Proteome Mapping of Human Skim Milk Proteins in Term and Preterm

Article pubs.acs.org/jpr

Proteome Mapping of Human Skim Milk Proteins in Term and Preterm Milk Claire E. Molinari,*,† Ylenia S. Casadio,† Ben T. Hartmann,‡ Andreja Livk,§ Scott Bringans,§ Peter G. Arthur,† and Peter E. Hartmann† †

School of Chemistry and Biochemistry, The University of Western Australia, Crawley, 6009, Australia Perron Rotary Express Milk Bank (PREM Bank) Neonatal Paediatrics, King Edward Memorial Hospital, Subiaco, 6008, Australia § Proteomics International, Perth, Western Australia, Australia ‡

S Supporting Information *

ABSTRACT: The abundant proteins in human milk have been well characterized and are known to provide nutritional, protective, and developmental advantages to both term and preterm infants. However, relatively little is known about the expression of the low abundance proteins that are present in human milk because of the technical difficulties associated with their detection. We used a combination of electrophoretic techniques, ProteoMiner treatment, and two-dimensional liquid chromatography to examine the proteome of human skim milk expressed between 7 and 28 days postpartum by healthy term mothers and identified 415 in a pooled milk sample. Of these, 261 were found in human skim milk for the first time, greatly expanding our understanding of the human skim milk proteome. The majority of the proteins identified were involved in either the immune response (24%) or in cellular (28%) or protein (16%) metabolism. We also used iTRAQ analysis to examine the effects of premature delivery on milk protein composition. Differences in protein expression between pooled milk from mothers delivering at term (38−41 weeks gestation) and preterm (28−32 weeks gestation) were investigated, with 55 proteins found to be differentially expressed with at least 90% confidence. Twenty-eight proteins were present at higher levels in preterm milk, and 27 were present at higher levels in term milk. KEYWORDS: human milk, protein, proteomics, ProteoMiner, iTRAQ, 2D LC−MS

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INTRODUCTION The importance of human milk proteins to the growth and development of breastfed infants is well established. They not only provide a digestible source of amino acids to infants, but also confer immunological protection and perform developmental and regulatory functions, exerting both long and short-term benefits compared to formula feeding.1,2 Human milk proteins are particularly important for infants who are born prematurely. Recent studies stress the importance of both the total amount of protein and the ratio of protein/ energy that preterm infants receive for their growth and development.3 Significantly, the protein composition of milk from preterm mothers is known to differ from that of term mothers. The concentration of total protein is typically higher in preterm milk;4 however, while some individual proteins are expressed at higher levels in preterm milk, others are present at lower concentrations.5−7 Hitherto, most studies investigating milk protein composition have focused upon the most abundant proteins present, resulting in their relative concentrations in term and preterm milk being well-defined.8−10 However, it is also important that the identity and behavior of the lower abundance proteins in © 2012 American Chemical Society

human milk be characterized. There are two main reasons for this. First, it is possible that these proteins play significant roles in infant growth and development. Second, knowledge of how the expression of low abundance proteins differs between term and preterm milk may be useful diagnostically, as a reflection of the developmental changes occurring in the mammary gland during pregnancy and lactation. Historically, there have been a number of technical challenges associated with characterizing the low abundance proteins in human milk. Initial studies using gel electrophoretic methods coupled with mass spectrometry were unable to detect more than 10 different gene products in either human or bovine milk, despite observing hundreds of distinct protein spots.11−13 This difficulty results from the fact that six proteins, α-lactalbumin, β-casein, secretory immunoglobulin A, lysozyme, lactoferrin, and secretory component, constitute over 90% of the total protein content in mature human milk,14 obscuring the detection of less abundant proteins of potential biological interest. Received: September 2, 2011 Published: February 7, 2012 1696

dx.doi.org/10.1021/pr2008797 | J. Proteome Res. 2012, 11, 1696−1714

Journal of Proteome Research

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(7−14 days lactation) and 16 term mothers (7−14 days lactation) were used. All milk samples were thawed, pooled, and centrifuged at 10000g for 10 min to remove the cream layer. A mammalian protease inhibitor cocktail containing 4-(2aminoethyl)benzenesulfonyl fluoride, E-64, bestatin, leupeptin, aprotinin, and sodium EDTA was added to each pooled sample, which was then depleted of casein using a previously described method.22 Briefly, to deplete the samples of casein, CaCl2 was added to a concentration of 60 mM, and the pH was adjusted to pH 4.3. Samples were then centrifuged at 189000g at 4 °C for 60 min, and the supernatant was collected. For the ProteoMiner-treated samples (Figure 1), caseindepleted skim milk was dialyzed against 10 volumes of 10 mM Tris, pH 7 at 4 °C, with three buffer changes at two-hour intervals using dialysis tubing with a MW cutoff of 3500 Da (Spectrapor Membrane Tubing, Spectrum Medical Industries, Rancho Domingues, CA). The samples were then lyophilized and reconstituted in water. The samples were analyzed for their protein content and diluted such that 1 mL of 50 g/L protein solution was loaded onto the hexaligand library beads (ProteoMiner Large Capacity Protein Enrichment Kit, BioRad, Gladesville, NSW, Australia), according to the manufacturer’s instructions. Briefly, after swelling the beads using the buffer provided, the samples were loaded onto individual ProteoMiner columns and rocked for 6 h at room temperature. For the pooled term milk samples (collected 15−28 days postpartum), the unbound proteins were then washed through the column, and the bound proteins were eluted using the acidic elution buffer provided in the kit. For the pooled term and preterm milk samples to be subsequently labeled with iTRAQ reagents (collected 7−14 days postpartum), the bound proteins were eluted sequentially using four different buffers: 1 M sodium chloride in 20 mM HEPES (pH 7.0), 0.2 M glycine (pH 2.4), 60% (w/v) ethylene glycol in water, and 33.3% (v/v) 2-propanol, 16.7% (v/v) acetonitrile, 0.1% (v/v) trifluoroacetic acid.

