Temporal Changes in Milk Proteomes Reveal Developing Milk Functions

Article pubs.acs.org/jpr

Temporal Changes in Milk Proteomes Reveal Developing Milk Functions Xinliu Gao,† Robert J. McMahon,† Jessica G. Woo,‡ Barbara S. Davidson,‡ Ardythe L. Morrow,‡ and Qiang Zhang*,† †

Mead Johnson Nutrition Pediatric Nutrition Institute, 2400 West Lloyd Expressway, Evansville, Indiana 47721, United States Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio 45213, United States

‡

S Supporting Information *

ABSTRACT: Human milk proteins provide essential nutrition for growth and development, and support a number of vital developmental processes in the neonate. A complete understanding of the possible functions of human milk proteins has been limited by incomplete knowledge of the human milk proteome. In this report, we have analyzed the proteomes of whey from human transitional and mature milk using ion-exchange and SDS-PAGE based protein fractionation methods. With a larger-than-normal sample loading approach, we are able to largely extend human milk proteome to 976 proteins. Among them, 152 proteins are found to render significant regulatory changes between transitional milk and mature milk. We further found that immunoglobulins sIgA and IgM are more abundant in transitional milk, whereas IgG is more abundant in mature milk, suggesting a transformation in defense mechanism from newborns to young infants. Additionally, we report a more comprehensive view of a complement system and associated regulatory apparatus in human milk, demonstrating the presence and function of a system similar to that found in the circulation but prevailed by alternative pathway in complement activation. Proteins involved in various aspects of carbohydrate metabolism are also described, revealing either a transition in milk functionality to accommodate carbohydrate-rich secretions as lactation progresses, or a potentially novel way of looking at the metabolic state of the mammary tissue. Lately, a number of extracellular matrix (ECM) proteins are found to be in higher abundance in transitional milk and may be relevant to the development of infants’ gastrointestinal tract in early life. In contrast, the ECM protein fibronectin and several of the actin cytoskeleton proteins that it regulates are more abundant in mature milk, which may indicate the important functional role for milk in regulating reactive oxygen species. KEYWORDS: milk proteome, immune system, carbohydrate metabolism, extracellular matrix proteins, mass spectrometry

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INTRODUCTION The proteins found in human milk play a number of critical roles for the developing neonate.1 While many are completely digested even in the weakly acidic environment of the neonatal gut to provide essential amino acids for growth and development, other proteins are either partially or poorly broken down and exert functionality related to its structure digestive products per se.2−4 For example, some milk proteins facilitate the digestion and uptake of other nutrients in milk,5 while others defend against pathogenic bacteria and viruses, help bolster the acquired immune system,5−7 influence cognitive development,7 affect metabolic development8−11 and aid the development12 and maturation of the gastrointestinal tract (GIT).4,13,14 In an effort to further explore the benefits that human milk can provide, numerous proteomic and nonproteomic studies15 have been carried out, investigating the proteins in milk whey and milk fat globule membrane (MFGM).16−24 Palmer et al. used immunoprecipitation to remove high-abundance milk proteins, which resulted in 152 milk proteins being identified.22 © 2012 American Chemical Society

Recently, Liao et al. used ProteoMiner to enrich low-abundance proteins where 115 milk proteins were found.19 More recently, Hettinga used SDS-PAGE to fractionate milk proteins, leading to 268 proteins being identified.16 The number of proteins characterized in human whey has increased steadily over the years. Despite this, however, there is still no comprehensive view of the milk proteome. As a result, the biology and f unctions of milk proteins remain incompletely understood. Defining in-depth the milk proteome and how it changes during the course of lactation is a key step toward an improved understanding of milk biology and function and has the potential to provide novel insights in this area. For instance, while the complement system found in blood is well-known to play an important role in host defense, its corresponding role in milk is still uncertain.25 The defense system has the potential to damage host tissue; however, its regulation and potential impact on breastfeeding women is largely undefined. While Received: April 30, 2012 Published: June 8, 2012 3897

dx.doi.org/10.1021/pr3004002 | J. Proteome Res. 2012, 11, 3897−3907

Journal of Proteome Research

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at a flow rate of 900 μL/min. A NaCl gradient was started with 100% A (5% MeOH with 20 mM NaCl) and ramped to 100% B (5% MeOH with 800 mM NaCl) over 35 min, followed by a gradient ramped from 30% C (5% MeOH with 800 mM NaClO4) to 100% C over 10 min and then held at 100% C for 15 min. UV absorption was monitored at both 220 and 280 nm (Supplementary Figure 1A). The eluent was manually collected to obtain individual samples that had particularly high abundance of protein material. Approximately 15 fractions with retention times ranging from 2 to 55 min were collected. Proteins fractionated by ion exchange chromatography were buffer-exchanged with 100 μL of 50 mM NH4HCO3 using a 10 kDa MWCO. PPS Silent surfactant in 50 mM NH4HCO3 was added to each fraction for a final concentration of 0.1%. DTT in 50 mM NH4HCO3 was added to each fraction for a final concentration of 5 mM. Protein fractions were incubated with DTT at 50 °C for 30 min and cooled to room temperature (r.t.), followed by incubation with fresh IAA (15 mM final concentration) at r.t. for 30 min in the dark. After alkylation with IAA, protein fractions were subjected to in vitro enzymatic digestion using trypsin (in 50 mM NH4HCO3; 1:50 protein-toenzyme ratio) at 37 °C overnight. To quench trypsin reactivity, HCl (200 mM final concentration) was added at 37 °C for 45 min at the end of digestion. The resulted tryptic peptides were stored at −80 °C until ready for liquid-chromatographic electrospray-ionization tandem mass-spectrometric (LC−ESI/ MS/MS) analysis.

