proteome mapping of human skim milk proteins in term and preterm milk

Proteome Mapping of Human Skim Milk Proteins in Term andPreterm MilkClaire 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 havebeen well characterized and are known to provide nutritional,protective, and developmental advantages to both term andpreterm infants. However, relatively little is known about theexpression of the low abundance proteins that are present inhuman milk because of the technical difficulties associated withtheir detection. We used a combination of electrophoretictechniques, ProteoMiner treatment, and two-dimensional liquidchromatography to examine the proteome of human skim milkexpressed between 7 and 28 days postpartum by healthy termmothers and identified 415 in a pooled milk sample. Of these,261 were found in human skim milk for the first time, greatlyexpanding 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

■ INTRODUCTIONThe importance of human milk proteins to the growth anddevelopment of breastfed infants is well established. They notonly provide a digestible source of amino acids to infants, butalso confer immunological protection and perform developmen-tal and regulatory functions, exerting both long and short-termbenefits compared to formula feeding.1,2

Human milk proteins are particularly important for infantswho are born prematurely. Recent studies stress the importanceof both the total amount of protein and the ratio of protein/energy that preterm infants receive for their growth and devel-opment.3 Significantly, the protein composition of milk frompreterm mothers is known to differ from that of term mothers.The concentration of total protein is typically higher in pretermmilk;4 however, while some individual proteins are expressed athigher levels in preterm milk, others are present at lower con-centrations.5−7

Hitherto, most studies investigating milk protein composi-tion have focused upon the most abundant proteins present,resulting in their relative concentrations in term and pretermmilk being well-defined.8−10 However, it is also important thatthe identity and behavior of the lower abundance proteins in

human milk be characterized. There are two main reasons forthis. First, it is possible that these proteins play significant rolesin infant growth and development. Second, knowledge of howthe expression of low abundance proteins differs between termand preterm milk may be useful diagnostically, as a reflection ofthe developmental changes occurring in the mammary glandduring pregnancy and lactation.Historically, there have been a number of technical

challenges associated with characterizing the low abundanceproteins in human milk. Initial studies using gel electrophoreticmethods coupled with mass spectrometry were unable to detectmore than 10 different gene products in either human orbovine milk, despite observing hundreds of distinct proteinspots.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% ofthe total protein content in mature human milk,14 obscuringthe detection of less abundant proteins of potential biologicalinterest.

Received: September 2, 2011Published: February 7, 2012

Article

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

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More recently, proteomic studies employing strategies todeplete the high abundance proteins or to extensively frac-tionate the sample prior to analysis have been more successfulat identifying low abundance proteins than the earlier gel-basedmethods.14−17 Two studies in particular have identified a largenumber of low abundance human milk proteins. Palmer et al.18

used immunodepletion columns to deplete the five mostabundant proteins in colostrum prior to 2D LC−MS/MS an-alysis and were able to identify 151 proteins. More recently,Liao et al.19 employed combinatorial hexapeptide ligandlibraries (ProteoMiner) to enrich the low abundance milkproteins before analysis by LC−MS/MS and identified 115proteins. Liao et al.19 also showed that many of these proteinschange in expression over the course of 12 months of lactation,highlighting the dynamic nature of milk composition. One ofthe advantages of using a ProteoMiner bead approach is that itdoes not require tailored antibodies or optimization. When asample is applied to the ligand library, the high abundance pro-teins saturate their ligands and the excess remains unbound,whereas the lower abundance proteins bind completely, result-ing in an overall compression of the dynamic range. Recentstudies have also shown the ProteoMiner treatment to be com-patible with downstream quantitative analyses of low abundanceproteins.20,21

The aim of the present study was two-fold. First, we aimed tofurther characterize the proteome of mature human skim milkfrom established lactation (7−28 days postpartum), using acombination of ultracentrifugation and ProteoMiner enrich-ment to compress the dynamic range of the proteome prior toanalysis by 2D LC−MS/MS. Second, we sought to quanti-tatively examine whether there are differences in proteinexpression between term and preterm milk that reflect thephysiological and metabolic effects of preterm delivery uponthe mammary gland.

■ EXPERIMENTAL PROCEDURES

Materials

Unless otherwise stated, all chemicals and reagents were ob-tained from Sigma-Aldrich (NSW, Australia).

Sample Collection

Term and preterm milk samples were obtained from healthylactating mothers at King Edward Memorial Hospital, Subiaco,Western Australia. Participating term mothers had deliveredbetween 38 and 41 weeks of gestation, and their infants had achronological age of between 7 and 28 days at the time ofsample collection. Participating preterm mothers had deliveredbetween 28 and 32 weeks of gestation, and their infants had achronological age of between 7 and 14 days at the time ofsample collection. All donors gave written informed consent fortheir donations to be used in this research, and this study wasapproved by the University of Western Australia, HumanResearch 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 °Cstorage within 3 days.

Sample Treatment

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

(7−14 days lactation) and 16 term mothers (7−14 days lactation)were used. All milk samples were thawed, pooled, and centri-fuged at 10000g for 10 min to remove the cream layer. Amammalian protease inhibitor cocktail containing 4-(2-aminoethyl)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 describedmethod.22 Briefly, to deplete the samples of casein, CaCl2 wasadded to a concentration of 60 mM, and the pH was adjustedto pH 4.3. Samples were then centrifuged at 189000g at 4 °Cfor 60 min, and the supernatant was collected.For the ProteoMiner-treated samples (Figure 1), casein-

depleted skim milk was dialyzed against 10 volumes of 10 mMTris, pH 7 at 4 °C, with three buffer changes at two-hour inter-vals using dialysis tubing with a MW cutoff of 3500 Da(Spectrapor Membrane Tubing, Spectrum Medical Industries,Rancho Domingues, CA). The samples were then lyophilizedand reconstituted in water. The samples were analyzed for theirprotein content and diluted such that 1 mL of 50 g/L proteinsolution was loaded onto the hexaligand library beads(ProteoMiner Large Capacity Protein Enrichment Kit, BioRad,Gladesville, NSW, Australia), according to the manufacturer’sinstructions. Briefly, after swelling the beads using the bufferprovided, the samples were loaded onto individual ProteoMinercolumns and rocked for 6 h at room temperature. For thepooled 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 elutionbuffer provided in the kit. For the pooled term and pretermmilk samples to be subsequently labeled with iTRAQ reagents(collected 7−14 days postpartum), the bound proteins wereeluted sequentially using four different buffers: 1 M sodiumchloride 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.

Protein Concentration Determination

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

Sodium Dodecyl Sulfate Polyacrylamide GelElectrophoresis (SDS-PAGE)

SDS-PAGE analysis was conducted using the HOEFER gelapparatus (HOEFER Scientific Instruments, San Francisco,CA) and the Laemmli gel system using 12.5% polyacrylamidegels.24 Gels were run using a constant current of 15 mA for16 h at 4 °C, fixed for 2 h in 50% methanol/10% trichloroaceticacid, destained using double deionized water, stained usingCoomassie Brilliant Blue R-250 overnight, and scanned usingan Epson Perfection V700 photographic flatbed color imagescanner (Epson, Nagano, Japan). The intensity of proteinbands were measured using the open access software packageImage J 1.410.25

Differential in-Gel Electrophoresis (DIGE)

Cy5 and Cy3 activated ester dyes were purchased fromLumiprobe (Lumiprobe Corp, FL). Skim milk samples beforeand after casein depletion and ProteoMiner treatment wereeach labeled with one of the dyes. The DIGE experiment wasconducted in duplicate, and the order of the dyes were swappedfor the duplicate experiment. Fifty micrograms of protein samplewas labeled according to the CyDye DIGE Flours (minimal dyes)