More recently, proteomic studies employing strategies to deplete the high abundance proteins or to extensively fractionate the sample prior to analysis have been more successful at identifying low abundance proteins than the earlier gel-based methods.14−17 Two studies in particular have identified a large number of low abundance human milk proteins. Palmer et al.18 used immunodepletion columns to deplete the five most abundant proteins in colostrum prior to 2D LC−MS/MS analysis and were able to identify 151 proteins. More recently, Liao et al.19 employed combinatorial hexapeptide ligand libraries (ProteoMiner) to enrich the low abundance milk proteins before analysis by LC−MS/MS and identified 115 proteins. Liao et al.19 also showed that many of these proteins change in expression over the course of 12 months of lactation, highlighting the dynamic nature of milk composition. One of the advantages of using a ProteoMiner bead approach is that it does not require tailored antibodies or optimization. When a sample is applied to the ligand library, the high abundance proteins saturate their ligands and the excess remains unbound, whereas the lower abundance proteins bind completely, resulting in an overall compression of the dynamic range. Recent studies have also shown the ProteoMiner treatment to be compatible with downstream quantitative analyses of low abundance proteins.20,21 The aim of the present study was two-fold. First, we aimed to further characterize the proteome of mature human skim milk from established lactation (7−28 days postpartum), using a combination of ultracentrifugation and ProteoMiner enrichment to compress the dynamic range of the proteome prior to analysis by 2D LC−MS/MS. Second, we sought to quantitatively examine whether there are differences in protein expression between term and preterm milk that reflect the physiological and metabolic effects of preterm delivery upon the mammary gland.

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EXPERIMENTAL PROCEDURES

Protein Concentration Determination

Materials

Protein concentrations of human milk samples were determined using a Bicinchoninic acid kit (Sigma-Aldrich, NSW, Australia), as described in a previous study.23

Unless otherwise stated, all chemicals and reagents were obtained from Sigma-Aldrich (NSW, Australia). Sample Collection

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Term and preterm milk samples were obtained from healthy lactating mothers at King Edward Memorial Hospital, Subiaco, Western Australia. Participating term mothers had delivered between 38 and 41 weeks of gestation, and their infants had a chronological age of between 7 and 28 days at the time of sample collection. Participating preterm mothers had delivered between 28 and 32 weeks of gestation, and their infants had a chronological age of between 7 and 14 days at the time of sample collection. All donors gave written informed consent for their donations to be used in this research, and this study was approved by the University of Western Australia, Human Research Ethics Committee and King Edward Memorial Hospital, Human Ethics Research Committee. All samples were frozen at −20 °C within an hour of expression and transferred to −80 °C storage within 3 days.

SDS-PAGE analysis was conducted using the HOEFER gel apparatus (HOEFER Scientific Instruments, San Francisco, CA) and the Laemmli gel system using 12.5% polyacrylamide gels.24 Gels were run using a constant current of 15 mA for 16 h at 4 °C, fixed for 2 h in 50% methanol/10% trichloroacetic acid, destained using double deionized water, stained using Coomassie Brilliant Blue R-250 overnight, and scanned using an Epson Perfection V700 photographic flatbed color image scanner (Epson, Nagano, Japan). The intensity of protein bands were measured using the open access software package Image J 1.410.25 Differential in-Gel Electrophoresis (DIGE)

Cy5 and Cy3 activated ester dyes were purchased from Lumiprobe (Lumiprobe Corp, FL). Skim milk samples before and after casein depletion and ProteoMiner treatment were each labeled with one of the dyes. The DIGE experiment was conducted in duplicate, and the order of the dyes were swapped for the duplicate experiment. Fifty micrograms of protein sample was labeled according to the CyDye DIGE Flours (minimal dyes)

Sample Treatment

The sample analysis workflow is described in Figure 1. For the purposes of protein identification in mature term milk, milk samples collected from 8 mothers (15−28 days lactation) were pooled. For the quantitative comparison between term and preterm milk, milk samples from 16 preterm mothers 1697



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Figure 1. Experimental workflow.

