 with some modifications. Briefly, the gel bands were sliced into approximately 1.0 mm cubes, dehydrated in acetonitrile for 15 min, and dried in vacuum centrifuge (MiVac Module Series Centrifuge Vacuum Concentrator; Gardiner, NY, USA) for 30 min at room temperature. The samples were then washed with 100 μL of a 100 mM ammonium bicarbonate buffer, pH 8.0 prior to reduction with 50 mL dithiothreitol (DTT, 10


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mM). Samples were then alkylated using 50 μL iodoacetamide (50 mM). Gel bands were rehydrated in 10 ng/μL of sequencing grade modified trypsin (Promega, Madison, WI) in 50 mM ammonium bicarbonate and allowed to digest on ice for 15 min. Excess solution was replaced with 25 μL of 50 mM ammonium bicarbonate and the samples were digested overnight at 37oC. Peptides were extracted with 50 μL of 50% acetonitrile and water containing 5% trifluoroacetic acid (TFA) followed by 50 μL of acetonitrile containing 5% TFA. Peptides were concentrated in a vacuum centrifuge to completion and reconstituted in 25 μL 0.1% TFA in dH2O. Extracted peptides were stored at -20°C until further analysis.

Nano-scale liquid chromatography tandem mass spectrometry (nLC-MS/MS) analysis: nLC-MS/MS analyses were carried out by injecting 5 µL of each sample into a nano flow HPLC instrument (Eksigent; Dublin, CA, USA), coupled to a linear ion trap LTQ Velos mass spectrometer (Thermo Fisher Scientific; Waltham, MA, USA). Peptides were loaded onto a nanoAcquity 100 μm x 100 mm C18 reverse phase ultra-performance liquid chromatography (UPLC) column (Waters; Milford, MA, USA). The mobile phases consisted of 0.1% formic acid in water (A) and acetonitrile (B), respectively. Separation was achieved through applying a linear gradient from 5─20% B in 15 min, 20─60% B in 50 min, 60─80% B in 60 min, and 8095% B in 70 min at 400 nL/min flow rate. The eluate was electrosprayed by applying 1.7 kV to the terminal PicoTip emitter (25mm id, 10mm id tip, New Objective, MA, USA). The LTQ Velos mass spectrometer was operated in positive ion mode, and spectra were acquired for 70 min in data-dependent tandem MS mode. For MS/MS spectra acquisition, the ten most intense ions in each MS survey scan (over the range 400 to 2000 m/z) were subjected to MS/MS by collision-induced dissociation (CID). Normalized collision energy was set to 35% and activation



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time to 10 ms. Singly charged ions, and ions for which no charge state could be determined, were excluded from the selection. Mass spectral peak lists were generated using Proteome Discoverer software (v. 1.2, Thermo Fisher Scientific) without smoothing or signal-to-noise thresholding. To minimize carryover, two consecutive 55 min blank injections were run between each sample.

Database Searching: For peptide identification, mass spectral peak lists were submitted to Mascot (v.2.3.0; Matrix Science, London, UK) searches of the Uniprot KB Swiss-Prot protein sequence library (v.57.15) using proteome Discoverer Daemon. Search parameters included: other mammalia, 1.3 Da precursor mass tolerance, 0.8 Da product ion mass tolerance. cleavages

was

used

as

the

enzyme

and

ESI-TRAP

Trypsin up to two missed as

the

instrument

type.

Carbamidomethylation of cysteine (+57.02) was set as fixed modification along with methionine oxidation (+15.99), serine, threonine, and tyrosine phosphorylation (+79.97) as variable modifications. All data were further filtered by peptide Ion Score greater than the Mascot Identity Threshold (at P < 0.05).


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RESULTS SDS-PAGE and Identification of Casein Phosphorylation Sites: Proteins in milk samples obtained from healthy goats before and after experimental induction of endotoxin mastitis were pre-fractionated using SDS-PAGE. To evaluate the quality of the electrophoretic separation, the gels were stained with Coomassie brilliant blue and subsequently visualized (Figure 1). The lanes in the molecular weight range expected for casein variants (20 kD – 30 kD) were excised from the gels, in-gel digested, and analyzed by nLC tandem mass spectrometry.