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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 pHgradient 3−10, length 18 cm (GE Health Care) and anIPGphor isoelectric focusing unit (IPGPhor, GE Health Care).The strips were actively rehydrated for 10 h at a constant volt-age of 200 V and a constant temperature of 20 °C. One hundredmicrograms of protein was loaded. Following rehydration, thestrips were exposed to a linear increase to 1000 V over 4 hfollowed by 8000 V until a total of 45000 V hr was reached.The second dimension separation was carried out in the darkaccording to the method of Lui, Lipscombe, and Arthur.26 Gelswere imaged using a Typhoon Trio scanner (GE Health Care),with the Cy5 and Cy3 labeled samples visible using 633/670 nmand 532/580 nm excitation/emission filters, respectively. Gelswere post stained for total protein content using CoomassieBrilliant Blue. DIGE gels were analyzed using the ProgenesisSameSpots software package (Nonlinear Dynamics Ltd., New-castle upon Tyne, U. K.). Spots with a normalized spot volumeof less than 500 were excluded from the analysis. All data ispresented as mean ± SEM.For the purposes of downstream mass spectrometry analysis,

a preparative 2D gel analysis was also conducted. Five hundredmicrograms of the ProteoMiner-treated skim milk sample wasloaded, and the first and second dimensions were carried out asabove. Gels were fixed and stained as described above. Co-omassie stained bands and spots of interest were cut from thegel, destained, and digested as described by Shevchenko et al.27

Mass spectrometry was conducted as described in a pre-vious study using an UltraFlex MALDI-TOF/TOF instrument(Bruker Daltonics, Bremen, Germany).23 MS/MS data was im-ported into the database search engine Mascot (Version 2.3.01,www.matrixscience.com) and searched against the Swiss-ProtMammalia database (49 887 sequences).

LC−MS/MS

Sample Preparation. Protein samples were precipitated byadding five volumes of cold acetone to the treated samples(Figure 1), incubating for 1 h at −20 °C, and pulse centrifugingfor 5−10 s. The protein pellets were resuspended in 0.5 Mtriethylammonium bicarbonate (TEAB) (pH 8.5) by shakingbefore reduction and alkylation according to the iTRAQprotocol (Applied Biosystems, Foster City, CA). A total of55 μg of each sample was digested overnight with 5.5 μg trypsinat 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 of10−45% acetonitrile, 0.1% trifluoroacetic acid over 165 min,using the Ultimate 3000 nano HPLC system (LC Packings-Dionex). Every 30 s, the eluent was mixed with matrix solution(5 mg/mL CHCA) and spotted onto a 384 well Opti-TOFplate (Applied Biosystems) using a Probot Micro FractionCollector (LC Packings-Dionex).

2D-LC. Peptides were desalted on a Strata-X 33 μm poly-meric reversed phase column (Phenomenex) and dissolved in abuffer containing 10 mM potassium hydrogen phosphate, pH 3in 10% acetonitrile, before separation by strong cation exchangechromatography on an Agilent 1100 HPLC system (AgilentTechnologies, 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 mMKCl. Eight fractions containing the peptides were collected anddesalted on Strata-X columns. Each peptide fraction was thenseparated and spotted onto a 384-well Opti-TOF plateaccording to the 1D-LC protocol described above, exceptingthat a 10−40% acetonitrile (0.1% trifluoracetic acid) gradientwas used.

iTRAQ. The tryptic digests were dried in a SpeedVac, re-suspended in 30 μL of 0.5 M TEAB, and labeled by addingiTRAQ reagents to preterm and term milk samples, respectively,according to the iTRAQ protocol (Applied Biosystems). Forthe iTRAQ experiment comparing term and preterm milksamples without ProteoMiner treatment (Figure 1), duplicatepooled preterm milk samples were labeled with iTRAQ reagents

Figure 1. Experimental workflow.



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114 and 116. Duplicate pooled term milk samples were labeledwith iTRAQ reagents 115 and 117. For the iTRAQ experimentcomparing term and preterm milk samples after ProteoMinertreatment (Figure 1), duplicate pooled preterm milk sampleswere labeled with iTRAQ reagents 116 and 117. Duplicatepooled term milk samples were labeled with iTRAQ reagents114 and 115. Excess iTRAQ reagent was quenched by adding1 mL of water; the samples were then combined, desalted on aStrata-X 33 μm polymeric reverse phase column (Phenomenex,Torrance, CA), and analyzed using the 2D-LC protocol de-scribed above.MALDI-MS/MS. Peptides were analyzed on a 5800 MALDI-

TOF/TOF mass spectrometer (Applied Biosystems) operatedin reflector positive mode. MS data were acquired over a massrange of 800−4000 m/z, and for each spectrum, a total of 400shots were accumulated. A job-wide interpretation methodselected the 20 most intense precursor ions above a signal/noise ratio of 20 from each spectrum for MS/MS acquisitionbut only in the spot where their intensity was at its peak. MS/MS spectra were acquired with 4000 laser shots per selected ionwith 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/MSspectra were searched against the Swiss-Prot human genomicdatabase (2011_3 for the 2D-LC analysis, 2011_5 for the 1D-LC analysis). Search parameters were as follows: Cys alkylation,MMTS; Digestion, trypsin; Instrument, 5800; Special factors,none; Species, none; Quantitate tab, unchecked; Detectedprotein threshold (unused ProtScore), 1.3, which correspondsto proteins identified with <95% confidence.For the iTRAQ experiment, MS/MS spectra were analyzed

as above with ProteinPilot 4.0.8085 software, with the addition

of the parameter, iTRAQ 4plex (peptide labeled) modification,and the Quantitate tab checked. MS/MS spectra were searchedagainst the Swiss-Prot human genomic database (2011_5). Forquantitation analysis, the duplicates were analyzed separately.Average protein ratios and p-values to indicate significant differ-ential expression were calculated by the software. To be con-sidered as being differentially expressed, proteins were requiredto have an unused protein score greater than 1.3, correspondingto a confidence interval of 95%, and have significantly differentprotein ratios in both replicates, also at a confidence level of95% (p < 0.05). Identified proteins for which a difference wasfound at a confidence level of 90% (p < 0.1) in both replicateswere also reported. The p values represent the variation in thereported iTRAQ ratios for all the peptides of the associatedprotein and do not relate to either biological variation ortechnical reproducibility.The false discovery rate was less than 1%, calculated using a

database containing reversed sequences. In order to categorizethe identified proteins, the results were analyzed using thesoftware program IPA (Ingenuity Databases) and the UniProtDatabase release 2011_6 (http://www.uniprot.org/). Resultswere compared to a recent comprehensive review publication28

and a subsequent research paper19 in order to determine whichproteins had not been previously identified.

■ RESULTSCasein Depletion

The SDS-PAGE gel analysis showed that casein depletion re-sulted in the removal of 67 ± 3% (n = 5) of the 30 kDa β-caseinband present in the pooled skim milk sample (Figure 2). The κ-casein protein band at 38 kDa and a number of other β-caseinbands in the 20−30 kDa region present in the skim milk sample

Figure 2. SDS-PAGE electrophoretograms and mass spectrometry identifications of milk protein fractions. Proteins (15 μg) from (A) skim milk, (B)skim milk after ProteoMiner treatment, (C) skim milk after casein depletion, and (D) skim milk after casein depletion and ProteoMiner treatmentwere analyzed (n = 4, 2 depicted). Significant protein identifications obtained using MALDI MS/MS are displayed adjacent to the correspondingband of the casein-depleted ProteoMiner-treated skim milk.



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also appear to be present at considerably lower levels in thecasein-depleted sample (Figure 2).

ProteoMiner Treatment: SDS-PAGE

The ProteoMiner treatment compressed the dynamic range ofthe proteins present in human milk. However, ProteoMinertreatment resulted in a far greater enrichment of lower abun-dance proteins when used after casein depletion, compared towhen it was performed on the delipidated skim milk alone(Figure 2). As a result, all samples were depleted of casein priorto further treatment and analysis (Figure 1). In the SDS-PAGEanalysis of the casein-depleted protein sample, the five mostintense protein bands constituted 52% before ProteoMinertreatment and 24% after treatment (p < 0.001) (Figure 2). Themajor protein bands of β-casein (30 kDa), α-lactalbumin(14 kDa), serum albumin (65 kDa), and sIgA α-chain C region(60 kDa) were all depleted after the combinatorial ligandlibrary treatment (p < 0.002) (Figure 2). It is also possible tosee the resulting enrichment of lower abundance proteins. Thexanthine dehydrogenase band at ∼170 kDa was enriched by 5.4fold (p < 0.001), and there were a number of additional bandsseen in the 30−50 kDa range and between 20 and 25 kDa(Figure 2).