LC−MS/MS

for Ettan DIGE system protocol (GE Healthcare, Pittsburgh, PA), and the samples were mixed together. Isoelectric focusing (IEF) and strip equilibration was performed according to the Ettan DIGE system protocol (GE Healthcare), using ready-to-use Immobiline DryStrip gel strips, linear pH gradient 3−10, length 18 cm (GE Health Care) and an IPGphor isoelectric focusing unit (IPGPhor, GE Health Care). The strips were actively rehydrated for 10 h at a constant voltage of 200 V and a constant temperature of 20 °C. One hundred micrograms of protein was loaded. Following rehydration, the strips were exposed to a linear increase to 1000 V over 4 h followed by 8000 V until a total of 45000 V hr was reached. The second dimension separation was carried out in the dark according to the method of Lui, Lipscombe, and Arthur.26 Gels were imaged using a Typhoon Trio scanner (GE Health Care), with the Cy5 and Cy3 labeled samples visible using 633/670 nm and 532/580 nm excitation/emission filters, respectively. Gels were post stained for total protein content using Coomassie Brilliant Blue. DIGE gels were analyzed using the Progenesis SameSpots software package (Nonlinear Dynamics Ltd., Newcastle upon Tyne, U. K.). Spots with a normalized spot volume of less than 500 were excluded from the analysis. All data is presented as mean ± SEM. For the purposes of downstream mass spectrometry analysis, a preparative 2D gel analysis was also conducted. Five hundred micrograms of the ProteoMiner-treated skim milk sample was loaded, and the first and second dimensions were carried out as above. Gels were fixed and stained as described above. Coomassie stained bands and spots of interest were cut from the gel, destained, and digested as described by Shevchenko et al.27 Mass spectrometry was conducted as described in a previous study using an UltraFlex MALDI-TOF/TOF instrument (Bruker Daltonics, Bremen, Germany).23 MS/MS data was imported into the database search engine Mascot (Version 2.3.01, www.matrixscience.com) and searched against the Swiss-Prot Mammalia database (49 887 sequences).

Sample Preparation. Protein samples were precipitated by adding five volumes of cold acetone to the treated samples (Figure 1), incubating for 1 h at −20 °C, and pulse centrifuging for 5−10 s. The protein pellets were resuspended in 0.5 M triethylammonium bicarbonate (TEAB) (pH 8.5) by shaking before reduction and alkylation according to the iTRAQ protocol (Applied Biosystems, Foster City, CA). A total of 55 μg of each sample was digested overnight with 5.5 μg trypsin at 37 °C in 0.5 M TEAB. 1D-LC. Peptides were separated on a C18 PepMap100, 3 μm column (LC Packings, Sunnyvale, CA) with a gradient of 10−45% acetonitrile, 0.1% trifluoroacetic acid over 165 min, using the Ultimate 3000 nano HPLC system (LC PackingsDionex). Every 30 s, the eluent was mixed with matrix solution (5 mg/mL CHCA) and spotted onto a 384 well Opti-TOF plate (Applied Biosystems) using a Probot Micro Fraction Collector (LC Packings-Dionex). 2D-LC. Peptides were desalted on a Strata-X 33 μm polymeric reversed phase column (Phenomenex) and dissolved in a buffer containing 10 mM potassium hydrogen phosphate, pH 3 in 10% acetonitrile, before separation by strong cation exchange chromatography on an Agilent 1100 HPLC system (Agilent Technologies, Palo Alto, CA) using a PolySulfethyl column (4.6 × 100 mm, 5 μm, 300 Å, Nest Group, Southborough, MA). Peptides were eluted with a linear gradient of 0−400 mM KCl. Eight fractions containing the peptides were collected and desalted on Strata-X columns. Each peptide fraction was then separated and spotted onto a 384-well Opti-TOF plate according to the 1D-LC protocol described above, excepting that a 10−40% acetonitrile (0.1% trifluoracetic acid) gradient was used. iTRAQ. The tryptic digests were dried in a SpeedVac, resuspended in 30 μL of 0.5 M TEAB, and labeled by adding iTRAQ reagents to preterm and term milk samples, respectively, according to the iTRAQ protocol (Applied Biosystems). For the iTRAQ experiment comparing term and preterm milk samples without ProteoMiner treatment (Figure 1), duplicate pooled preterm milk samples were labeled with iTRAQ reagents 1698



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of the parameter, iTRAQ 4plex (peptide labeled) modification, and the Quantitate tab checked. MS/MS spectra were searched against the Swiss-Prot human genomic database (2011_5). For quantitation analysis, the duplicates were analyzed separately. Average protein ratios and p-values to indicate significant differential expression were calculated by the software. To be considered as being differentially expressed, proteins were required to have an unused protein score greater than 1.3, corresponding to a confidence interval of 95%, and have significantly different protein ratios in both replicates, also at a confidence level of 95% (p < 0.05). Identified proteins for which a difference was found at a confidence level of 90% (p < 0.1) in both replicates were also reported. The p values represent the variation in the reported iTRAQ ratios for all the peptides of the associated protein and do not relate to either biological variation or technical reproducibility. The false discovery rate was less than 1%, calculated using a database containing reversed sequences. In order to categorize the identified proteins, the results were analyzed using the software program IPA (Ingenuity Databases) and the UniProt Database release 2011_6 (http://www.uniprot.org/). Results were compared to a recent comprehensive review publication28 and a subsequent research paper19 in order to determine which proteins had not been previously identified.

114 and 116. Duplicate pooled term milk samples were labeled with iTRAQ reagents 115 and 117. For the iTRAQ experiment comparing term and preterm milk samples after ProteoMiner treatment (Figure 1), duplicate pooled preterm milk samples were labeled with iTRAQ reagents 116 and 117. Duplicate pooled term milk samples were labeled with iTRAQ reagents 114 and 115. Excess iTRAQ reagent was quenched by adding 1 mL of water; the samples were then combined, desalted on a Strata-X 33 μm polymeric reverse phase column (Phenomenex, Torrance, CA), and analyzed using the 2D-LC protocol described above. MALDI-MS/MS. Peptides were analyzed on a 5800 MALDITOF/TOF mass spectrometer (Applied Biosystems) operated in reflector positive mode. MS data were acquired over a mass range of 800−4000 m/z, and for each spectrum, a total of 400 shots were accumulated. A job-wide interpretation method selected the 20 most intense precursor ions above a signal/ noise ratio of 20 from each spectrum for MS/MS acquisition but only in the spot where their intensity was at its peak. MS/ MS spectra were acquired with 4000 laser shots per selected ion with a mass range of 60 to the precursor ion −20. Data Analysis. Protein identification was performed using ProteinPilot 4.0.8085 Software (Applied Biosystems). MS/MS spectra were searched against the Swiss-Prot human genomic database (2011_3 for the 2D-LC analysis, 2011_5 for the 1DLC analysis). Search parameters were as follows: Cys alkylation, MMTS; Digestion, trypsin; Instrument, 5800; Special factors, none; Species, none; Quantitate tab, unchecked; Detected protein threshold (unused ProtScore), 1.3, which corresponds to proteins identified with 95% and in the other replicate at a confidence level >90%. eProteins that were found to be differentially expressed in both replicates at a confidence level >90%. 1710