milk proteins are also observed to promote gastrointestinal development,4,13 the protein candidates that account for the maturation of the GIT in young infants remain an open question. Here, we use a fractionation approach with larger-thannormal sample loads to explore less abundant proteins. We report the comprehensive whey proteome of human transitional and mature milk. We further describe the significant and consistent changes in protein abundances between these two types of milk samples. On the basis of our data, we delineate biological processes and signaling pathways that are novel or whose importance in milk is not yet understood.

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MATERIALS AND METHODS

Chemicals

Liquid chromatography mass spectrometric- and proteomicgrade sodium chloride (NaCl), sodium perchlorate (NaClO4), ammonium bicarbonate (NH4HCO3), formic acid (FA), hydrogen chloride (HCl), methanol (MeOH), acetonitrile (ACN), H2O, dithiothreitol (DTT), iodoacetamide (IAA) and trypsin were purchased from Sigma-Aldrich (St. Louis, MO). PPS Silent surfactant was purchased from Protein Discovery (Knoxville, TN). Criterion Tris-HCl gradient gel, Laemmli sample buffer (4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromophenol blue, and 0.125 M TrisHCl, pH 6.8), Tris/Glycine/SDS running buffer (25 mM Tris, 192 mM glycine, and 0.1% SDS, pH 8.3), Coomassie blue stain, and Immun-Star Western C chemiluminescent reagent were purchased from Bio-Rad Laboratory (Hercules, CA).

SDS-PAGE Fractionation and In-Gel Digestion

In a parallel setup, samples were fractionated with SDS-PAGE. Approximately 15 μL, representing 500 μg of protein, was mixed with an equal volume of Laemmli sample buffer. The mixtures were heated to 65 °C for 10 min and loaded immediately on a 10−20% gradient gel. The cathodic and anodic compartments were filled with Tris/Glycine/SDS running buffer. Electrophoresis was carried out at 200 V constant power until the dye front reached the bottom of the gel. Coomassie blue was used to visualize the protein bands and the SDS-PAGE gel was scanned using a ChemiDoc imaging system (Bio-Rad) (Supplementary Figure 1B). The SDS-PAGE gels were cut to separate the fractions for each sample lane (transitional milk or mature milk), followed by in-gel tryptic digestion and peptide extraction.26 The volume of the peptide extracts was reduced to 5 μL under vacuum and 1% FA was added for a final volume of 30 μL. The resulted tryptic peptides were stored at −80 °C until ready for LC−ESI/MS/MS analysis.

Milk Samples

Human milk samples were obtained from three healthy donors at approximately the first week of lactation (day 8, 7, and 6 for donors 1, 2, and 3, respectively; termed transitional milk) and 3 months after the onset of lactation (termed mature milk). All samples were collected at the Cincinnati Children’s Hospital (Cincinnati, OH) using protocols approved by institutional review boards. The healthiness of donors was assessed and verified each time prior to milk donation. The samples were collected from one breast and frozen and stored in plastic containers at −80 °C. Samples from each donor were analyzed independently without pooling. When ready for use, milk was thawed at 4 °C, then ultracentrifuged at 4 °C (100 000g for 60 min) so that samples had pellet casein micelles at the bottom, a fat layer on the top, and de-lipidated whey supernatant in the middle. To obtain protein samples, the whey layer was filtered using a 10 kDa molecular-weight cutoff (MWCO) device (Millipore, Billerica, MA) and subjected to buffer-exchange with water.

Protein Identification by Nanocapillary Chromatography and Mass Spectrometry

Nanocapillary LC−ESI/MS/MS analysis was conducted on an LTQ/OrbitrapXL hybrid mass spectrometer (Thermo-Fisher Scientific, San Jose, CA) coupled with a nano UltraHPLC (Eksigent, Dublin, CA). For each fraction, 5 μL of tryptic digest was loaded into a 100 μm i.d. × 2.5 cm reversed-phase (RP) trap column packed with 5-μm C18 particles (IDEX, Oak Harbor, WA). Peptide separations were carried out using an approximately 15-cm-long uncoated 75-μm i.d., 15-μm nanotip fused-silica column (New Objectives, Woburn, MA) packed inhouse with 3-μm C18 particles (Michrom Bioresources, Auburn, CA). The separation was started with 98% mobile phase A (H2O/0.1% FA), 2% B (ACN/0.1% FA) for 2 min and then an 88 min linear gradient to 30% B, followed by an 80% B wash for 20 min with a flow rate of 300 nL/min. Full-scan mass