ProteoMiner Treatment: 2D-DIGE

The compression of the protein dynamic range caused by thecasein depletion and ProteoMiner treatment was visible to agreater extent in the 2D-DIGE analysis. There were 308 and320 spots present on the duplicate skim milk gels, compared to359 and 364 spots on the duplicate gels of the casein-depleted,ProteoMiner-treated samples (Figure 3). Over two-thirds(68 ± 5%) of the spots were present at a relatively higher in-tensity after casein depletion and ProteoMiner treatment com-pared to in the skim milk. Similarly, 28 ± 3% of the spots in thetreated sample occupied a 2-fold greater percentage intensity,and 20 ± 0.5% of spots were present at a 3-fold higher per-centage intensity relative to the skim milk.In the skim milk gels, 60 ± 4% of the total gel intensity was

occupied by the β-casein and α-lactalbumin spot clusters at 25−30 kDa and 14.4 kDa, respectively. Casein depletion andProteoMiner treatment reduced the dominance of the abun-dant proteins, with only 40 ± 3% of the total gel intensity beingoccupied by the same β-casein and α-lactalbumin spots aftertreatment (Figure 3). In the skim milk gels, 54 ± 0.1% of spotswere present at very low levels, each occupying less than 0.04%of the total gel intensity. In the treated sample, however, only29 ± 0.5% of the spots on the gel were present at an intensityless than this 0.04% threshold.For the preparative 2DE analysis, 110 spots were processed

for MALDI-MS/MS analysis, and 61 of these spots werepositively identified. Many of the spots that were not identifiedwere of extremely low abundance and were only faintly visibleusing Coomassie Brilliant Blue staining. There was a great dealof redundancy, with the 61 spots corresponding to only21 gene products (Supporting Information Data Files 2 and 3).

Protein Identifications

A total of 415 proteins were identified at a confidence level of95% in human milk collected between 7 and 28 days post-partum from term mothers, 261 of which had not previouslybeen identified in human skim milk (Table 1). With regard tomethodology, the majority of these proteins were identified inthe 2D LC−MS/MS analyses after ProteoMiner treatment,with 15 proteins being identified in only the 1D LC−MS/MS

analysis, and 29 proteins being identified in the iTRAQ experi-ment without ProteoMiner treatment (Figure 4A,B). Withregard to the stage of lactation, 174 proteins were identified inboth the pooled term milk collected 7−14 days postpartum andin the pooled term milk collected 15−28 days postpartum. Therewere 141 proteins unique to the pool collected earlier in lactation,and 100 proteins were identified only in the pooled term milkcollected 15−28 days postpartum (Figure 4C). The 415 proteinswere categorized by both their subcellular location and theirfunctions, according to their annotations in the UniProt Databaserelease 2011_6 (http://www.uniprot.org/) (Figure 5). Themajority of the proteins found in the present study were cyto-plasmic (46%) or extracellular space proteins (38%). Functionally,the majority of the proteins identified were involved in either inthe immune response (24%) or in cellular metabolism andcellular growth (28%). See Supporting Information Data File 4for a description of how each protein was categorized.

Figure 3. Two-dimensional fluorescent differential in-gel expression(DIGE) experiment. (A) Skim milk protein fraction, (B) skim milkprotein fraction after casein depletion and ProteoMiner treatment.The gels depicted are one pair out of a set of duplicate experiments.



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Table 1

protein name accession no. unuseda % covb peptides methodc

1 14-3-3 protein epsilond P62258 2 20 3 3, 42 14-3-3 protein gammad P61981 2 20.2 2 43 14-3-3 protein zeta/delta P63104 11.01 40.4 6 3, 4, 64 40S ribosomal protein S10d P46783 6 27.9 4 65 40S ribosomal protein S12d P25398 1.68 17.4 1 46 40S ribosomal protein S14d P62263 2.88 22.5 2 4, 67 40S ribosomal protein S19d P39019 19.09 55.9 9 4, 68 40S ribosomal protein S20d P60866 6.04 37.8 4 69 40S ribosomal protein S25d P62851 4.63 27.2 3 4, 610 40S ribosomal protein S28d P62857 1.42 17.4 1 511 40S ribosomal protein S3d P23396 3.24 14.4 3 4, 612 40S ribosomal protein S3ad P61247 4.25 16.3 3 613 40S ribosomal protein S5d P46782 1.44 4.4 1 614 40S ribosomal protein SAd P08865 1.63 8.5 3 615 45 kDa calcium-binding proteind Q9BRK5 6 9.7 4 4, 616 4F2 cell surface antigen heavy chaind P08195 1.55 1.6 1 617 6-phosphogluconate dehydrogenase, decarboxylatingd P52209 5.67 21.7 7 3, 4, 618 6-phosphoglucolactonased O95336 4.54 22.1 2 619 60S acidic ribosomal protein P0-liked Q8NHW5 2 15.1 3 420 60S acidic ribosomal protein P1 P05386 1.82 14 1 621 60S acidic ribosomal protein P2d P05387 2 28.7 1 422 60S ribosomal protein L12d P30050 2 22.4 2 623 60S ribosomal protein L23d P62829 1.82 20.7 2 624 60S ribosomal protein L24d P83731 3.5 22.9 2 625 60S ribosomal protein L31d P62899 2.02 22.4 2 626 78 kDa glucose-regulated protein P11021 23.53 41.3 19 2, 4, 627 Acetyl-CoA acetyltransferase, cytosolicd Q9BWD1 4 13.9 2 4, 628 Acid sphingomyelinase-like phosphodiesterase 3b Q92485 5.09 12.1 4 429 Actin-related protein 2/3 complex subunit 1Bd O15143 2 8.3 2 4, 630 Actin, cytoplasmic 2 P63261 25.01 57.3 36 1, 2, 3, 4, 5, 631 Acyl-CoA binding proteind P07108 3.07 20.7 2 532 ADP-ribosylation factor 1d P84077 2 17.7 1 433 ADP ribosylation factor 5d P84085 1.66 9.4 2 634 Adenine phosphoribosyltransferased P07741 1.44 18.9 3 635 Alanine aminotransferase 1d P24298 2.02 3.4 2 636 Alcohol dehydrogenase [NADP+] P14550 12.62 35.7 8 4, 637 Alpha-(1,3)-fucosyltransferased P51993 2.24 12 2 638 Alpha-1-acid glycoprotein P02763 7.07 28.9 6 539 Alpha-1-antichymotrypsin P01011 22.54 39.2 21 4, 5, 640 Alpha-1-antitrypsin P01009 44.3 61.7 70 3, 4, 5, 641 Alpha-1B-glycoprotein P04217 4.47 11.5 2 642 Alpha-2-antiplasmind P08697 5.32 10 4 4, 643 Alpha-2-HS-glycoprotein P02765 7.07 20.4 5 444 Alpha-2-macroglobulind P01023 7.93 7.3 6 4, 645 Alpha-actinin-4 O43707 2 8 1 346 Alpha-aminoadipic semialdehyde dehydrogenased P49419 2 5.2 1 447 Alpha-amylase 2Bd P19961 2.01 5.9 2 648 Alpha-enolase P06733 18.01 45.2 14 2, 3, 4, 5, 649 Alpha-lactalbumin P00709 31.46 82.4 71 1, 2, 3, 4, 5, 650 Alpha-S1-casein P47710 38.86 81.1 71 1, 2, 3, 4, 5, 651 Amyloid beta A4 protein P05067 4.02 16.2 6 4, 652 Angiopoietin-related protein 4d Q9BY76 7.87 20.4 6 4, 653 Antileukoproteinased P02647 3.88 37.9 3 4, 654 Antithrombin-III P01008 9.92 39.4 9 4, 655 Apolipoprotein A-I P02647 29.14 67.8 30 1, 2, 3, 4, 5, 656 Apolipoprotein A-II P02652 9.75 41 9 3, 4, 657 Apolipoprotein A-IV P06727 17.91 54.3 14 3, 4, 658 Apolipoprotein B-100d P04114 63.34 13.2 32 3, 659 Apolipoprotein D P05090 8.43 30.7 7 4, 660 Apolipoprotein E P02649 15.73 46.1 13 3, 461 Arylsulfatase Ad P15289 3.42 9.9 3 4, 6