MS/MS analysis of the resulting peptides confirmed that the excised bands

corresponded to κ-casein (21441 kD), αS1-casein (24290 kD), β-casein (24865 kD), and αS2casein (26389 kD). The overall protein sequence coverage varied; however, on average better than 75% sequence coverage was obtained for each of the caseins. All the predicted tryptic phosphopeptides in αS1, αS2, and β-casein were detected (Table 1), with the exception of one hexa-phosphopeptide in αS1-casein. These included characterization of 18 different phosphorylation sites from a series of mono- and multi-phosphopeptides as shown in Table 1 column 4.

The sites of phosphorylation were accurately assigned in three mono

phosphopeptide despite the presence of additional S or T residues in the sequences. Additionally, the conserved SSSEE sequence motif was detected as one tetra-phosphopeptide in β-casein and two multi-phosphopeptides in αS2-casein.

Analysis of phosphopeptides in β-casein: The β-casein present in goat milk has five reported phosphorylation sites at serine residues that result in two different tryptic, one mono- and one tetra-phosphopeptide.

The mono-

phosphopeptide (F48–K63) with amino acid sequence FQSEEQQQTEDELQDK was detected as



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doubly charged ion (MH2+= 1032.21) and was subsequently selected for tandem MS analysis (Figure 2a). The MS/MS spectrum indicated an intense neutral loss evidenced at m/z 982.77, corresponding to the doubly charged molecular ion with loss of phosphoric acid. Despite the presence of another threonine residue in the peptide, the detection of majority of y-ions, specifically y8 and y14, clearly indicated that phosphorylation occurred on S50, rather than on T56. Similarly as shown in Figure 2b, the tetra-phosphopeptide (E17–K43) containing the conserved

sequence

motif

(SSSEE)

with

the

full

amino

acid

sequence

EQEELNVVGETVESLSSSEESITHINK was detected as triply charged ion [MH3+= 1103.10]. Regardless of the presence of multiple T and S residues within the peptide, the sites of phosphorylation were accurately assigned at S32, S33, S34, and S37, rather than at T27, S30, and T39.

Specifically, observing intact threonine (y5) ruled out phosphorylation at T39.

Likewise, detecting phosphorylated serine at (y7, y11, and y12) established the presence of all four phosphorylation sites. The same two β-casein phosphopeptides were also accurately identified in mastitic milk samples obtained from the same goats following the induction of endotoxin mastitis.

Phosphorylation of αS1-casein: Three tryptic phosphopeptides, including one mono- one di-, and one hexa-phosphopeptide have been reported in αS1-casein, two of which were identified in the current study. The monophosphopeptide with the amino acid sequence KYNVPQLEIVPKSAEEQLHSMK was detected as a triply charged ion (MH3+= 888.79), with two abundant ion clusters due to the presence of proline residues in the sequence (Figure 3a).

The site of phosphorylation was accurately

assigned at S130 by detecting neutral loss (98 Da) from singly charged y10 and (49 Da) from


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doubly charged ion. All the ions in this spectrum were detected with 16 Da mass shifts due to the oxidation of the M residue. Similarly, the di-phosphopeptide (D58–K73) was detected in αS1-casein as doubly charged ion (MH2+= 951.32) corresponding to (MH+= 1901.64). The phosphorylation sites were accurately determined at the fourth and sixth amino acid residue from the N-terminus in the DIGSESTEDQAMEDAK sequence (Figure 3b).

These sites were

confirmed by observing a series of y-ions, specifically y10, y11, and y14 ions, which eliminated the possibility of phosphorylation on T64 and demonstrated attachment of one and two phosphate moieties on S63, and S61. Both of these αS1-casein phosphopeptides were also detected in mastitic goat milk samples. We did not detect the predicted hexa-phosphopeptide in the current analysis of goat milk in either of our sample sets, before and following experimental induction of endotoxin mastitis.