Mixed-Bed Ion Exchange Fractionation and In-Solution Tryptic Digestion

Transitional and mature-milk whey proteins so obtained were diluted 1:1 with 40 mM NaCl in 10% MeOH for injection into a 4 mm i.d. × 10 mm WAX guard column (PolyWAX LP, particle size 5 μm, pore size 1000 Å, PolyLC, Inc., Columbia, MD) connected to a 4.6 mm i.d. × 200 mm mixed-bed ion exchange (IEX) column (PolyCAT A and PolyWAX LP 1:1, particle size 5 μm, pore size 1000 Å, PolyLC, Inc.). The amount of sample loaded corresponded to 4 mg of protein material as determined with the Dumas combustion methodology using an FP-2000 analyzer (LECO, St. Joseph, MI). Protein separation was carried out using HPLC (U3000, Dionex, Sunnyvale, CA) 3898



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spectra were acquired in the LTQ/Orbitrap mass spectrometer in the mass-to-charge ratio (m/z) range of 350−2000 Da and with the resolution set to 60 000, followed by three datadependent MS/MS scans of the most abundant ions from the full scan with a normalized collision energy setting of 35 and minimum signal threshold of 1000 counts. The maximum injection time was 500 ms for parent-ion analysis and 100 ms for product-ion analysis. Target ions already selected for MS/ MS were dynamically excluded for 60 s. Three LC/MS/MS analyses were carried out for each fraction.

abundance ratios were determined with Partek by comparing the number of spectral counts observed for a particular protein in transitional milk and in mature milk. The total spectral counts of each protein obtained from SDS-PAGE fractionation were used to assess relative protein abundance. The spectral counts were pooled across runs and samples. To make total spectral counts comparable among proteins, each sample lane was cut into 12 slices at a similar manner and equal number of analysis was performed for each of the transitional and mature milk samples.

Database Searches and Protein Report

Bioinformatics Analysis

All MS/MS spectra with charges +2 and +3 were analyzed using Mascot (Matrix Science, London, U.K.; version 2.2.06). Mascot was set up to search against the human UniprotKB database (20 319 entries; version 2011_08) assuming the digestion enzyme was trypsin with a maximum of 1 missed cleavage allowed. Fragment b- and y-ions were used in peptide sequencing. The searches were performed with a fragment ion mass tolerance of 0.6 Da and a parent ion tolerance of 10 ppm. Iodoacetamide derivatization of cysteine was specified in Mascot as a fixed modification. Pyro-glu from E of the Nterminus, S-carbamoylmethylcysteine cyclization of the Nterminus, deamidation of asparagine and glutamine, oxidation of methionine, acetylation of the N-terminus and phosphorylation of serine, threonine, and tyrosine were specified in Mascot as variable modifications. The Scaffold program (Proteome Software, Inc., Portland, OR; version 3_00_07) was used to validate MS/MS-based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95% probability as specified by the Peptide Prophet algorithm.27 Protein identifications were accepted if they could be established at greater than 99% probability and contained at least two identified peptides by the Protein Prophet algorithm.28 Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Technical false-discovery rate (FDR) was estimated by search against a decoy database, resulting in a 2% FDR at the protein level.

Gene Ontology (GO) analysis of biological processes, cellular components, and molecular functions and KEGG pathway analysis for the milk proteome were carried out using the Database for Annotation, Visualization and Integrated Discovery (DAVID) software using the Homo sapiens genome as reference.32,33 Both GO and pathway analysis were reported with a Benjamini (FDR corrected) p-value < 0.05.34 Protein interaction networks were analyzed using Search Tool for the Retrieval of Interacting Genes/Proteins (STRING; version 9.0).35 STRING networks were calculated with a highest confidence score of 0.900 for the entire set of milk proteins and a high confidence score of 0.700 for carbohydrate metabolism and ECM protein subsets. Tightly connected protein interactions were clustered using MCL algorithm in STRING. Immunoblotting Analysis

Beta-1,4-galactosyltransferase 1 (B4GALT1), fructose-1,6-bisphosphatase 1 (FBP1), fructose-bisphosphate aldolase A (ALDOA), pyruvate kinase isozymes M1/M2 (PKM2), and acetyl-CoA acetyltransferase, cytosolic (ACAT2) antibodies were purchased from Santa Cruz Biotechnology, Inc. Immunoblotting was performed for a series of whey proteins extracted from transitional and mature milk (B4GALT1, FBP1, PKM2, ACAT2, and ALDOA). Samples containing 36.9 μg of each protein (or 184.5 μg in the case of ALDOA) were loaded onto a Criterion polyacrylamide gel. Electrophoreses were run for 50 min, transferred onto nitrocellulose membranes according to the instructions for the Trans-Blot Turbo transfer system (Bio-Rad), blocked 1 h in SuperBlock (Thermo-Pierce) at r.t., and incubated overnight with primary antibodies at various dilutions at 4 °C and secondary antibodies (1:5000) 1 h at r.t. Bands were detected using Immun-Star Western C chemiluminescent reagent and quantified using ChemiDoc (Bio-Rad).