Table 1. continued


62 Beta-1,4-galactosyltransferase 1 P15291 15.28 53.8 24 3, 4, 663 Beta-2-microglobulin P61769 7.41 32.8 5 4, 564 Beta-casein P05814 140.3 82.3 195 1, 2, 3, 4, 5, 665 Bifunctional purine biosynthesis protein PURHd P31939 2 2.7 2 666 Bile salt-activated lipase P19835 64.64 52.5 96 1, 3, 4, 5, 667 Biotinidase P43251 2.99 12.3 6 4, 568 Bone morphogenetic protein 1d P13497 2.01 2.5 2 669 Butyrophilin subfamily 1 member A1 Q13410 39.81 45.8 48 1, 3, 4, 5, 670 C-1-tetrahydrofolate synthase, cytoplasmicd P11586 3.83 6.2 2 671 C-type lectin domain family 11 member A Q9Y240 4 16.1 2 672 C−C motif chemokine 28d Q9NRJ3 2 11.8 1 473 C-X-C motif chemokine 2 P19875 4.28 38.3 3 674 C4b-binding protein alpha chain P04003 9.6 17.3 10 4, 675 Cadherin-1d P12830 3.18 7.8 5 4, 676 Calmodulin P62158 7.74 38.3 4 4, 677 Calmodulin-like protein 5d Q9NZT1 4.57 37 4 3, 4, 678 Calreticulin P27797 7.74 17.3 5 4, 679 Calumenind O43852 2 7 2 480 Carbonic anhydrase 2d P00918 2.08 16.9 2 681 Carbonic anhydrase 6 P23280 19.66 46.8 19 3, 4, 682 Carbonyl reductase [NADPH] 3d O75828 1.66 9.7 3 4, 683 Carboxypeptidase B2d Q96IY4 2 3.8 1 484 Cathepsin Bd P07858 15.51 42.8 14 4, 685 Cathepsin S P25774 4.12 13.3 4 486 Cation-independent mannose-6-phosphate receptord P11717 1.8 2.1 2 4, 687 CD9 antigen P21926 2.23 16.7 13 588 CD81 antigend P60033 2 8.5 1 589 Cell division control protein 42 homologued P60953 5.92 29.3 4 4, 690 Ceruloplasmin P00450 13.06 20.2 16 3, 4, 691 Chitinase-3-like protein 1 P36222 1.32 2.9 1 492 Chloride intracellular channel protein 1d O00299 4.01 27.4 4 4, 5, 693 Chordin-like protein 2 Q6WN34 34.41 47.6 33 3, 4, 5, 694 Clusterin P10909 46.61 64.6 87 1, 2, 3, 4, 5, 695 Cofilin-1 P23528 14.3 55.4 8 3, 4, 696 Coiled-coil domain containing protein 38d Q502W7 1.89 3.4 3 597 Complement C1q tumor necrosis factor-related protein 1d Q9BXJ1 2.33 17.8 3 498 Complement C2 P06681 2.39 7.5 3 699 Complement C3 P01024 113.44 49.3 126 3, 4, 5, 6100 Complement C4-A P0C0L4 2 54.8 92 4101 Complement C4-B P0C0L5 130.07 54.8 146 3, 4, 5, 6102 Complement C5 P01031 1.51 2.1 1 4103 Complement component C9 P02748 6.73 13.8 5 4104 Complement factor B P00751 25.91 36.1 26 4, 6105 Corticosteroid-binding globulin P08185 2.91 9.6 2 4106 Cyclic AMP-dependent transcription factor ATF-6 betad Q99941 1.51 1.7 1 6107 Cystatin-Bd P04080 2 33.7 1 4108 Cystatin-C P01034 4.75 40.4 3 4, 5, 6109 Cysteine desulfurase, mitochondriald Q9Y697 1.62 2.8 2 5110 Cysteine-rich motor neuron 1 proteind Q9NZV1 4 6.4 2 3111 Cytoplasmic aconitate hydratased P21399 2.09 16.7 3 4112 Cytoplasmic dynein 1 light intermediate chain 2d O43237 2 6.1 1 6113 Cytosol aminopeptidased P28838 4.59 11 3 4114 Cytosolic nonspecific dipeptidased Q96KP4 16.12 44.6 20 3, 4, 6115 D-dopachrome decarboxylased P30046 2 21.2 2 4116 Destrind P60981 3.01 27.9 3 3, 4117 Dihydropyrimidinase-related protein 2d Q16555 4.18 20.6 7 3, 4, 6118 Dihydropyrimidinase-related protein 3d Q14195 11.99 36.8 13 3, 4, 6119 Dipeptidyl peptidase 1 P53634 4.56 11.7 4 3, 4120 DNA-binding protein Ad P16989 8.15 30.4 8 4, 6121 Double stranded RNA-binding protein Staufen homologue 1d O95793 2 1.7 1 6122 Dyslexia-associated protein KIAA0319-like proteind Q8IZA0 1.52 4.2 2 4, 6



Table 1. continued


123 Dystroglycan Q14118 1.34 5.8 2 4124 EGF-containing fibulin-like extracellular matrix protein 1 Q12805 6.28 19.9 10 4, 6125 Elongation factor 1-betad P24534 2 16.4 2 4126 Elongation factor 1-gammad P26641 1.64 7.3 2 6127 Elongation factor 2 P13639 18.49 21.7 18 3, 4, 6128 Enoyl-CoA hydratase domain-containing protein 1d Q9NTX5 2 3.6 1 6129 Eosinophil cationic proteind P12724 2 7.5 1 6130 Epithelial discoidin domain-containing receptor 1 Q08345 3.74 11.2 5 4, 6131 Eukaryotic peptide chain release factor subunit 1d P62495 2 7.6 2 6132 Eukaryotic translation initiation factor 5A-1d P63241 2.03 25.3 2 6133 Ezrin P15311 6.75 21.5 8 3, 5134 F-actin-capping protein subunit alpha-1d P52907 2 8 2 4135 Farnesyl pyrophosphate synthased P14324 4 14.6 4 4136 Fatty acid-binding protein P05413 12.62 53.4 9 5, 6137 Fatty acid synthase P49327 16.71 13.3 15 1, 3, 4, 6138 FERM domain-containing protein 4Bd Q9Y2L6 1.72 7 1 3139 Ferritin heavy chaind P02794 6.01 24 9 3, 4, 6140 Fibrinogen alpha chaind P02671 6.02 18.7 5 4, 6141 Fibrinogen beta chain P02675 11.25 30.6 14 4, 6142 Fibrinogen gamma chain P02679 4.95 23.4 5 4, 6143 Fibroblast growth factor-binding protein 1 Q14512 3.25 15 2 6144 Fibronectind P02751 48.84 31.5 55 3, 4, 6145 Fibulin-1d P23142 3.51 5.4 3 4146 Ficolin-2d Q15485 2 8.9 2 6147 Filamin-Bd O75369 2.62 4.7 5 4, 6148 Flavin reductased P30043 6.44 36.4 5 4, 6149 Follistatin-related protein 1 Q12841 5.72 11.4 3 4, 6150 Fructose-1,6-bisphosphatase 1d P09467 4.32 13.3 4 4, 6151 Fructose-bisphosphate aldolase A P04075 32.69 62.1 21 3, 4, 6152 Fructose-bisphosphate aldolase Cd P09972 2.02 25.3 6 4153 G-protein coupled receptor family C group 5 member B Q9NZH0 2 3 2 5154 Galactose-1-phosphate uridylyltransferased P07902 1.68 6.9 2 6155 Galectin-3-binding protein Q08380 32.14 36.9 46 1, 3, 4, 5, 6156 Gamma-glutamyltranspeptidase 1 P19440 6.09 14.4 3 4, 6157 Gamma-glutamyltranspeptidase 2 P36268 1.73 13.5 7 5158 Ganglioside GM2 activatord P17900 1.47 16.1 2 4159 Gelsolin P06396 6.35 20.5 11 3, 4, 6160 Glucose-6-phosphate 1-dehydrogenased P11413 4.29 16.8 2 6161 Glutathione peroxidase 3d P22352 5.2 28.3 6 3, 4, 6162 Glutathione S-transferase omega-1d P78417 2 10 2 6163 Glyceraldehyde-3-phosphate dehydrogenase P04406 17.65 59.4 15 3, 4, 6164 Glycerol-3-phosphate dehydrogenase [NAD+], cytoplasmicd P21695 2.02 9.2 2 6165 Glyoxalase domain-containing protein 4d Q9HC38 1.6 7.3 2 4166 Golgi-associated plant pathogenesis-related protein 1 Q9H4G4 2 16.9 2 4, 6167 Granulinsd P28799 15.9 31.9 19 4, 6168 Gremlin-2d Q9H772 2 10.7 2 4, 6169 Group 3 secretory phospholipase A2d Q9NZ20 4 8.1 2 6170 Haptoglobin P00738 34.87 55.9 30 4, 5, 6171 Heat shock 70 kDa protein 4d P34932 2.02 6.4 2 6172 Heat shock 70 kDa protein 6d P17066 6 12 8 6173 Heat shock 70 kDa protein 13d P48723 2 5.5 1 4174 Heat shock 70 kDa protein 1A/1Bd P08107 12.58 27.3 13 4175 Heat shock cognate 71 kDa protein P11142 21.49 35.1 19 4, 6176 Heat shock protein beta-1d P04792 4.02 36.6 2 3, 4177 Heat shock protein HSP 90-alphad P07900 4.07 14.9 5 4, 6178 Heat shock protein HSP 90-betad P08238 2 20.2 6 3, 4, 6179 Heat shock-related 70 kDa protein 2d P54652 6.14 23.6 4 3180 Heme-binding protein 1 Q9NRV9 5.29 28 6 4, 6181 Hemoglobin subunit betad P68871 2.83 19.6 2 6182 Hemopexin P02790 2 4.1 2 5183 Heterogeneous nuclear ribonucleoprotein Qd O60506 2 6.3 2 4, 6