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Figure 6. SDS-PAGE analysis of differences in protein composition of pooled term (T) and preterm (PT) milk samples. (A) Representative SDSPAGE electrophoretograms of PT and T milk. Each sample was run in triplicate (n = 3). (B) The intensity of the protein bands corresponding to bile-salt stimulated lipase (BSSL), lactoferrin (LF), serum albumin (SA), sIgA, β-casein, and α-lactalbumin (ALA) were expressed as percentages of the total intensity of each lane. Differences between the two samples were analyzed using a Student’s t-test. All significant differences between groups are indicated (p < 0.05).

during episodes of mastitis.35,37 A recent study in bovine milk also identified a number of immunological proteins as being upregulated during Escherichia coli infection.38 Of the proteins we identified, 120 (24% of total) were associated with immune and inflammatory pathways and may have the potential to act as sensitive biomarkers of mammary gland inflammation. Promising candidates include eight heat shock proteins that we identified in human milk for the first time (Table 1), which are known to respond to cellular insults.39 In addition to yielding information regarding mammary gland function, the expansion of the human milk proteome in the present study is also of potential interest from an infant centered perspective, in that many of the proteins may be involved in providing nutritional, protective, and developmental advantages to breastfed infants. With respect to infant growth and development, we identified 33 proteins (7%) as being involved in tissue development, including a number of proteins identified for the first time, such as granulin (GRN), cysteinerich motor neuron protein (CRIM1), gremlin-2 (GREM2), nephronectin (NPNT), and bone morphogenetic protein 1 (BMP1). GRN is a growth factor with marked functional similarities to the epidermal growth factor family and has been shown to regulate cellular proliferation, particularly in the hematopoietic and reproductive systems.40 Similarly, GREM2 also regulates cellular differentiation and is involved in the Wnt signaling pathways.41 NPNT, BMP1, and CRIM1 have been shown to interact with several growth factors and to play important roles in the differentiation of osteoclasts, chondrocytes, and motor neurons, respectively, although more general roles in development have also been proposed.42−44 While further research is required to assess whether these proteins retain their activity upon digestion, it is known that human-milk-fed infants experience developmental advantages over formula-fed infants,45−47 and it is possible that proteins within this group may be partly responsible. Proteins involved in immunity and inflammation constitute 24% of the proteins identifed (Figure 5), signifying the critical

role that human milk plays in protecting infants from infection. Indeed, Vorbach et al.48 argued that it was immune protection rather than the provision of nutrition that was the original function of the mammary gland in premammals. Although there is an abundance of literature describing the protective advantages conferred upon infants through breastfeeding, the mechanisms involved are far from delineated.49 Individual proteins have been shown to exert protective effects through multiple pathways, even after proteolytic digestion.50,51 Furthermore, there is a great deal of interaction between different proteins involved in the immune response, as well as with the infant’s own defenses.49 The identification of additional potential immune proteins present in human milk (Table 1) may be of use in further elucidating these complex protective mechanisms. Immune response proteins that we identified in human milk for the first time include C1q and tumor necrosis factor related protein-1 (CTRP1), peroxiredoxins-1, -2, and -6 (PRDX1, PRDX2, PRDX6), glutathione peroxidase 3 (GPX3), and HLA class I and II histocompatibility antigens (HLA-A, HLA-DRA). CTRP1 is a cytokine that possesses both immunomodulatory and metabolic functionality, inhibiting common pro-inflammatory pathways, as well as being involved in glucose and insulin regulation.52,53 PRDX1, PRDX2, PRDX6, and GPX3 are all involved in systems of oxidative stress regulation, protecting cells, enzymes, and other proteins from oxidative damage,54 whereas HLA-A and HLA-DRA are both membrane proteins, involved in presenting foreign antigens to the immune system.55 While the presence of these proteins in human milk does not indicate that they are functionally active in the infant gut, they nonetheless provide useful targets for further investigation. To investigate whether the human milk proteome undergoes detectable changes at different stages of mammary gland development, a pilot study involving iTRAQ analysis was used to compare the protein composition of term and preterm milk. Although iTRAQ experiments are designed to detect differences in the fractional composition of protein samples, in the present study the original protein concentration of each pooled 1711