Label-Free Semiquantification

Proteins fractionated by SDS-PAGE were used for quantification. Each sample lane was cut into 12 slices at a similar manner for both transitional and mature milk. Label-free spectralcounting semiquantification29 was performed with Scaffold program by comparing the number of MS/MS spectra identifying peptides of a given protein with an accepted identity (greater than 99% protein probability with at least two unique peptides identified) across three LC/MS/MS replicates. Peptide identifications that can be assigned to more than one protein were removed to avoid errors in protein quantification. Only proteins with number of spectral counts greater than or equal to 4 per LC/MS run were used for quantification to avoid widening of confidence intervals of spectral counting.30,31 Using the software program Partek (St. Louis, MO), a nested ANOVA that accounts for the data hierarchy including both biological and technical replicates was carried out to assess the statistical significance of differences in protein abundance between transitional milk and mature milk. Samples were grouped into two categories, transitional milk and mature milk. For each category, the technical replicates were nested into the biological replicates. Proteins with p-values less than 0.05 were considered to have significantly altered abundance. Relative

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RESULTS AND DISCUSSION

Characterization of the Milk Proteome

As described in the Materials and Methods, we employed both mixed-bed IEX technique36 and SDS-PAGE to fractionate proteins for identification. Proteins in fractionated milk samples were subjected to tryptic digestion and the resulting peptides identified by nano LC/MS/MS analysis. We found that the less abundant proteins could be identified with IEX and SDS-PAGE using larger-than-normal sample loads. With this approach, we expanded the previously known human milk proteins15,16,18,19,22 to 976 unique gene products (Supplementary Table 1). Of these protein entries, 90% were found in both transitional milk and mature milk (Supplementary Figure 2 inset), indicating a consistency in the proteome between these two lactation stages. Supplementary Figure 2 shows a plot of MS/MS spectral count versus probability ranking for each of 3899



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Figure 1. Enrichment analysis. (A) GO distributions for the human milk proteome in biological processes (blue), cellular location (green), and molecular function (orange). For each category, the factor of enrichment compared to the human average is shown adjacent to data bar. (B) KEGG pathways for the human milk proteome. Pathways with Benjamini p-values (adjusted p-values) < 0.05 were considered to be significantly enriched.

upregulation was observed for 65 proteins in transitional milk and 87 in mature milk (Supplementary Table 2).

the 976 proteins. The 126 most probable proteins contribute to 90.0% of the total spectral counts, while the 391 least probable proteins account for 1.0% of the total counts. Correlation between protein concentration obtained from ELISA analysis and their spectral counts shows that higher protein concentration is associated in general with higher spectral counts.37 The spectral counts of each protein were used to assess relative protein abundance in milk. We further used label-free spectral-counting semiquantification to determine differences in protein expression between transitional milk and mature milk (Materials and Methods). With minimal spectral count ≥4 per LC/MS run being taken for quantitation,

Transitional Milk and Mature Milk Favor Distinct Modes of Defense

GO analysis (Figure 1A) shows that milk proteins related to immune function are overrepresented (106 genes, Benjamini pvalue = 7.9 × 10−8), and in response to stimulus (276 genes, Benjamini p-value = 4.2 × 10−7), compared to human genome. These findings are consistent with the defensive roles that milk provides through immunological processes upon bacterial or viral stimulations. It is well-known that milk contains antibodies 3900



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Figure 2. (A) Relative-abundance distribution of IgM (IGHM), sIgA (IGHA1, IGHA2, and PIGR) and IgG (IGHG1 and IGHG2) in transitional milk and mature milk from three individual donors. (B) Spectral-count distribution of complement components and complement component-related proteins in milk. Proteins are listed in the order of descending spectral counts. (C) Immunoblotting validation of carbohydrate metabolism-related proteins expressed in human transitional milk and mature milk from three individual donors. T, transitional milk; M, mature milk; B4GALT1, beta1,4-galactosyltransferase; FBP1, fructose-1,6-bisphosphatase 1; ALDOA, fructose-bisphosphate aldolase A; PKM2, pyruvate kinase isozymes M1/ M2; ACAT2, acetyl-CoA acetyltransferase, cytosolic.

IgG, IgA, and IgM that provide a means for mothers to confer immunity to their offspring.38 Immunoglobulins are produced by B cells and start with formation of pentameric IgM. Transformation of IgM to other isotypes, for example, monomeric IgG and dimeric IgA, is regulated by environmental effector functions, that is, stress of pathogen invasion.39 Ig heavy chains, IGHA, IGHG, and IGHM, are characteristic to IgA, IgG, and IgM, respectively, and were used as measures of Ig’s in bottom-up proteomics. IgA in milk takes the secretory form (sIgA), which binds to a portion of the polymeric Ig receptor (PIGR).40 To account for the secretory form of IgA, PIGR was further taken as an indicator of sIgA in milk. Immunoglobulins are traditionally thought to be present at higher levels in transitional milk than in mature milk so as to provide a higher degree of protection to newborns whose defense systems are naive and immature compared to young infants. Consistent with that concept, we found that sIgA