Multi-phosphorylation sites of αS2-casein: All four predicted tryptic phosphopeptides, two of which contained the conserved SSSEE sequence motifs, were detected in αS2-casein. These included 10 different phosphorylation sites at S23, S24, S25, S72, S73, S74, S77, S145, S147, and S159. A mono-phosphopeptide (T154– K165) with the amino acid sequence TIDMESTEVFTK was detected as doubly charged ion (MH2+= 749.32, supplementary data). The phosphorylation site was identified at S159 rather than at any of the three T residues present in this phosphopeptide, and was confirmed specifically by intact y2, y6, and y7.

Furthermore, from the N-terminus, detection of intact b3 and b4

eliminated the possibility of phosphorylation at T154.

Likewise, a di-phosphopeptide was

detected as a doubly charged ion (MH2+= 1283.54) in the miscleavage amino acid sequence



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(N131–K152; supplementary data). The phosphorylation sites at S145 and S147 were similarly confirmed in the amino acid sequence (NAGPFTPTVNREQLSTSEENSK). Two additional multi-phosphopeptides containing the conserved SSSEE sequence were also detected in αS2-casein.

A tri-phosphopeptide (M19–K37) with corresponding amino acid

sequence MEHVSSSEEPINIFQEIYK was detected as a doubly charged ion (MH2+= 1269.22, Figure 4a). Each ion exhibited 16 Da shifts in mass from the N-terminus which were due to M oxidation. Three dominant neutral loss peaks were identified as doubly charged ions at m/z 1219.71, m/z 1170.78, and m/z 1121.81, corresponding to the loss of one, two, and three H3PO4, respectively.

Regardless of strong neutral loss ions, some small but measurable signals

associated with y-type and b-type ions were also detected which confirmed the sequence identity and localization of the modification at S23, S24, and S25 residues. Despite lower apparent abundance, this multi-phosphopeptide was also detected in milk samples obtained from animals which were experimentally-induced by endotoxin mastitis. However, a tetra-phosphopeptide (N62–K86) with the amino acid sequence NANEEEYSIRSSSEESAEVAPEEIK in αS2-casein was never observed in the mastitic goat milk samples. This tetra-phosphopeptide was detected as triply charged ion (MH3+ = 1039.89) corresponding to (MH+ = 3117.08) in milk of healthy animals, (Figure 4b). After inspection of the MS/MS data, the sites of phosphorylation were assigned to S72, S73, S74, and S77.

Miscleavage was presumably occurred due to the

attachment of multiple phosphate moieties blocking the trypsin cleavage site. Instead of this miscleavage in the mastitic goat milk, two very strong tryptic peptides were observed. The first portion in the amino acid sequence (NANEEEYSIR) was detected as an intense doubly charged ion (MH2+= 614.18 Da) corresponding to (MH+= 1227.36 Da). The second dephosphorylated segment (SSSEESAEVAPEEIK) was detected as doubly charged ion (MH2+= 796.68 Da)


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corresponding to (MH+= 1592.36 Da). These observations clearly indicated that the peptide remained dephosphorylated in mastitic goat milk.

DISCUSSIONS Historically, detection of multi-phosphopeptides has been difficult both from separation and mass analysis perspective.28 Low-abundant peptides may co-elute with high-abundant peptides during chromatographic separation and cannot be distinguished following MS/MS.

Highly

abundant proteins generate an overwhelming number of peptides following proteolytic digestion, and tend to mask the detection of low-abundant modified peptides in the sample. Phosphorylation typically occurs at low stoichiometric rates because at any given time, only a small fraction of the proteins present in a given proteome are phosphorylated. Subsequently investigators often utilize strategies such as immobilized metal affinity chromatography (IMAC) and Titanium Dioxide (TiO2) chromatography to enrich for phosphopeptides and reduce sample complexity. Depending on the number of phosphorylated moieties, TiO2 has a preference for mono-phosphopeptides and IMAC is generally biased towards multi-phosphopeptides.29-30 In this study enrichment efficiency for all phosphopeptides and potential of missing phosphorylated species in a single gel band was of concern and thus no enrichment strategies were used. The detection and characterization of casein phosphopeptides is further complicated by the existence of the conserved SSSEE sequence domain, as the presence of the glutamic acid residues increases the hydrophilic nature of the multi-phosphopeptides. Despite their large size, majority of these tryptic phosphopeptides eluted early during chromatographic separation. Due to the large size of the multi-phosphopeptides, many were either not detected following database