Table 1. continued


184 Histamine N-methyltransferased P50135 1.77 8.2 1 4185 Histone H12d P16403 6 13.6 3 6186 Histone H2A type 1-Jd Q99878 2.65 27.3 2 4187 Histone H2B type 1-Ad Q96A08 2.02 15.8 2 6188 HLA class I histocompatibility antigen, A-69 alpha chaind P10316 3.03 30.1 3 4, 6189 HLA class II histocompatibility antigen, DR alpha chaind P01903 2.2 13.8 3 4190 Hornerind Q86YZ3 1.38 4.1 2 4191 Hsp90 cochaperone Cdc37d Q16543 1.66 5.6 2 6192 Hyaluronan and proteoglycan link protein 3 Q96S86 21.07 49.4 14 3, 4, 6193 Hypoxia up-regulated protein 1d Q9Y4L1 15.42 23.9 15 3, 4, 6194 Ig alpha-1 chain C region P01876 33.39 58.6 42 1, 2, 3, 4, 5, 6195 Ig alpha-2 chain C region P01877 6.03 58.2 28 2, 3, 4, 5, 6196 Ig delta chain C regiond P01880 4 19.8 4 4197 Ig gamma-1 chain C region P01857 3.1 21.5 3 5198 Ig gamma-2 chain C regiond P01859 2.05 16.9 2 4, 5199 Ig heavy chain V−I region HG3 P01743 2.35 21.4 3 5200 Ig heavy chain V−I region V35d P23083 3.14 20.5 2 6201 Ig heavy chain V−III region BRO P01766 3.96 30.8 3 5, 6202 Ig heavy chain V−III region TEId P01777 2 16 2 3, 4203 Ig heavy chain V−III region TILd P01765 2.83 26.1 3 5204 Ig heavy chain V−III region TURd P01779 2 30.2 2 6205 Ig kappa chain C region P01834 11.82 67 24 1, 2, 3, 4, 5, 6206 Ig kappa chain V−I region BANd P04430 2.03 26.9 3 6207 Ig kappa chain V−I region EUd P01598 2.5 38.9 2 6208 Ig kappa chain V−I region Haud P01600 2 26.9 3 6209 Ig kappa chain V−I region Mevd P01612 2 16.5 2 6210 Ig kappa chain V−I region WEAd P01610 2 30.6 1 4211 Ig kappa chain V−I region Wesd P01611 2 16.7 1 4212 Ig kappa chain V−II region GM607 (Fragment)d P06309 4 37.6 3 4, 6213 Ig kappa chain V−II region RPMI 6410d P06310 1.77 9.8 2 5214 Ig kappa chain V−III region CLLd P04207 2.63 22.5 2 5, 6215 Ig kappa chain V−III region HICd P18136 3.09 50.4 4 2, 4, 5, 6216 Ig kappa chain V−III region VG P04433 4 33 3 5, 6217 Ig kappa chain V−IV region B17d P06314 2.01 23.9 2 6218 Ig lambda chain V−I region HA P01700 2.04 16.1 2 5, 6219 Ig lambda chain V−I region NEWMd P01703 2 16.5 1 3220 Ig lambda chain V−I region WAHd P04208 2 11.9 1 4221 Ig lambda chain V−III region LOI P80748 4 41.4 3 4, 5, 6222 Ig lambda chain V−III region SH P01714 2 25 2 4, 6223 Ig lambda chain V−IV region Hil P01717 2.07 39.3 3 4, 5, 6224 Ig lambda chain C regions P01842 8 60 4 3225 Ig lambda-1 chain C regions P0CG04 2 46.2 9 4226 Ig lambda-2 chain C regions P0CG05 8.79 67 12 4227 Ig lambda-3 chain C regionsd P0CG06 8.13 67.9 11 5, 6228 Ig mu chain C region P01871 25.61 35.4 21 4, 5, 6229 Immunity-related GTPase family Q proteind Q8WZA9 3.07 12.8 2 6230 Immunoglobulin J chain P01591 10.14 56 12 2, 3, 4, 5, 6231 Immunoglobulin lambda-like polypeptide 5d B9A064 4.07 43.5 12 6232 Insulin-like growth factor-binding protein 2 P18065 6.83 23.4 7 4, 5, 6233 Interalpha-trypsin inhibitor heavy chain H2d P19823 2.29 6.3 2 4, 6234 Interalpha-trypsin inhibitor heavy chain H4d Q14624 9.17 15.5 12 4, 6235 Isocitrate dehydrogenase [NADP] cytoplasmicd O75874 6.39 23 4 3, 6236 Isopentenyl-diphosphate Delta-isomerase 1d Q13907 3.85 13.2 2 4237 Kallistatind P29622 3.6 11.7 2 6238 Kappa-casein P07498 66.42 57.7 135 1, 2, 3, 4, 5, 6239 Keratin, type I cytoskeletal 10d P13645 18.69 23.6 12 3, 4, 6240 Keratin, type I cytoskeletal 14d P02533 2.23 13.8 3 6241 Keratin, type I cytoskeletal 20d P35900 1.8 6.4 1 4242 Keratin, type I cytoskeletal 9d P35527 4.91 21.2 10 3, 4, 6243 Keratin, type II cytoskeletal 1d P04264 27.12 48.9 21 3, 4, 6244 Keratin, type II cytoskeletal 2 epidermald P35908 6.57 25.2 8 3, 4, 6