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were differentially expressed in term and preterm milk, with BSSL present at a higher level in preterm milk and both LPL and BTD found at a higher level in term milk. The presence of these enzymes in human milk is thought to compensate for a lack of endogenous enzymes in the immature pancreatic juice of newborns, enabling the efficient digestion of triacylglycerols and biotin, respectively.59−61 Given the importance of the absorption of fat and biotin to an infant’s growth and development, it is likely that the delivery of these enzymes is of particular importance to preterm infants. Again, their pattern of expression defies teleological explanation, in that while the higher levels of BSSL in preterm milk may promote additional growth in preterm infants, the lower levels of BTD and LPL may be of detriment. Identifying proteins that are differentially expressed in term and preterm milk may also be useful diagnostically. There have been a number of studies investigating the differences in mammary gland physiology and metabolism after preterm birth; however, the link between these physiological differences and preterm milk composition largely remain unknown.4,34,62 The differential expression of proteins in preterm milk may highlight potential regulatory and metabolic pathways that are disrupted after preterm delivery. For example, the higher levels of serum albumin that we found in preterm milk may be due to the persistence of an open paracellular pathway after delivery in preterm mothers, allowing the flow of serum proteins into the mammary alveoli. Indeed, levels of serum albumin in milk have been used previously as a marker of an open paracellular pathway.63,64 Similarly, the low level of prolactin-inducible protein in preterm milk is interesting from a diagnostic perspective, in that it may reflect low levels of circulating maternal prolactin. Indeed, it has been shown that preterm mothers are more likely to have lower levels of serum prolactin, which may be responsible in part for lower levels of milk production in preterm mothers.65 Tenascin is another protein that was found to be present at much lower levels in preterm milk compared to term milk (Table 2). Having been previously implicated in mammary gland development and differentiation,66,67 its concentration in milk may also potentially be useful as a marker of mammary gland development after preterm delivery. It is also of interest that distinct sets of proteins were identified in the iTRAQ analysis of pooled term milk collected 7−14 days postpartum and in the 2D-LC analysis of pooled term milk collected 15−28 days postpartum, with over 100 proteins unique to each sample (Figure 4C). Although no quantitative comparison was conducted comparing these samples, it is likely that many of these proteins change in relative abundance in term milk over this time period and therefore may also represent proteins of interest with regard to mammary gland development throughout lactation. In summary, the present study represents the most comprehensive study to date of the human milk proteome, identifying 261 novel proteins, as well as documenting changes in the relative abundance of proteins in term and preterm milk. Knowledge obtained from this characterization of the proteins in human milk will provide insights into the regulatory mechanisms involved in the synthesis and secretion of human milk, particularly after preterm delivery, as well as identifying potential proteins in human milk responsible for the nutritional, immunological, and developmental advantages conferred onto the breastfed infant.

sample was equivalent. This means that the differences in fractional composition also directly correspond to differences in the concentration of individual proteins between term and preterm milk. In order to detect differentially expressed proteins of both high and low abundance in term and preterm milk we adopted a workflow including parallel iTRAQ experiments. In a similar manner to a recent publication,20 iTRAQ comparisons of pooled term and preterm milk samples were conducted both with and without prior ProteoMiner treatment. This enabled us to distinguish between high abundance proteins, those identified without ProteoMiner treatment, and low abundance proteins, those identified only when ProteoMiner treatment was used prior to iTRAQ analysis. We found that ProteoMiner treatment did significantly alter the relative quantitation of the high abundance proteins, and therefore in the iTRAQ analysis of ProteoMiner-treated samples, only the relative abundance ratios of the low abundance proteins were considered to be accurate. SDS-PAGE analysis was performed to verify the iTRAQ quantitative analysis of high abundance proteins. The direction of the differences in expression of five proteins, bile-salt-stimulated lipase, lactoferrin, β-casein, sIgA, and serum albumin, in term and preterm milk were consistent between the two analytical methods; however, the iTRAQ experiment overestimated the magnitude of the difference. Alpha-lactalbumin was found to be differentially expressed in the iTRAQ experiment but not by SDS-PAGE. It is likely that these differences between the two methods are due to an overestimation of the level of background contamination of the iTRAQ spectra by the analysis software.56 Although previous studies report the quantitative accuracy of iTRAQ analysis of low abundance proteins after ProteoMiner-treated samples,20 these results were not verified in the present study. Despite these limitations, these results provide support for the use of an iTRAQ approach to identify differentially expressed proteins in human milk and illustrate how ProteoMiner treatment can be incorporated within a quantitative analysis. We found 28 proteins that had significantly higher expression levels in preterm milk compared to term milk and 27 proteins that had significantly lower levels of expression (Table 2). A number of these differentially expressed proteins such as lactoferrin, lysozyme, polymeric immunoglobulin receptor, lactadherin, prolactin inducible protein, Ig heavy chain, mucin-4, vitronectin, and complement C3 are associated with the immune response, protecting infants against infection.57 A number of targeted studies have found higher levels of specific immunologic factors in preterm milk and have accounted for these differences on a teleological basis, arguing that the composition of preterm milk differs from term milk to render it more suited to the protection of vulnerable preterm infants.10,58 Our data contradicts this assertion, in that we found no concerted difference in the expression of immunological proteins between term and preterm milk, with some present at higher levels in preterm milk and vice versa (Table 2). This suggests that differences in protein composition between term and preterm milk are due to physiological differences in the mammary gland affecting protein synthetic and transport pathways, rather than being the result of differing infant requirements, and supports the idea that as preterm infants have only been able to survive in recent years, there has not been any selection pressure to encourage an increase in protective proteins in preterm milk.4 Similarly, three digestive enzymes, biotinidase (BTD), lipoprotein lipase (LPL), and bile-salt-stimulated lipase (BSSL) 1712