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searching, or were assigned very low peptide score. Consequently, manual inspection of the MS/MS spectra to validate the identifications was necessary. Accurate database searching related to peptide size were also evidenced for the multiphosphopeptides EQEELNVVGETVESLSSSEESITHINK, KYNVPQLEIVPKSAEEQLHSMK, and NAGPFTPTVNREQLSTSEENSK. For each of these peptides, more than one potential phosphorylation site exist. As expected, manual inspection was time-consuming; nonetheless, extensive searching of the RAW files eliminated false identification and accurately annotated the correct sites of phosphorylation that might otherwise have gone undetected. In a large scale phosphoproteome analysis, manual inspection of individual phosphopeptide would not be possible, and this issue will remain an obstacle pending further augmentation and expansion of available databases for different species as well as associated bioinformatics tools. In αs1-casein, one mono (S130) and one di-phosphopeptide (S61, S63) were successfully identified. As predicted by database similarity, the αs1-casein present in goat milk should have an additional multi-phosphopeptide, which corresponds to six phosphorylation sites (S79, S80, S81, S82, S83, and S90).31 This hexa-phosphopeptide containing the conserved SSSEE motif was not observed, most likely due to the extreme hydrophilic nature, the instability, or in-source decay. Different phosphorylation patterns have been reported for milk αs1-casein of different mammalian species. In human, 2-D electrophoresis combined with MALDI-TOF/TOF MS revealed that αs1-casein has 8 phosphorylation sites.32

Similarly, two to six different sites for

αs1-casein were observed in equine.33 Reports of casein phosphopeptides in sheep have likewise detailed different phosphorylation forms of αS1-, αS2-, and β-casein within the same amino acid sequence.14 All of these analyses point to the fact that the phosphorylation pattern of caseins is highly variable within and among different casein genetic variants of different mammalian


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species. However, finding deviations within different genetic variants was not the focus of our study. Rather, the primary interest was to obtain a comprehensive profile of the goat milk casein phosphoproteome and to determine if the phosphorylation pattern(s) of goat milk caseins changed as a result of disease. Caseins in milk samples obtained from goats following experimental-induction of endotoxin mastitis were analyzed and despite lower apparent abundance, several multi-phosphopeptides were detected. However, the tetra-phosphopeptide in αS2-casein was never observed in the mastitic goat milk samples. Instead two separate tryptic peptides were observed implying that phosphorylation at S72, S73, S74, and S77 induced this particular miscleavage in the αS2-casein found in milk collected from healthy goats. The absence of this multi-phosphopeptide in the mastitic milk samples was a result of disease, and therefore, could potentially be used as a marker for milk quality.

A direct link between αs2-casein phosphorylation patterns and

modulation due to disease has not yet been established. Prior studies regarding allergic reactions and intolerance of milk proteins have revealed that IgE binding to αs2-casein in bovine milk could be partially or completely inhibited by two variants of dephosphorylated αs2-casein peptides.34 This report has also indicated that some of the phosphorylated regions of αs2-casein, including residues S73-S77, form part of the IgE epitope, and the phosphates are involved in antibody recognition. The most direct evidence of mastitis affecting milk casein phosphorylation patterns could perhaps be credited to early reports of increased phosphatase and phosphorylated esters levels in the milk of cows with clinical mastitis.35 Later reports indicated that mineral contents in bovine milk are altered during mastitis, and that phosphorous levels in particular were diminished during infection, presumably due to the influx of blood constituents into mammary gland.36 Similarly,



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calcium and phosphorus concentrations were shown to be negatively affected by the presence of mastitis-causing pathogens and as well as by increased somatic cell counts.37-38 The current observation provides further evidence that phosphorylation patterns of milk caseins may be modulated as a result of mastitis infections. Nonetheless, additional investigations are needed to discern the role of kinases and phosphatases, as well as the exact mechanisms that govern the phosphorylation of goat milk caseins, and apparent modulations in casein phosphorylation during mastitis. Despite the need for further investigations to elucidate the potential disease-specificity of casein phosphorylation patterns, the current study represents the first comprehensive analysis of the goat milk casein phosphoproteome, including the accurate determination of the sites of phosphorylation of all of the major goat milk caseins.