(IGHA1, IGHA2, and PIGR) and IgM (IGHM) are upregulated in transitional milk. Strikingly, however, IgG (IGHG1 and IGHG2) is demonstrated to be consistently in higher abundance in mature milk (Figure 2A). The higher abundance of sIgA and IgM in transitional milk and IgG in mature milk suggests that milk may function differently in the defense of newborns in comparison to older infants. sIgA is thought to play a major role in neutralizing pathogens found in the intestinal lumen, while in contrast, IgG binds to antigens that may be found in the intestinal lumen and transfers them across the epithelial layer via binding to the epithelial neonatal Fc receptor (FcRn), which recognizes the Fc region of IgG. Antigens so transported are then released into the basal propria underlying epithelial layer, which subsequently induce B-cell proliferation, immune activation, or tolerance.40 The upregulation of sIgA in transitional milk and IgG in mature milk suggests a transformation in milk’s function from 3901



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supporting direct pathogen-killing conferred by sIgA in early lactation toward antigen intake facilitated by IgG that aims to develop an infant’s own immunity as lactation progresses. It is worthwhile to further note that levels of IgG in newborns are comparable to those of their mothers because of transplacental transport of maternal IgG. Infants delay production of their own IgG until about 6 months after birth. The combination of late production of endogenous IgG and catabolism of maternal IgG leads to a transient deficiency of IgG in infants during the period from birth to one year of age.41 The increasing supply of IgG from transitional milk to mature milk may provide a means to complement the synchronous decrease in IgG during early infancy.

Several APPs were previously identified in the whey of cow’s milk. 44 In the example of C-reactive protein (CRP, Supplementary Table 1) identified in this work, its binding to microbial polysaccharides provides a secondary binding site for CFH, resulting in amplification of the alternative pathway.45 Altogether, the higher levels of CFB, CFI, and CFH relative to C1 and C2, and the CRP-mediated CFH activation, suggest a predominant role for the alternative pathway compared to the C1- and C2-mediated classical pathway. Initiation of the complement system by pathogen binding leads to activation of a series of proteases, which eventually leads to the formation of C3 and C5 convertases that activate C3 and C5, respectively.39,41 Alternatively, in vitro studies suggest that thrombin (F2), plasmin (PLG), and lysosomal proteases can also act as natural C3 and C5 convertases that are independent of the established pathways.46,47 Given its capacity for killing cells, the complement system has the potential to damage host tissue and must be tightly regulated. Vitronectin (VTN), clusterin (CLU), and CD59, which were previously found in milk,15 can prevent the binding of C5−C9 to cell surfaces to form the membrane attack complex (MAC) and, in that way, inhibit complement-mediated cytolysis.48 CFI and CFH inhibit the formation of C3 convertase in the alternative pathway.41 We further identified several complement component-related proteins, including complement C1q tumor necrosis factor-related protein 1 (C1QTNF1) and complement factor H-related protein 1, 2, and 3 (CFHR1, -2, and -3), that were not previously known to exist in milk (Figure 2B). CFHR1, for example, appears to play a protective role in protecting the host from complement attack. It inhibits the complement cascade by blocking C5 convertase activity and interfering with C5b surface deposition during MAC formation.49 Serine protease inhibitors (SERPINs) are another class of proteins that are key components in regulating the complement system (complement network in Supplementary Figure 3 and KEGG complement and coagulation cascades). For example, plasma protease C1 inhibitor (SERPING1) inhibit C1s, whereas SERPINF2, -C1, -A1, and alpha-2-macroglobulin (A2M) inhibit complement-activating PLG. Among them, SERPING1, SERPINA1, and A2M are in higher abundance in transitional milk. A total of 45 genes were classified as peptidase inhibitors by GO analysis, which represents an enrichment by a factor of 5.2 (Benjamini p-value = 2.6 × 10−17) compared to the whole human genome (Figure 1A). Among the peptidase inhibitors identified, 28 were SERPINs, representing an enrichment by a factor of 5.3 (Benjamini pvalue = 8.2 × 10−11). Additional SERPINs found upregulated in transitional milk include pregnancy zone protein (PZP) and SERPINA3, by factors of 4.2 and 3.5, respectively. The diverse array of SERPINs and their upregulation in transitional milk indicates their significance in complement regulation in human milk, particularly during early lactation.