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(4) Atkinson, S. A. Effects of gestational stage at delivery on human milk components. In Handbook of Milk Composition; Jensen, R. G., Ed.; Academic Press: San Diego, 1995; pp 222−237. (5) Montagne, P.; Cuilliere, M. L.; Mole, C.; Bene, M. C.; et al. Immunological and nutritional composition of human milk in relation to prematurity and mothers’ parity during the first 2 weeks of lactation. J. Pediatr. Gastroenterol. Nutr. 1999, 29, 75−80. (6) Bielicki, J.; Huch, R.; von Mandach, U. Time-course of leptin levels in term and preterm human milk. Eur. J. Endocrinol. 2004, 151, 271−276. (7) Dvorak, B.; Fituch, C. C.; Williams, C. S.; Hurst, A. N. M.; et al. Increased epidermal growth factor levels in human milk of mothers with extremely premature infants. Pediatr. Res. 2003, 54, 15−19. (8) Montagne, P.; Cuillere, M.; Mole, C.; Bene, M.;et al., Changes in lactoferrin and lysozyme levels in human milk during the first twelve weeks of lactation. In Bioactive Components of Human Milk; Newburg, D., Ed.; Springer: New York, 2001; Vol. 1. (9) Kunz, C.; Lonnerdal, B. Re-evaluation of the whey protein/casein ratio of human milk. Acta Paediatr. 1992, 81, 107−112. (10) Grosse, S. J.; Buckley, R. H.; Wakil, S. S.; McAllister, D. C.; et al. Elevated IgA concentration in milk produced by mothers delivered of preterm infants. J. Pediatr. 1981, 99, 389−393. (11) Galvani, M.; Hamdan, M.; Righetti, P. G. Two-dimensional gel electrophoresis/matrix-assisted laser desorption/ionisation mass spectrometry of a milk powder. Rapid Commun. Mass Spectrom. 2000, 14, 1889−1897. (12) Galvani, M.; Hamdan, M.; Righetti, P. G. Two-dimensional gel electrophoresis/matrix-assisted laser desorption/ionization mass spectrometry of commercial bovine milk. Rapid Commun. Mass Spectrom. 2001, 15, 258−264. (13) Holland, J. W.; Deeth, H. C.; Alewood, P. F. Proteomic analysis of kappa-casein micro-heterogeneity. Proteomics 2004, 4, 743−752. (14) Mange, A.; Bellet, V.; Tuaillon, E.; Van de Perre, P.; et al. Comprehensive proteomic analysis of the human milk proteome: Contribution of protein fractionation. J. Chromatogr., B 2008, 876, 252−256. (15) Yamada, M.; Murakami, K.; Wallingford, J. C.; Yuki, Y. Identification of low-abundance proteins of bovine colostral and mature milk using two-dimensional electrophoresis followed by microsequencing and mass spectrometry. Electrophoresis 2002, 23, 1153−1160. (16) Picariello, G.; Ferranti, P.; Mamone, G.; Roepstorff, P.; et al. Identification of N-linked glycoproteins in human milk by hydrophilic interaction liquid chromatography and mass spectrometry. Proteomics 2008, 8, 3833−3847. (17) Panchaud, A.; Kussmann, M.; Affolter, M. Rapid enrichment of bioactive milk proteins and iterative, consolidated protein identification by multidimensional protein identification technology. Proteomics 2005, 5, 3836−3846. (18) Palmer, D. J.; Kelly, V. C.; Smit, A.-M.; Kuy, S.; et al. Human colostrum: Identification of minor proteins in the aqueous phase by proteomics. Proteomics 2006, 6, 2208−2216. (19) Liao, Y.; Alvarado, R.; Phinney, B.; Lonnerdal, B. Proteomic characterization of human milk whey proteins during a twelve-month lactation period. J. Proteome Res. 2011, 10, 1746−1754. (20) Bandhakavi, S.; Van Riper, S. K.; Tawfik, P. N.; Stone, M. D.; et al. Hexapeptide libraries for enhanced protein PTM identification and relative abundance profiling in whole human saliva. J. Proteome Res. 2011, 10, 1052−1061. (21) Hartwig, S.; Czibere, A.; Kotzka, J.; Pablack, W.; et al. Combinatorial hexapeptide ligand libraries (ProteoMiner): An innovative fractionation tool for differential quantitative clinical proteomics. Arch. Physiol. Biochem. 2009, 115, 155−160. (22) Kunz, C.; Lonnerdal, B. Human-milk proteins: Analysis of casein and casein subunits by anion exchange chromatography, gel electrophoresis, and specific staining methods. Am. J. Clin. Nutr. 1990, 51, 37−46. (23) Molinari, C. E.; Casadio, Y. S.; Arthur, P. G.; Hartmann, P. E. The effect of storage at 25 °C on proteins in human milk. Int. Dairy J. 2011, 21, 286−293.

CONCLUSIONS We investigated the effectiveness of a number of different analytical techniques to analyze the human milk proteome. Dynamic range compression of human skim milk by the depletion of casein and ProteoMiner treatment followed by 2D LC−MS/MS was the most successful approach. In total, 415 proteins were identified, over half of which had not been found before in human milk. In addition, iTRAQ analysis was used to identify differentially expressed proteins between term and preterm milk, providing insights into metabolic differences in the mammary gland after preterm birth in comparison to term birth.

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ASSOCIATED CONTENT

S Supporting Information *

Supporting Information Data File 1 contains individual annotated MS/MS spectra of each of the proteins for which only one peptide was identified. Supporting Information Data File 2 displays the preparative 2DE gel of the skim milk after casein depletion and ProteoMiner treatment and each of the spots that was analyzed by MS. Supporting Information Data File 3 contains tables displaying all of the peptides used for identifying proteins in the 1D-LC, 2D-LC, iTRAQ, 1D SDSPAGE, and 2DE experiments and their corresponding identification strengths. Supporting Information File 4 is a table displaying the classification of the proteins according to their function and location. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +61 6488 4428. Fax: +61 8 6488 7086. E-mail: 10224872@ student.uwa.edu.au.