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Moreno, F.J. Detection of two minor phosphorylation sites for bovine κ-casein macropeptide by reversed-phase liquid chromatography-tandem mass spectrometry. J. Agric. Food Chem. 2011, 59 (20) 10848-10853. (9)

Schmidt, A.; Csaszar, E.; Ammerer, G.; Mechtler, K. Enhanced detection and

identification of multiply phosphorylated peptides using TiO2 enrichment in combination with MALDI TOF/TOF MS. Proteomics 2008, 8 (21) 4577–4592. (10) Miquel, E.; Gómez, J.A.; Alegría, A.; Barberá, R.; Farré, R.; Recio, I. Identification of casein phosphopeptides released after simulated digestion of milk-based infant formulas. J Agric Food Chem. 2005, 53 (9) 3426-3433.



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(17) D'Auria, E.; Agostoni, C.; Giovannini, M.; Riva, E.; Zetterström, R.; Fortin, R.; Greppi, G.F.; Bonizzi, L.; Roncada, P. Proteomic evaluation of milk from different mammalian species as a substitute for breast milk. Acta Paediatr. 2005, 94 (12) 1708-1713. (18) Boehmer, J.L.; Bannerman, D.D.; Shefcheck, K.J.; Ward, J.L. Proteomic analysis of differentially expressed proteins in bovine milk during experimentally induced Escherichia coli mastitis. J. Dairy Sci. 2008, 91 (11) 4206-4218. (19) Boehmer, J.L.; Ward, J.L.; Peters, R.R.; Shefcheck, K.J.; McFarland, M.A.; Bannerman, D.D. Proteomic analysis of the temporal expression of bovine milk proteins during coliform mastitis and label-free relative quantification. J. Dairy Sci. 2010, 93 (2) 593-603. (20) Boehmer, J.L.; DeGrasse, J.A.; McFarland, M.A.; Tall, E.A.; Shefcheck, K.J.; Ward, J.L.; Bannerman, D.D. The Proteomic Advantage: Label- Free Quantification of Proteins Expressed in Bovine Milk during Experimentally Induced Coliform Mastitis. Vet. Immunol. Immunop. 2010, 138 (4) 252-266. (21) Smolenski, G.; Haines, S.; Kwan, F. Y.; Bond, J.; Farr, V.; Davis, S.R.; Stelwagen, K.; Wheeler, T.T. Characterization of host defense proteins in milk using a proteomic approach. J. Proteom Res.2007, 6 (1) 207-215. (22) Danielsen, M.; Codrea, M.C.; Ingvartsen, K.L.; Friggens, N.C.; Bendixen, E.; Røntved, C.M. Quantitative milk proteomics - Host responses to lipopolysaccharide-mediated inflammation of bovine mammary gland. Proteomics 2010, 10 (12) 2240-2249.