The Complement System Is Present in Milk in Fighting against Pathogens

Another important component of the immune system is the complement system. There are two major pathways that can activate the system. In what is known as the “classical” pathway, the complement system works in conjunction with antibodies that recognize the surface of pathogens. In the “alternative” pathway, the microbial surfaces directly activate the complement system and the presence of antibodies is not required.41 The possible role of complement components in breast milk has received comparatively little attention, which may be ascribed to the technical difficulty of detecting them using traditional approaches and controversy findings obtained thus far. Assays for assessing serum complement have been unreliable when adapted to milk, leading to a large degree of variation.25 With the proteomic approach used here, we are able to provide a more comprehensive view of complement components, which includes proteins that have already been detected in milk or are reported in this study for the first time. A number of complement components and related regulatory proteins were found (Figure 2B). The spectral counts span from approximately 6000 counts for C3 to 2 counts for C1S, suggesting a likely broad dynamic range of concentration of complement components in milk. KEGG analysis (Materials and Methods) reveals that the complement cascade is among the most significantly enriched pathways (31 genes, Benjamini p-value = 8.9 × 10−13) in human milk (Figure 1B). The relative contribution of the classical and alternative activation pathways in the human milk complement system has been unclear. sIgA, a poor mediator of the classical pathway,42 presents at high abundance in human milk,6 whereas IgM and IgG, the dominant mediators of the classical pathway,39 are found in lower abundance.43 It has been considered controversial that C2, a characteristic component of C3 and C5 convertases in the classical pathway, was previously measured at a level comparable to that of C3.25 This present proteomic analysis confirms the higher abundance of sIgA in transitional milk in comparison to IgM and IgG (Figure 2A). Furthermore, C3 is found to be among the most abundant complement components, and C2 is among the least abundant (Figure 2B). C1r, C1s, and C2 are specific to the classical pathway, whereas CFB, CFI, and CFH are characteristic to the alternative pathway (KEGG complement and coagulation cascades). Proteomic analysis suggests higher abundance for CFB, CFI, and CFH than for C1r, C1s, and C2 (Figure 2B). We further identified 15 acute phase proteins (APPs) according to GO analysis. APPs are a class of proteins which play an important role during complement activation and exhibit significant abundance changes in response to inflammation.

Carbohydrate Metabolism Is Transformed To Meet the Developmental Needs of Infants

Carbohydrate is an important source of energy for infants during breastfeeding. Beta-1,4-galactosyltransferase 1 (B4GALT1) participates in lactose biosynthesis50 and is in higher abundance in transitional milk (Figure 2C, Supplementary Table 2). Its higher abundance occurs concurrently with a relatively higher level of lactose in early lactation. Extended GO analysis of biological processes reveals that the carbohydrate metabolic process is strongly overrepresented in 3902



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Figure 3. Analysis of interaction networks of (A) carbohydrate metabolism proteins and (B) extracellular matrix proteins using STRING. Densely connected networks were clustered and color-coded. Proteins upregulated in transitional milk and mature milk are labeled with blue and red arrows, respectively. The upregulation of pyruvate kinase (PKM2) was determined using protein immunoblot.

milk (80 genes, Benjamini p-value = 5.7 × 10−12). Twenty-four out of the 80 proteins were found to render significant regulatory changes, with 20 being upregulated in mature milk (Figure 3A, Supplementary Table 2). Pathway analysis shows that proteins involved in pentose, glucose, pyruvate, amino/ nucleotide sugar, and glycan metabolism are all significantly

enriched in milk (Figure 1B). The protein−protein interactions in various carbohydrate metabolism processes are illustrated using network analysis in Figure 3A. A breakdown of the 20 mature-milk upregulated proteins confirms that 18 of them are distributed in various pathways of carbohydrate metabolism (Supplementary Table 3). 3903



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through ECM proteins that play a vital role in the differentiation, proliferation, and migration of cells through adhesion receptors, that is, integrins.53,54 Pathway analysis (Figure 1B) shows that “ECM-receptor interaction” is significantly enriched (22 genes, Benjamini pvalue = 2.5 × 10−4). Among the interaction proteins, tenascin (TNC), thrombospondin-1 (THBS1), and osteopontin (SPP1) are more abundant in transitional milk (Supplementary Table 2). In the example of TNC, upon binding to an integrin receptor, TNC initiates a counteradhesion signal which results in cell migration, proliferation, and differentiation.55 The physiological importance of ECM proteins in triggering cell proliferation and the role of milk in providing support for optimal growth and development of infants are well understood. The observation of high relative abundance of ECM proteins in transitional milk suggests that ECM proteins may be relevant to growing infants whose GITs undergo pronounced growth, morphological changes, and functional maturation postnatally and require rapid cell proliferation.4,13 The elevated developmental roles provided by milk are further evident from the overrepresentation of biological process in development (227 genes, Benjamini p-value = 2.0 × 10−3, Figure 1A). To correlate developmental functions and molecular pathways, the proteins involved in developmental process were further subjected to pathway analysis. The ECM-receptor pathway was found to be the most significantly enriched among the developmental protein subset (Supplementary Figure 4). The breakdown result confirms that a significant portion of the developmental functions milk provides is associated with ECM−receptor interactions. In addition to contributing to developmental functions through the ECM−receptor interactions, the ECM acts as a reservoir of cytokines which are vital in developmental processes.53 While cytokines are used in protecting the host, they are also transferred by the immune system to newborns and infants to help them defend against infection. In the example of milk cytokines,38 they contribute to acquired immune response vital to the intestinal host defense in young infants.4 A number of cytokines and cytokine-regulatory proteins have now been identified in milk (Supplementary Table 4). In the examples of tumor necrosis factor ligand superfamily members 11B, -13 (TNFSF11B, -13) and nicotinamide phosphoribosyltransferase (NAMPT), GO analysis shows that they play an important role in immune cell development.