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ACKNOWLEDGMENTS The MS analyses were performed in facilities provided by the Lotterywest State Biomedical Facility-Proteomics node at the Western Australian Institute for Medical Resesarch. This study was funded by an unrestricted grant from Medela AG (Switzerland) to the University of Western Australia. C.E. Molinari was supported by a scholarship from the Western Australian Women’s Service Guild (2009−2011), and an Australian Postgraduate Award (2009−2011).

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ABBREVIATIONS 1D-LC, one-dimensional liquid chromatography; 2DE, twodimensional gel electrophoresis; 2D-LC, two-dimensional liquid chromatography; iTRAQ, isobaric tags for relative and absolute quantitation; MALDI, matrix-assisted laser desorption/ionization; MS, mass spectrometry; PAGE, polyacrylamide gel electrophoresis; sIgA, secretory IgA; SDS, sodium dodecyl sulfate; TOF, time-of-flight

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REFERENCES

(1) Lonnerdal, B. Nutritional and physiologic significance of human milk proteins. Am. J. Clin. Nutr. 2003, 77, 1537S−1543. (2) Srinivasan, M.; Patel, M. S. Metabolic programming in the immediate postnatal period. Trends Endocrinol. Metab. 2007, 19, 146− 152. (3) Rigo, J.; Senterre, J. Nutritional needs of premature infants: Current issues. J. Pediatr. 2006, 149, S80−S88. 1713



Article

(45) Dvorak, B. Milk epidermal growth factor and gut protection. J. Pediatr. 2010, 156, S31−S35. (46) Lucas, A.; Morley, R.; Cole, T. J.; Lister, G.; et al. Breast milk and subsequent intelligence quotient in children born preterm. Lancet 1992, 339, 261−264. (47) Quinn, P. J.; O’Callaghan, M.; Williams, G. M.; Najman, J. M.; et al. The effect of breastfeeding on child development at 5 years: A cohort study. J. Pediatr. Child Health 2001, 37, 465−469. (48) Vorbach, C.; Capecchi, M.; Penninger, J. Evolution of the mammary gland from the innate immune system? Bioessays 2006, 28, 606−616. (49) Xanthou, M. Immune protection of human milk. Biol. Neonate 1998, 74, 121−133. (50) Lonnerdal, B.; Iyer, S. Lactoferrin: Molecular structure and biological function. Annu. Rev. Nutr. 1995, 15, 93−110. (51) Strate, B. v. d.; Belijaars, L.; Molema, G.; Harmsen, M.; et al. Antiviral activities of lactoferrin. Antiviral Res. 2001, 52, 225−239. (52) Davis, K.; Scherer, P. Adiponectin: No longer the lone soul in the fight against insulin resistance? Biochem. J. 2008, 416, e7−e9. (53) Nayak, A.; Pedenekar, L.; Reid, K. B. M.; Kishore, U. Complement and non-complement activating functions of C1q: A prototypical innate immune molecule. Innate Immun. 2011. (54) Rhee, S. G.; Yang, K. S.; Kang, S. W.; Woo, H. A.; et al. Controlled elimination of intracellular H2O2: Regulation of peroxiredoxin, catalase, and glutathione peroxidase via post-translational modification. Antioxid. Redox Signaling 2005, 7, 619−26. (55) Garcia-Lora, A.; Algarra, I.; Garrido, F. MHC class I antigens, immune surveillance, and tumor immune escape. J. Cell. Physiol. 2003, 195, 346−355. (56) Karp, N. A.; Huber, W.; Sadowski, P. G.; Charles, P. D.; et al. Addressing accuracy and precision issues in iTRAQ quantitation. Mol. Cell. Proteomics 2010, 9, 1885−1897. (57) Field, C. J. The immunological components of human milk and their effect on immune development in Infants. J. Nutr. 2005, 135, 1−4. (58) Chandra, R. K. Immunoglobulin and protein levels in breast milk produced by mothers of preterm infants. Nutr. Res. (N. Y., NY, U. S.) 1982, 2, 27−30. (59) Bernback, S.; Blackberg, L.; Hernell, O. The complete digestion of human milk triacylglycerol in vitro requires gastric lipase, pancreatic colipase-dependent lipase, and bile salt-stimulated lipase. J. Clin. Invest. 1990, 85, 1221−1226. (60) Oizumi, J.; Hirano, M.; Hayakawa, K.; Daimatsu, T.;et al. The significance of breast milk biotinidase. Pediatr. Int. 1989, 31. (61) Suormala, T.; Wick, H.; Baumgartner, E. Low biotinidase activity in plasma of some preterm infants: Possible source of falsepositive screening results. Eur. J. Pediatr. 1988, 147, 478−480. (62) Anderson, G. The effect of prematurity on milk composition and its physiological basis. Fed. Proc. 1984, 43, 2438−2442. (63) Hartmann, P. E.; Kulski, J. K. Changes in the composition of the mammary secretion of women after abrupt termination of breast feeding. J. Physiol. 1978, 275, 1−11. (64) Stelwagen, K. Mammary epithelial cell tight junction integrity and mammary blood flow during an extended milking interval in goats. J. Dairy Sci. 1994, 77, 426−432. (65) Hill, P. D.; Aldag, J. C.; Demirtas, H.; Naeem, V.; et al. Association of serum prolactin and oxytocin with milk production in mothers of preterm and term infants. Biol. Res. Nurs. 2009, 10, 340− 349. (66) Jones, F. S.; Jones, P. L. The tenascin family of ECM glycoproteins: Structure, function, and regulation during embryonic development and tissue remodeling. Dev. Dyn. 2000, 218, 235−259. (67) Jones, P. L.; Boudreau, N.; Myers, C. A.; Erickson, H. P.; et al. Tenascin-C inhibits extracellular matrix-dependent gene expression in mammary epithelial cells. Localization of active regions using recombinant tenascin fragments. J. Cell Sci. 1995, 108, 519−527.