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(23) Bellioni-Businco, B.; Paganelli, R.; Lucenti, P.; Giampietro, P. G.; Perborn, H.; Businco, L. Allergenicity of goat’s milk in children with cow’s milk allergy. J. Allergy Clin. Immunol. 1999, 103 (6) 1191-1194. (24) McCullough, F. S. W. Nutritional evaluation of goat’s milk. Br. Food J. 2003, 105 (45) 239-251. (25) Sanz Ceballos, L.; Sanz Sampelayo, M.R.; Gil Extremera, F.; Rodriguez Osorio, M. Evaluation of the allergenicity of goat milk, cow milk, and their lactosera in a guinea pig model. J. Dairy Sci. 2009, 92 (3) 837-846. (26) Olumee-Shabon, Z.; Swain, T.; Smith, E.A.; Tall, E.; Boehmer, J.L. Proteomic Analysis of Differentially Expressed Proteins in Caprine Milk during Experimentally Induced Endotoxin Mastitis. manuscript accepted J. Dairy Sci. 2012. (27) Shevchenko, A.; Jensen, O.N.; Podtelejnikov, A.V.; Sagliocco, F.; Wilm, M.; Vorm, O.; Mortensen, P.; Shevchenko, A.; Boucherie, H.; Mann, M. Linking genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two dimensional gels. Proc. Natl. Acad. Sci. U. S. A. 1996, 93 (25) 14440-14445. (28) Mann, M.; Ong, S.E.; Grønborg, M.; Steen, H.; Jensen, O.N.; Pandey, A. Analysis of protein phosphorylation using mass spectrometry: deciphering the phosphoproteome. Trends Biotechnol. 2002, 20 (6) 261-268. (29) Larsen, M.R.; Thingholm, T.E.; Jensen, O.N.; Roepstorff, P.; Jorgensen, T.J. Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol Cell Proteomics 2005, 4 (7) 873–886.


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(30) Thingholm, T.E.; Larsen, M.R. The use of titanium dioxide micro-columns to selectively isolate phosphopeptides from proteolytic digests. Methods Mol Biol. 2009, 527, 57– 66. (31) Brignon, G.; Mahe, M-F.; Grosclaude, F.; Ribadeau-Dumas, B. Sequence of caprine alpha s1-casein and characterization of those of its genetic variants which are synthesized at a high level, alpha s1-CnA, B and C. Protein Seq. Data Anal. 1989, 2 (3) 181-188. (32) Sørensen, E.S.; Møller, L.; Vinther, M.; Petersen, T.E.; Rasmussen, L.K. The phosphorylation pattern of human alphas1-casein is markedly different from the ruminant species. Eur. J. Biochem. 2003, 270 (17) 3651-3655. (33) Mateos, A.; Miclo, L.; Molle, D.; Dary, A.; Girardet, J.M.; Gaillard, J.L. Equine alpha S1-casein: characterization of alternative splicing isoforms and determination of phosphorylation levels. J. Dairy Sci. 2009, 92 (8) 3604-3615. (34) Bernard, H.; Meisel, H.; Creminon, C.; Wal, J.M. Post-translational phosphorylation affects the IgE binding capacity of caseins. FEBS Lett. 2000, 467 (2-3) 239-244. (35) Chanda, R.; Owen, E.C.; Watts, P.S. The Effect of Mastitis on the Phosphatase and Phosphorylated Esters in Cow's Milk. Biochem. J. 1951, 50 (2) ii. (36) Chanda, R. The effect of mastitis on the carotenoids, vitamin A and phosphorus compounds of milk. Biochem. J. 1953, 54 (1) 68–77.



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Table 1. Phosphopeptides obtained in goat milk caseins Fragment 58-73 119-139 19-37 62-86 131-152 154-165 17-43 48-63

Peptide Sequence

Protein

DIGSESTEDQAMEDAK KYNVPQLEIVPKSAEEQLHSMK MEHVSSSEEPINIFQEIYK NANEEEYSIRSSSEESAEVAPEEIK NAGPFTPTVNREQLSTSEENSK TIDMESTEVFTK EQEELNVVGETVESLSSSEESITHINK FQSEEQQQTEDELQDK

α-S1 casein α-S1 casein α-S2 casein α-S2 casein α-S2 casein α-S2 casein β- casein β- casein


Site 2 1 3 4 2 1 4 1

m/z

[M+H]+

951.32 888.79 1269.22 1039.89 1283.54 749.32 1103.10 1032.21

1901.64 2664.31 2537.15 3117.08 2566.08 1497.63 3307.29 2063.42


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Marker

0h

24h

200.0 116.0 97.4 66.0 45.0 31.0

21.5

14.5 6.5

Figure 1. Samples were separated by one-dimensional SDS-PAGE and stained with Coomassie.Blue. Gel bands at 20-30 kD correspond to different casein isoforms were excised and in-gel digested prior to nLC-MS/MS analysis.