Table 1. continued


245 Keratin, type II cytoskeletal 5 P13647 2.61 15.3 7 4, 6246 Kininogen-1d P01042 16.2 17.6 12 4, 6247 L-lactate dehydrogenase A chaind P00338 2.33 10.2 2 6248 L-lactate dehydrogenase B chain P07195 4.76 26.1 5 3, 4, 6249 L-xylulose reductased Q7Z4W1 2.75 23 4 3, 4, 6250 Lactadherin Q08431 33.43 63.6 42 1, 3, 4, 5, 6251 Lactotransferrin P02788 162.54 89.2 325 1, 2, 3, 4, 5, 6252 Left-right determination factor 2d O00292 5.88 23.2 3 6253 Legumaind Q99538 3.05 20.3 2 3254 Leucine-rich alpha-2-glycoprotein P02750 9.82 30.6 13 4, 5, 6255 Lipolysis-stimulated lipoprotein receptord Q86 × 29 2 6.6 2 6256 Lipoprotein lipase P06858 12.74 28.2 16 3, 4, 5, 6257 Lysozyme C P61626 19.74 80.4 50 2, 3, 4, 5, 6258 Lysyl oxidase homologue 4d Q96JB6 2.6 6.2 2 6259 Macrophage mannose receptor 1 P22897 2 4.4 2 6260 Macrophage mannose receptor 1-like protein 1 Q5VSK2 26.97 14.9 22 3, 4, 5261 Macrophage migration inhibitory factor P14174 4.02 17.4 4 4, 6262 Malate dehydrogenase, cytoplasmicd P40925 10.48 21 6 3, 4, 6263 Mannose-6-phosphate receptor-binding protein 1 O60664 10.01 27.9 8 3264 Mannosyl-oligosaccharide 1,2-alpha-mannosidase 1Ad P33908 2.03 11.8 4 6265 Matrilin-3d O15232 3.56 19.8 7 4266 Matrix-GIa proteind P08493 2 15.5 2 6267 Midkined P21741 3.05 27.3 3 4, 6268 Moesin P26038 1.92 9.9 3 6269 Monocyte differentiation antigen CD14 P08571 32.65 60.8 41 2, 3, 4, 5, 6270 Mucin-1 P15941 9.28 6.1 9 4, 5, 6271 Mucin-4 Q99102 7.33 7.9 9 4, 5, 6272 Multiple inositol polyphosphate phosphatase 1d Q9UNW1 2 8.4 2 4273 Myosin-IXad B2RTY4 2.28 7.8 3 3274 N-acetyl-D-glucosamine kinased Q9UJ70 2 6.7 2 6275 N-acetylglucosamine-1-phosphotransferase subunit gammad Q9UJJ9 3.6 11.2 2 4, 6276 N-acetylmuramoyl-L-alanine amidased Q96PD5 4.02 14.1 4 4277 N(G),N(G)-dimethylarginine dimethylaminohydrolase 1d O94760 11.43 26 8 3, 4, 6278 Na(+)/H(+) exchange regulatory cofactor NHE-RF1d O14745 2.12 14.8 2 4, 6279 Nephronectind Q6UXI9 12.89 22.5 8 4, 6280 Nicotinamide phosphoribosyltransferased P43490 2.28 5.7 2 4, 6281 Nidogen-1d P14543 4.36 6.6 3 6282 Nuclease-sensitive element-binding protein 1d P67809 4 31.8 6 4283 Nucleobindin-1 Q02818 34.55 58.8 34 2, 3, 4, 6284 Nucleobindin-2 P80303 38.52 62.6 49 1, 2, 3, 4, 6285 Nucleolind P19338 2.02 3.1 4 6286 Nucleoside diphosphate kinase Bd P22392 2.53 25 4 4, 6287 Nucleotide exchange factor SIL1 Q9H173 4.85 20.2 8 4, 6288 Obg-like ATPase 1d Q9NTK5 2.01 7.6 2 4289 Osteopontin P10451 14.15 36.9 32 3, 4, 5, 6290 Parathyroid hormone-related protein P12272 2 17.5 4 4, 6291 Peptidyl-prolyl cis−trans isomerase A P62937 16.36 63 11 3, 4, 6292 Peptidyl-prolyl cis−trans isomerase B P23284 9 38.4 10 4, 5, 6293 Peptidyl-prolyl cis−trans isomerase Cd P45877 3.92 42.9 3 4294 Peptidyl-prolyl cis−trans isomerase FKBP1Ad P62942 3.8 25 3 4, 6295 Perilipin-2 Q99541 10 18.5 5 6296 Perilipin-3 O60664 6.63 22.6 8 4, 6297 Peroxiredoxin-1d Q06830 3.34 9.5 3 6298 Peroxiredoxin-2d P32119 5.49 18.7 3 4299 Peroxiredoxin-6d P30041 6.76 25.9 5 4, 6300 Phosphatidylethanolamine-binding protein 1 P30086 4.38 27.8 5 3, 4, 6301 Phosphoglucomutase-1d P36871 15.09 32.2 11 3, 4, 6302 Phosphoglycerate kinase 1d P00558 15.64 34.1 10 3, 4, 6303 Phosphoglycolate phosphatased A6NDG6 2 15.3 2 6304 Phospholipid transfer protein P55058 6.69 19.3 4 3, 4305 Pigment epithelium-derived factord P36955 4.22 16.8 2 6



Table 1. continued


306 Plasma protease C1 inhibitor P05155 13.26 18.2 8 4, 5, 6307 Plasminogen P00747 9.29 19.6 6 4, 6308 Plastin-2 P13796 2.23 7.3 2 4309 Platelet-derived growth factor Cd Q9NRA1 2 3.5 1 6310 Platelet glycoprotein 4 P16671 3.11 19.1 9 4, 5311 Poly(rC)-binding protein 1d Q15365 8 23 4 6312 Polyadenylate-binding protein 1d P11940 5.3 13.8 4 6313 Polyadenylate-binding protein 3d Q9H361 1.57 12.8 4 4314 Polymeric immunoglobulin receptor P01833 54.01 48 68 2, 3, 4, 5, 6315 Polypeptide N-acetylgalactosaminyltransferase 2d Q10471 5.4 12.3 3 6316 Polyubiquitind P0CG48 5.5 74 6 5317 Prefoldin subunit 4d Q9NQP4 2 11.2 1 4318 Pro-epidermal growth factor P01133 22.61 21.6 20 3, 4, 6319 Proactivator polypeptide P07602 44.5 58.6 62 3, 4, 6320 Procollagen-lysine,2-oxoglutarate 5-dioxygenase 1d Q02809 3.51 9.5 5 4, 6321 Programmed cell death 6-interacting proteind Q8WUM4 2.72 6.5 5 4, 6322 Prolactin-inducible protein P12273 3.4 33.6 4 4, 5, 6323 Proline synthase cotranscribed bacterial homologue proteind O94903 2 2.9 1 6324 Proliferation-associated protein 2G4d Q9UQ80 2 9.9 1 4325 Prostaglandin reductase 1d Q14914 3.6 10 2 4326 Proteasome activator complex subunit 1d Q06323 4.86 17.3 4 3, 4327 Proteasome subunit alpha type-7-liked Q8TAA3 2.65 17.6 2 4328 Proteasome subunit alpha type-5d P28066 1.72 6.2 2 6329 Proteasome subunit beta type-1d P20618 8.88 44 9 4, 6330 Proteasome subunit beta type-2d P49721 2 7.5 1 4331 Protein CutAd O60888 2 13.4 2 6332 Protein disulfide-isomerase A3 P30101 3.17 15.3 5 4, 6333 Protein disulfide-isomerase A6 Q15084 4.01 11.8 3 4334 Protein disulfide-isomerase P07237 33.62 56.5 27 4, 6335 Protein DJ-1d Q99497 2 21.2 2 4336 Protein ERGIC-53d P49257 1.57 8.4 2 4337 Protein FAM150Ad Q6UXT8 2.7 31.8 2 6338 Protein NDRG2d Q9UN36 2.51 15.6 2 4339 Protein OS-9d Q13438 4.35 5.7 4 4340 Protein S100-A1 P23297 2 38.3 2 3, 4341 Protein S100-A9 P06702 2 18.2 1 3342 Protein TFGd Q92734 2 6.5 1 4343 Prothrombin P00734 4.2 7.6 3 4, 6344 Purine nucleoside phosphorylased P00491 3.85 20.8 3 4345 Putative 40S ribosomal protein S26-like 1d Q5JNZ5 2 7.8 1 6346 Putative elongation factor 1-alpha-like 3d Q5VTE0 10.38 22.7 11 3, 4, 6347 Putative tropomyosin alpha-3 chain-like proteind A6NL28 3.56 17 2 6348 Putative trypsin-6d Q8NHM4 2 4 1 4349 Pyruvate kinase isozymes M1/M2d P14618 4.7 15.8 4 3, 4, 6350 Quinone oxidoreductased Q08257 2 12.2 2 4351 Rab GDP dissociation inhibitor beta P50395 10.81 39.1 13 4, 6352 Radixind P35241 6.73 16 7 4353 Ras-related C3 botulinum toxin substrate 1d P63000 4.23 29.2 3 4354 Ras-related protein Rap-1Ad P62834 2 6.5 1 3355 Ras-related protein Rab-11Bd Q15907 1.56 8.3 2 6356 Ras-related protein Rab-1Ad P62820 7.25 27.3 5 6357 Ras-related protein Rab-1Bd Q9H0U4 2.5 18.9 3 4358 Ras-related protein Rab-21d Q9UL25 4 23.1 2 4359 Ras-related protein Rab-5Cd P51148 3.68 17.6 3 4360 Ras-related protein Rab-7ad P51149 8 29.5 4 4, 6361 Ras-related protein Rab-8Ad P61006 1.83 31.4 3 6362 Receptor-type tyrosine-protein phosphatase Fd P10586 4.43 5.6 2 6363 Retinoid-inducible serine carboxypeptidased Q9HB40 4.01 7.1 2 4364 Rho-related GTP-binding protein RhoBd P62745 2 30.1 2 3365 Ribonuclease 4d P34096 2.13 23.1 3 4366 Ribonuclease inhibitord P13489 1.77 3.3 1 4