(24) Laemmli, U. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227, 680−685. (25) Abramoff, M. D.; Magelhaes, P. J.; Ram, S. J. Image processing with ImageJ. Biophoton Int. 2004, 11, 36−42. (26) Lui, J. K. C.; Lipscombe, R.; Arthur, P. G. Detecting changes in the thiol redox state of proteins following a decrease in oxygen concentration using a dual labeling technique. J. Proteome Res. 2009, 9, 383−392. (27) Shevchenko, A.; Tomas, H.; Havlis, J.; Olsen, J.; et al. In-gel digestion for mass spectrometric characterisation of proteins and proteomes. Nat. Protoc. 2007, 1, 2856−2860. (28) D’Alessandro, A.; Scaloni, A.; Zolla, L. Human milk proteins: An interactomics and updated functional overview. J. Proteome Res. 2010, 9, 3339−3373. (29) Mouton-Barbosa, E.; Roux-Dalvai, F.; Bouyssie, D.; Berger, F.; et al. In-depth exploration of cerebrospinal fluid by combining peptide ligand library treatment and label-free protein quantification. Mol. Cell. Proteomics 2010, 9, 1006−1021. (30) Roux-Dalvai, F.; Gonzalez de Peredo, A.; Sime, C.; Guerrier, L.; et al. Extensive analysis of the cytoplasmic proteome of human erythrocytes using the peptide ligand library technology and advanced mass spectrometry. Mol. Cell. Proteomics 2008, 7, 2254−2269. (31) Guerrier, L.; Pier Giorgio, R.; Egisto, B. Reduction of dynamic protein concentration range of biological extracts for the discovery of low-abundance proteins by means of hexapeptide ligand library. Nat. Protoc. 2008, 3, 883−890. (32) Patton, S.; Huston, G. Incidence and characteristics of cell pieces on human milk fat globules. Biochim. Biophys. Acta 1988, 965, 146−153. (33) Brooker, B. E. The epithelial cells and cell fragments in human milk. Cell Tissue Res. 1980, 210, 321−332. (34) Cregan, M. D.; De Mello, T. R.; Kershaw, D.; McDougall, K.; et al. Initiation of lactation in women after preterm delivery. Acta Obstet. Gynecol. Scand. 2002, 81, 870−877. (35) Fetherston, C. M.; Lai, C. T.; Mitoulas, L. R.; Hartmann, P. E. Excretion of lactose in urine as a measure of increased permeability of the lactating breast during inflammation. Acta Obstet. Gynecol. Scand. 2006, 85, 20−25. (36) Arthur, P. G.; Kent, J.; Hartmann, P. E. Metabolites of lactose synthesis in milk from diabetic and non-diabetic women during lactogenesis II. J. Pediatr. Gastroenterol. Nutr. 1994, 19, 100−108. (37) Semba, R. D.; Kumwenda, N.; Taha, T. E.; Hoover, D. R.; et al. Mastitis and immunological factors in breast milk of lactating women in Malawi. Clin. Diagn. Lab. Immunol. 1999, 6, 671−674. (38) Boehmer, J. L.; Bannerman, D. D.; Shefcheck, K.; Ward, J. L. Proteomic analysis of differentially expressed proteins in bovine milk during experimentally induced escherichia coli mastitis. J. Dairy Sci. 2008, 91, 4206−4218. (39) Kaufmann, S. H. E. Heat shock proteins and the immune response. Immunol. Today 1990, 11, 129−136. (40) Bateman, A.; Bennett, H. P. Granulins: The structure and function of an emerging family of growth factors. J. Endocrinol. 1998, 158, 145−51. (41) Klapholz-Brown, Z.; Walmsley, G.; Nusse, Y.; Brown, P. Transcriptional program induced by Wnt protein in human fibroblasts suggest mechanisms for cell cooperativity in defining tissue microenvironments. PLoS One 2007, 2, e945. (42) Kahai, S.; Lee, S.-C.; Seth, A.; Yang, B. B. Nephronectin promotes osteoblast differentiation via the epidermal growth factorlike repeats. FEBS Lett. 2010, 584, 233−238. (43) Kolle, G.; Georgas, K.; Holmes, G. P.; Little, M. H.; et al. CRIM1, a novel gene encoding a cysteine-rich repeat protein, is developmentally regulated and implicated in vertebrate CNS development and organogenesis. Mech. Dev. 2000, 90, 181−193. (44) Anderson, H.; Hodges, P.; Aguilera, X.; Missana, L.; et al. Bone morphogenetic protein (BMP) localization in developing human and rat growth plate, metaphysis, epiphysis, and articular cartilage. J. Histochem. Cytochem. 2000, 48, 1493−1502. 1714


Proteome Mapping of Human Skim Milk Proteins in Term and Preterm

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