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Figure 2. Tandem mass spectrum of a mono-phosphopeptide in β-casein was detected as a doubly charged ion (MH2+=1032.21) which allowed to identify the amino acid sequence and phosphorylation site (a). Loss of water molecule was observed from the y-ions due to the presence of multiple Q residues. MS/MS spectrum of a large tetra-phosphopeptide was detected as a triply charged ion (MH3+=1103.10) in β-casein (b), indicating the site of phosphorylation at four serine residues shown in bold. The y- and b-ions are marked along with the most prominent peaks in the spectrum due to the loss of phosphoric acids from the precursor ion. The sequences of the phosphopeptides are reported on top of the spectra. 24 ACS Paragon Plus Environment


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Figure 3. MS/MS spectrum at (MH2+=888.79) generated form a mono-phosphopeptide in αS1casein (a). In addition to strong neutral loss ions, abundant peaks due to the presence of P residues (y12, y18, b4, and b10), were also observed. Tandem mass spectrum of a diphosphopeptide in αS1-casein was also detected (b). The most prominent peaks in this spectrum were observed at m/z=902.65 and m/z=853.70 corresponding to doubly charged ions due to the loss of one and two phosphoric acids from the precursor ion, respectively.


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Figure 4. Tandem mass spectrum of a tri-phosphopeptide detected in αS2-casein with three abundant peaks due to the losses of one, two, and three phosphoric acids (a). The relatively lower abundant y- and b-ions confirmed the amino acid sequence reported on top of the spectrum. MS/MS spectrum of the triply charged ion (MH3+=1039.89) detected as a miscleavage tetra-phosphopeptide (b). The y- and b- ion series are marked in the spectrum and clearly identify this multi-phosphopeptide in αS2-casein. This tetra-phosphopeptide was only observed in milk samples obtained from healthy animals.



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Marker

0h

24h

200.0 116.0 97.4 66.0 45.0 31.0

21.5

14.5 6.5

Figure 1. Samples were separated by one-dimensional SDS-PAGE and stained with Coomassie.Blue. Gel bands at 20-30 kD correspond to different casein isoforms were excised and in-gel digested prior to nLC-MS/MS analysis.


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Figure 2. Tandem mass spectrum of a mono-phosphopeptide in β-casein was detected as a doubly charged ion (MH2+=1032.21) which allowed to identify the amino acid sequence and phosphorylation site (a). Loss of water molecule was observed from the y-ions due to the presence of multiple Q residues. MS/MS spectrum of a large tetra-phosphopeptide was detected as a triply charged ion (MH3+=1103.10) in β-casein (b), indicating the site of phosphorylation at four serine residues shown in bold. The y- and b-ions are marked along with the most prominent peaks in the spectrum due to the loss of phosphoric acids from the precursor ion. The sequences of the phosphopeptides are reported on top of the spectra.



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Figure 3. MS/MS spectrum at (MH2+=888.79) generated form a mono-phosphopeptide in αS1casein (a). In addition to strong neutral loss ions, abundant peaks due to the presence of P residues (y12, y18, b4, and b10), were also observed. Tandem mass spectrum of a diphosphopeptide in αS1-casein was also detected (b). The most prominent peaks in this spectrum were observed at m/z=902.65 and m/z=853.70 corresponding to doubly charged ions due to the loss of one and two phosphoric acids from the precursor ion, respectively.


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Figure 4. Tandem mass spectrum of a tri-phosphopeptide detected in αS2-casein with three abundant peaks due to the losses of one, two, and three phosphoric acids (a). The relatively lower abundant y- and b-ions confirmed the amino acid sequence reported on top of the spectrum. MS/MS spectrum of the triply charged ion (MH3+=1039.89) detected as a miscleavage tetra-phosphopeptide (b). The y- and b- ion series are marked in the spectrum and clearly identify this multi-phosphopeptide in αS2-casein. This tetra-phosphopeptide was only observed in milk samples obtained from healthy animals.



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Graphical Abstract 254x190mm (300 x 300 DPI)


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Detection of Casein Phosphopeptides in Goat Milk - American

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