Compatibility of ProteoMiner Treatment with DownstreamiTRAQ Quantitation

Low abundance proteins retain their relative abundance afterProteoMiner treatment, whereas high abundance proteins donot.20,21,29 As described in previous studies,20,30 we conductedtwo parallel iTRAQ experiments in order to distinguish

between proteins of high and low abundance: one in whichcasein-depleted skim milk samples were subjected to Proteo-Miner treatment prior to iTRAQ analysis and one in whichcasein-depleted skim milk samples were left untreated (Figure 1).The 80 proteins identified in the untreated milk samples wereclassified as proteins of high-abundance. The iTRAQ analysis of

Table 1. continued


367 Ribose-phosphate pyrophosphokinase 2d P11908 3.72 9.7 2 3, 4, 6368 S-acyl fatty acid synthase thioesterase, medium chaind Q9NV23 4.75 24.2 3 4, 6369 Sclerostin domain-containing protein 1 Q6 × 4U4 12.78 39.8 15 3, 4, 6370 Secreted frizzled-related protein 1d Q8N474 6.14 18.8 4 4, 6371 Selenium-binding protein 1 Q13228 27.49 51.3 25 3, 4, 5, 6372 Semaphorin-4Bd Q9NPR2 2.21 7.6 3 4373 Serum albumin P02768 41.02 44 51 1, 2, 3, 4, 5, 6374 Serum amyloid A-4 proteind P35542 3.5 38.5 2 6375 Sialic acid synthased Q9NR45 2 5 1 4376 Sortilin Q99523 2.74 3.6 3 4377 SPARC-like protein 1 Q14515 1.77 6.9 2 5378 StAR-related lipid transfer proteind Q9P2P6 2.01 2.5 8 5379 Sulfhydryl oxidase 1 O00391 43.94 51.3 44 3, 4, 5, 6380 Synaptic vesicle membrane protein VAT-1 homologued Q99536 11.88 35.9 11 4, 6381 Syntaxin-binding protein 2d Q15833 2.16 11.5 2 6382 Syntenin-1 O00560 6.91 26.2 5 4383 T-cell immunomodulatory proteind Q8TB96 2.47 5.4 2 4384 T-complex protein 1 subunit alphad P17987 3.65 6.6 3 4, 6385 Tenascin P24821 49.29 27.7 48 4, 6386 Thrombospondin-1 P07996 19.15 13.6 11 4, 6387 Thyroxine-binding globulind P05543 2 2.9 2 6388 Tissue alpha-L-fucosidased P04066 4.26 14.8 6 4389 Transforming protein RhoAd P61586 6.07 42.5 5 4, 6390 Transketolased P29401 15.81 27.3 16 3, 4, 6391 Transmembrane protease serine 13 Q9BYE2 2 18.6 4 5392 Transthyretin P02766 6.01 41.5 6 2, 3, 4, 5, 6393 Triosephosphate isomerase P60174 9.46 56.2 12 3, 4, 6394 Tropomyosin beta chaind P07951 1.7 24.7 4 6395 Trypsin-1d P07477 4.78 19.4 7 6396 Tryptophanyl-tRNA synthetase, cytoplasmicd P23381 8 31.2 9 4397 Tubulointerstitial nephritis antigen-liked Q9GZM7 1.47 2.8 1 6398 Tumor necrosis factor ligand superfamily member 13d O75888 4.01 16.8 4 4, 6399 Tumor necrosis factor receptor superfamily member 11B O00300 2 9.5 1 4, 6400 Ubiquitin carboxyl-terminal hydrolase isozyme L3d P15374 2 14.4 2 4, 6401 Ubiquitin-60S ribosomal protein L40d P62987 2 18.8 2 4402 UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 3d Q9Y2A9 2.17 11 2 6403 UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 7d Q8NFL0 5.6 16.7 4 4, 6404 UPF0764 protein C16orf89d Q6UX73 1.52 20.4 5 4405 UMP-CMP kinased P30085 2.15 15.8 2 6406 UTP-glucose-1-phosphate uridylyltransferase Q16851 13.22 32.7 8 3, 4, 6407 UV excision repair protein RAD23d P54727 1.62 3.2 2 6408 V-type proton ATPase subunit S1d Q15904 2 6.2 1 4409 Vinculind P18206 1.37 6.7 2 6410 Vitamin D-binding protein P02774 17.3 40.1 12 3, 4, 6411 Vitronectin P04004 24.59 46.7 28 1, 3, 4, 5, 6412 Von Willebrand factor A domain containing protein 1 Q6PCB0 1.33 9.2 2 5, 6413 Xanthine dehydrogenase/oxidase P47989 110.65 59.4 154 1, 3, 4, 5, 6414 Zinc-alpha-2-glycoprotein P25311 23.66 50.7 16 5415 Zymogen granule protein 16 homologue B Q96DA0 4 25.5 3 3, 4, 6

aUnused ProtScore is a measure of the protein confidence for a detected protein. An Unused ProtScore of 1.3 corresponds to 95% confidence, witha higher score representing a higher level of confidence. bThe percentage of the total protein sequence covered by the identified peptides. cTheexperiment in which the protein was identified. (1) SDS-PAGE, (2) 2D DIGE, (3) 1D LC−MALDI, (4) 2D LC−MALDI, (5) iTRAQ withoutProteoMiner treatment, (6) iTRAQ after ProteoMiner treatment. When the protein was identified using more than one experiment, the reportedresults are from the experiment that gave the highest Unused ProtScore. dProteins that have not previously been identified in human skim milk.



the untreated samples enabled accurate quantitation of thesehigh abundance proteins, whereas the iTRAQ analysis of theProteoMiner-treated samples enabled quantitation of the lowabundance proteins.To assess whether ProteoMiner treatment does in fact affect

the relative abundance ratios of the high abundance proteins,

the quantitative information from the 51 proteins identified inboth iTRAQ experiments (Figure 4) was compared. Of the 11proteins found to be differentially expressed in the iTRAQexperiment without ProteoMiner treatment, only four were alsodifferentially expressed after ProteoMiner treatment. Of the 40proteins found not to be differentially expressed in the iTRAQexperiment without ProteoMiner treatment, 14 were found tobe differentially expressed after ProteoMiner treatment. Of thetotal 51 proteins, only 28 displayed homodirectional changes inthe two iTRAQ experiments. These results indicate that theProteoMiner treatment does introduce significant error into therelative abundance ratios of these higher abundance proteins.Therefore, in accordance with previous studies,20 only therelative abundance ratios of the low abundance proteins (thosenot also identified in the iTRAQ analysis of untreated samples)were considered to be accurate in the iTRAQ analysis ofProteoMiner-treated samples.