Fructose-6-phosphate (F-6-P) and glyceraldehyde-3-phosphate (G-3-P) are the final products of the pentose phosphate pathway and serve as key reaction intermediates during a wide variety of carbohydrate metabolic processes. The formation of F-6-P from fructose-1,6-bisphosphate (F-1,6-BP) during gluconeogenesis is catalyzed by fructose-1,6-bisphosphatase 1 (FBP1). Aldolase (ALDO, isoform A or C) converts F-1,6-BP to G-3-P during glycolysis. Transketolase (TKT) catalyzes the formation of F-6-P and G-3-P in pentose phosphate pathway. At the final step of glycolysis, pyruvate kinase (PKM2) catalyzes the formation of pyruvate that can be used to produce acetyl CoA, which can later enter the tricarboxylic acid (TCA) cycle under aerobic condition and serves as the main step in energy generation during carbohydrate metabolism. Metabolism of two molecules of acetyl CoA by cytosolic acetyl-CoA acetyltransferase (ACAT2) leads to the formation of acetoacetyl CoA that is essential for cholesterol and bile acid synthesis and steroidogenesis. FBP1, ALDOA, TKT, and ACAT2 are found significantly upregulated in mature milk with spectral counting and the upregulation of FBP1, ALDOA, and ACAT2 was further confirmed with protein immunoblot (Figure 2C). PKM2, which has low spectral counts and cannot be quantified with spectral counting was validated using protein immunoblot. It is known that milk is rich in carbohydrate content that increases from transitional milk to mature milk.51 Milk enzymes are often thought to be relatively stable during gastric digestion and to be capable of aiding in metabolic processes in infants’ intestines.1,12 Although the mechanism remains unclear, milk whey proteins have been shown to have an effect in reducing the postprandial glycemia and stimulating the insulin secretion in adults.8−10 The carbohydrate-lowering and insulinotropic effects may be even more critical for nursing infants, given the cellular machinery of their GIT is immature in terms of its functions in ingestion, digestion, absorption, and metabolism.52 The diverse array of milk proteins involved in carbohydrate metabolism that have been identified in this work illustrates the importance of carbohydrate content regulation, for example, glucose homeostatis, in milk. The upregulation of metabolic proteins in various carbohydrate pathways in mature milk reflects the transforming metabolic activities in breastfeeding women who provide increased milk carbohydrate content to their babies during later lactation. In addition, these proteins may also aid metabolic processes in the intestine or bloodstream of nursing infants and, in that way, contribute to the previously found carbohydrate-lowering and insulinotropic effects conferred by milk proteins. Interestingly, another interpretation of this data is that the human milk proteome may be a window by which the metabolic state of the mammary tissue may be observed noninvasively, which may provide additional information on how the mammary gland regulates the composition of milk over time.

Co-regulation of Fibronectin and Actin Organization in Mature Lactation May Underlie the Milk Function in Regulating Reactive Oxygen Species

Cell adhesion and locomotion are key steps in regulating cell motility through the organization of actin skeleton.56 GO analysis demonstrates that biological processes related to adhesion, locomotion, and cellular location of the actin cytoskeleton are all overrepresented in milk (Figure 1A). We found G protein Cdc42, Ras-related C3 botulinum toxin substrate 1 (RAC1), transforming protein RhoA and RhoG (RHOA and -G) (Supplementary Table 1) that can be activated through binding to G-protein coupled receptors (GPRs) and play a central role in controlling actin organization.39 Several G-protein subtypes of α-, β-, and γsubunits that modulate a variety of intercellular signaling57 were also identified, including αs, αsXL, αs11, β1, β2, β4, γ5, and γ12. The GPRs GPR56, -126, -C5B, and -C5C are also present in milk.

Milk Is Enriched for Developmental Functions

GO analysis of cellular location shows that milk proteins associated with extracellular regions (296 genes, Benjamini pvalue = 1.6 × 10−60) are overrepresented in milk compared to the corresponding extracellular region of entire human genome. An important subcategory of the extracellular proteins consists of those associated with the ECM (53 genes, Benjamini p-value = 9.8 × 10−10, Figure 1A). Among the 53 ECM proteins, 12 were found to be more abundant in transitional milk (Figure 3B). The ECM proteins act as a structural support for cells and connective tissue.53 In addition, cellular signaling occurs 3904



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and its associated regulatory system may provide insight into the ongoing development and restructuring of the mammary tissue during development. Indeed, while the most striking morphological changes in the mammary tissue occur in preparation for lactation, with development of alveoli and the associated changes in the underlying tissue, the mammary tissue remains an extraordinarily active tissue throughout lactation and in the cessation of breastfeeding and lactation.

