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 pro- teome. 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 ■ INTRODUCTION human milk be characterized. There are two main reasons for The importance of human milk proteins to the growth and this. First, it is possible that these proteins play significant roles development of breastfed infants is well established. They not in infant growth and development. Second, knowledge of how only provide a digestible source of amino acids to infants, but the expression of low abundance proteins differs between term also confer immunological protection and perform developmen- and preterm milk may be useful diagnostically, as a reflection of tal and regulatory functions, exerting both long and short-term the developmental changes occurring in the mammary gland benefits compared to formula feeding.1,2 during pregnancy and lactation. Human milk proteins are particularly important for infants Historically, there have been a number of technical who are born prematurely. Recent studies stress the importance challenges associated with characterizing the low abundance of both the total amount of protein and the ratio of protein/ proteins in human milk. Initial studies using gel electrophoretic energy that preterm infants receive for their growth and devel- methods coupled with mass spectrometry were unable to detect opment.3 Significantly, the protein composition of milk from more than 10 different gene products in either human or bovine milk, despite observing hundreds of distinct protein preterm mothers is known to differ from that of term mothers. 11−13 The concentration of total protein is typically higher in preterm spots. This difficulty results from the fact that six proteins, α β milk;4 however, while some individual proteins are expressed at -lactalbumin, -casein, secretory immunoglobulin A, lysozyme, higher levels in preterm milk, others are present at lower con- lactoferrin, and secretory component, constitute over 90% of − 14 centrations.5 7 the total protein content in mature human milk, obscuring Hitherto, most studies investigating milk protein composi- the detection of less abundant proteins of potential biological tion have focused upon the most abundant proteins present, interest. resulting in their relative concentrations in term and preterm − milk being well-defined.8 10 However, it is also important that Received: September 2, 2011 the identity and behavior of the lower abundance proteins in Published: February 7, 2012 © 2012 American Chemical Society 1696 dx.doi.org/10.1021/pr2008797 | J. Proteome Res. 2012, 11, 1696−1714 Journal of Proteome Research Article More recently, proteomic studies employing strategies to (7−14 days lactation) and 16 term mothers (7−14 days lactation) deplete the high abundance proteins or to extensively frac- were used. All milk samples were thawed, pooled, and centri- tionate the sample prior to analysis have been more successful fuged at 10000g for 10 min to remove the cream layer. A at identifying low abundance proteins than the earlier gel-based − mammalian protease inhibitor cocktail containing 4-(2- methods.14 17 Two studies in particular have identified a large aminoethyl)benzenesulfonyl fluoride, E-64, bestatin, leupeptin, number of low abundance human milk proteins. Palmer et al.18 aprotinin, and sodium EDTA was added to each pooled sample, used immunodepletion columns to deplete the five most which was then depleted of casein using a previously described − 22 abundant proteins in colostrum prior to 2D LC MS/MS an- method. Briefly, to deplete the samples of casein, CaCl2 was alysis and were able to identify 151 proteins. More recently, added to a concentration of 60 mM, and the pH was adjusted 19 Liao et al. employed combinatorial hexapeptide ligand to pH 4.3. Samples were then centrifuged at 189000g at 4 °C libraries (ProteoMiner) to enrich the low abundance milk for 60 min, and the supernatant was collected. proteins before analysis by LC−MS/MS and identified 115 For the ProteoMiner-treated samples (Figure 1), casein- 19 proteins. Liao et al. also showed that many of these proteins depleted skim milk was dialyzed against 10 volumes of 10 mM change in expression over the course of 12 months of lactation, Tris, pH 7 at 4 °C, with three buffer changes at two-hour inter- highlighting the dynamic nature of milk composition. One of vals using dialysis tubing with a MW cutoff of 3500 Da the advantages of using a ProteoMiner bead approach is that it (Spectrapor Membrane Tubing, Spectrum Medical Industries, does not require tailored antibodies or optimization. When a Rancho Domingues, CA). The samples were then lyophilized sample is applied to the ligand library, the high abundance pro- and reconstituted in water. The samples were analyzed for their teins saturate their ligands and the excess remains unbound, protein content and diluted such that 1 mL of 50 g/L protein whereas the lower abundance proteins bind completely, result- solution was loaded onto the hexaligand library beads ing in an overall compression of the dynamic range. Recent (ProteoMiner Large Capacity Protein Enrichment Kit, BioRad, studies have also shown the ProteoMiner treatment to be com- Gladesville, NSW, Australia), according to the manufacturer’s patible with downstream quantitative analyses of low abundance 20,21 instructions. Briefly, after swelling the beads using the buffer proteins. provided, the samples were loaded onto individual ProteoMiner The aim of the present study was two-fold. First, we aimed to columns and rocked for 6 h at room temperature. For the further characterize the proteome of mature human skim milk pooled term milk samples (collected 15−28 days postpartum), from established lactation (7−28 days postpartum), using a the unbound proteins were then washed through the column, combination of ultracentrifugation and ProteoMiner enrich- and the bound proteins were eluted using the acidic elution ment to compress the dynamic range of the proteome prior to buffer provided in the kit. For the pooled term and preterm analysis by 2D LC−MS/MS. Second, we sought to quanti- milk samples to be subsequently labeled with iTRAQ reagents tatively examine whether there are differences in protein − expression between term and preterm milk that reflect the (collected 7 14 days postpartum), the bound proteins were physiological and metabolic effects of preterm delivery upon eluted sequentially using four different buffers: 1 M sodium the mammary gland. 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, ■ EXPERIMENTAL PROCEDURES 16.7% (v/v) acetonitrile, 0.1% (v/v) trifluoroacetic acid. Protein Concentration Determination Materials Protein concentrations of human milk samples were deter- Unless otherwise stated, all chemicals and reagents were ob- mined using a Bicinchoninic acid kit (Sigma-Aldrich, NSW, tained from Sigma-Aldrich (NSW, Australia). Australia), as described in a previous study.23 Sample Collection Sodium Dodecyl Sulfate Polyacrylamide Gel Term and preterm milk samples were obtained from healthy Electrophoresis (SDS-PAGE) lactating mothers at King Edward Memorial Hospital, Subiaco, Western Australia. Participating term mothers had delivered SDS-PAGE analysis was conducted using the HOEFER gel between 38 and 41 weeks of gestation, and their infants had a apparatus (HOEFER Scientific Instruments, San Francisco, chronological age of between 7 and 28 days at the time of CA) and the Laemmli gel system using 12.5% polyacrylamide gels.24 Gels were run using a constant current of 15 mA for sample collection. Participating