Rho GDP-dissociation inhibitor 1 and 2 (ARHGDIA and -B), Ras GTPase-activating-like protein (IQGAP1), Rho GTPaseactivating protein 29 (ARHGAP29), and Rho-guanine nucleotide exchange factors (RGNEF) are responsible for regulation of G protein activation and inactivation. With an understanding of cell motility, the functional roles of milk proteins profilin (PFN1); cofilin-1 and -2 (CFL1 and -2); thymosin-b4 and -b10 (TMSB4X and -10); actin-capping protein CAPZA1, CAPZA2, CAPZB, and CAPG; actin-related protein 2/3 subunit 1B, 2, 3, and 4 (ARPC1B, -2, -3, and -4); and T-complex 1 subunit β, δ, ε, η, and θ (CCT2, 4, 5, 7, and 8) can be understood given that they are essential in actin treadmilling, nucleation of branched filament assembly,39 and folding.58 In addition, several tetraspanin (TSPAN) proteins are found to be present in milk, including CD9, CD81, CD63, and TSPAN6. Tetraspanins are known to complex with integrins and to be involved in cell adhesion, motility, and signal transduction.59 Several proteins in the actin pathway (KEGG organization of actin cytoskeleton pathway) were found upregulated in mature milk, including PFN1, RHOA, and gelsolin (GSN) (Supplementary Table 2). Although many ECM proteins are upregulated in transitional milk (previous section), ECM protein fibronectin (FN1), which plays a role in cell adhesion, is conversely upregulated in mature milk (Supplementary Table 2). FN1 regulates the formation, contractility, and motility of the actin cytoskeleton via binding to integrins.53 This upregulation of FN1 in mature milk is, however, in accordance with the regulatory change of actincytoskeleton proteins. The actin cytoskeleton functions in a variety of essential cellular biological processes. It has been suggested that the contractile force obtained through the organization of the actin cytoskeleton has an important role in expelling reactive oxygen species (ROS).60 We noted that xanthine dehydrogenase/oxidase (XDH), which catalyzes the formation of H2O2, and lactoperoxidase (LPO), which has a bactericidal effect through the conversion of H2O2 to ROS, are upregulated in mature human milk, similar to the observations found in cow’s milk.36 The increased activities in actin organization during mature lactation are in accordance with the upregulation of ROS-generating XDH and LPO in mature milk. Excessive ROS generation could, however, damage tissue in both breastfeeding women and their nursing infants. GO analysis shows that molecular function in antioxidant activity is overrepresented in milk (16 genes, Benjamini p-value = 1.0 × 10−7, Figure 1). Pathway analysis demonstrates that glutathione metabolism is enriched (14 genes, Benjamini p-value = 4.2 × 10−3, Figure 1B). The overrepresentation of antioxidant activity and enrichment of glutathione metabolism reveals the important milk function in regulating oxidative stress. Examples of antioxidative enzymes include glutathione peroxidase 3, 4 (GPX3, -4), peroxiredoxin 1 to 6 (PRDX1−6) and superoxide dismutase 1 to 3 (SOD1−3) which retroconvert H2O2 to H2O. Among them, PRDX5 is upregulated in mature milk. With levels of bactericidal sIgA decreased from transitional milk to mature milk, the elevated levels of ROS generated through increased LPO and XDH may represent a primary means of killing pathogens during mature lactation. The concurrent upregulation of LPO, XDH, PRDX, FN1, and actin cytoskeleton proteins in mature milk highlights the comprehensive milk function in regulating ROS. As with the proteins identified in human milk related to carbohydrate metabolism, the presence of extracellular matrix

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CONCLUSIONS In summary, the establishment of the milk proteome can provide a platform for the search of milk proteins that play vital physiological roles in growing healthy infants. The higher abundance of sIgA and IgM in transitional milk compared to IgG in mature milk suggests the transforming milk function from direct pathogen-killing in breastfed newborns to promoting independent immune systems in young infants. A comprehensive proteomic approach reveals the importance of the milk complement system, in particular the alternative pathway, in protecting infants from pathogenic infections. The increased activities in actin organization and hydrogen peroxide regulation in mature milk reveals the elevated milk function in amending bactericidal ROS during mature lactation. It can also be concluded that carbohydrate metabolism evolves from transitional milk to mature milk to accommodate the transforming nutritional requirements of the growing infants. We further conjecture that ECM proteins enriched in milk may contribute to the development of the infant gastrointestinal tract.

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ASSOCIATED CONTENT

* Supporting Information S

IEX and SDS-PAGE of transitional milk and mature milk proteins; protein interaction networks in complement system; list of 976 milk proteins; list of proteins with significant regulatory changes; list of mature-milk upregulated proteins involved in carbohydrate metabolism; list of cytokines and cytokine receptors. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Address: Mead Johnson Nutrition, 2400 W. Lloyd Expressway, Evansville, IN. Tel.: 812-429-5440, Fax: 812-647-8206. Email: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank reviewers for their invaluable comments and suggestions. Q. Z. thanks A. J. Alpert at PolyLC and C. J. Carpenter at UC-Santa Barbara for helpful discussions and suggestions.

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REFERENCES

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Temporal Changes in Milk Proteomes Reveal Developing Milk Functions

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