Quantitative Comparison of Term and Preterm Milk

The protein concentration of the pooled term and preterm milksamples were similar both before casein depletion (15.9 mg/mLand 16.0 mg/mL, respectively) and afterward (7.1 mg/mL and7.0 mg/mL, respectively). There were 80 abundant proteinsidentified in the iTRAQ experiment of the non-ProteoMiner-treated samples (Table 1). All of the 80 proteins were found inboth the term and preterm milk samples and in each duplicate.Five proteins constituted a significantly greater proportion of theprotein content in the pooled preterm milk, and 10 proteins

Figure 4. Protein identifications in human term milk using differentanalytical techniques. (A) Human term milk (38−41 weeks gestation)was collected from 8 mothers between 15 and 28 days postpartum,pooled together, treated with ProteoMiner beads to deplete the mostabundant proteins, and then analyzed using different techniques. 274proteins were identified in total. (B) Sixteen term (38−41 weeksgestation) and 16 preterm (28−32 weeks gestation) mothers donatedmilk samples between 7 and 14 days postpartum. Samples in each groupwere pooled. Two iTRAQ experiments were conducted: one comparingthe protein expression in term and preterm milk without ProteoMinertreatment and one comparing the protein expression after ProteoMinertreatment. 315 proteins were identified in total in the iTRAQ experi-ments. (C) Combining the results from the experiments described in (A)and (B), 415 proteins were identified in total from human term milk(38−41 weeks gestation), expressed between 7 and 28 days postpartum.

Figure 5. Protein classifications. (A) Subcellular location of all 415proteins identified in the 1D LC−MALDI and 2D LC−MALDIanalyses. (B) Functional categorization of 415 proteins identified inthe 1D LC−MALDI and 2D LC−MALDI analyses. A number ofproteins are included in multiple categories. The number of proteins ineach category is indicated in parentheses.



were present at a significantly higher level in the pooled termmilk (Table 2). The remaining 65 proteins did not differ inabundance between the two samples.In the iTRAQ analysis of the ProteoMiner-treated pooled

term and preterm milk samples, 286 proteins were identified.Fifty-one of these proteins were also identified in the iTRAQexperiment without ProteoMiner treatment and were thusclassed as high abundance proteins. Of the 235 low abundanceproteins identified, 40 differed in abundance between the pooledterm and preterm milk samples (Table 2). Twenty-three of theselow abundance proteins were present at higher levels in pretermmilk, and 17 were present at higher levels in term milk. The re-maining 195 low abundance proteins were present at similarlevels in both term and preterm milk.SDS-PAGE analysis of the pooled preterm and term samples

was conducted to confirm the iTRAQ results of the highlyabundant proteins (Figure 6). The identity of protein bandswas confirmed using MALDI-MS/MS in a previous study.23

Bile salt-stimulated lipase, lactoferrin, and serum albumin werepresent at significantly higher levels in preterm milk (p < 0.05).Levels of β-casein and α-lactalbumin were similar in the termand preterm milk.

■ DISCUSSIONIn the present study, 415 proteins were identified using anumber of separation and identification techniques, 261 ofwhich had not been previously found in human skim milk. Themajority of the identified proteins (371 of 415) were foundusing a combination of casein depletion, ProteoMiner treat-ment, and a 2D LC separation (Figure 4). The efficacy of theProteoMiner treatment was highlighted by the iTRAQ experi-ment, in that when using a 2D LC separation alone withoutprior ProteoMiner treatment, only 80 proteins were identifiedin human skim milk, much fewer than the 286 proteins identi-fied in the corresponding analysis after ProteoMiner treatment.Similarly, the 2D LC separation provided a far greater level ofproteome coverage compared to a 1D LC separation or gel-based approach (Figure 4A). Indeed, we identified over threetimes as many proteins as a recent study that coupled ProteoMinertreatment to a 1D LC separation.19

ProteoMiner treatment was found to be reproducible, with ahigh degree of consistency observed in both the SDS-PAGEand 2D-DIGE analysis of the samples treated in duplicate(Figures 2, 3). However, it was apparent in the SDS-PAGEanalysis that not all proteins of equivalent initial concentrationwere depleted or enriched to the same extent. For example,α-lactalbumin was nearly completely removed, whereas lacto-ferrin remained at a relatively high concentration afterProteoMiner treatment, despite both being present at similarlevels in milk samples (Figure 2). Furthermore, 29 of the 80proteins that were identified in the casein-depleted skim milkby 2D LC−MS/MS in the iTRAQ experiment were not foundin the corresponding ProteoMiner-treated sample (Figure 4C).This indicates that some proteins of medium abundance mayhave a very low binding affinity for the hexapeptide ligandlibrary and are not retained on the ProteoMiner beads. Indeed,the different binding affinities of proteins for the ProteoMinerpeptide library have been described as one of the major limita-tions of the technique, with an estimated 5−15% of the proteinsin any given mixture not binding at all.31 Therefore, while our re-sults represent a considerable expansion of the known proteomeof human milk, it is likely that many other milk proteins remainto be identified, with some proteins escaping capture by the

ProteoMiner beads, and others present at levels too low to bedetected.The protein identification study yielded no direct informa-

tion about the relative concentration of the proteins in humanmilk. However, comparing the results to those of previousstudies in which fewer proteins were identified suggests that thecytoplasmic proteins are among those of least abundance inhuman milk. Liao et al.19 found that 23.5% of the 115 proteinsthey identified were cytoplasmic. Similarly, of the 80 proteinsidentified in the iTRAQ experiment without ProteoMinertreatment in the present study, only 15% were cytoplasmic. Bycontrast, nearly half (46%) of the total 415 proteins we identi-fied were of cytoplasmic origin. As these additional cytoplasmicproteins were only identified after an extensive enrichment andseparation process, it is likely that they represent the proteins ofleast abundance in milk. This is consistent with the notion thatsome cytoplasmic proteins are present in human milk as anincidental consequence of the secretory process rather thanbeing under regulatory control and are thus found in onlytrace amounts. Indeed, Patton and Huston32 reported thepresence of cytoplasmic crescents within milk fat globules,having being entrained within the fat globule membraneduring the secretory process. Alternatively, the cytoplasmicproteins found in human milk may be derived from thebreakdown of its cellular content. Histological studies havefound significant levels of cellular debris in human milk,mostly derived from secretory epithelial cells.33 It is likely thatcytoplasmic proteins enter human milk through both pro-cesses, and thus it may be possible to view them as a reflectionof the protein content of the cytosol of mammary secretoryepithelial cells.One of the challenges associated with proteomic mapping

experiments is processing and applying the large amounts ofinformation generated within a particular biological context.One way in which the expanded proteome of human milkmay be useful is if the proteins identified reveal any infor-mation about either the metabolism or function of the mam-mary gland. Selected metabolites in human milk are currentlyused in this manner as markers of mammary gland develop-ment and health. For example, levels of milk citrate, sodium,and lactose are used to indicate successful secretory differentia-tion and activation of the mammary gland.34 Similarly, milklactose and glucose can be used as markers of mastitis35 andmetabolites in the lactose synthesis pathway used to identifydiabetic mothers.36 In the present study, 140 of the proteinsidentified (28% of total) are constitutively involved in cellularmetabolism and normal cellular function. This includes 44proteins of the glycolysis, pentose phosphate, citrate, and carbo-hydrate metabolism pathways, many of which such as pyruvatekinase (PKM2), transketolase (TKT), malate dehydrogenase(MDH1), and fructose-1,6-bisphosphatase (FBP) were identi-fied for the first time in human milk. While further researchis required to investigate the expression of these proteinsthroughout lactation, it is possible that they may prove usefulas markers of events that involve alterations to the meta-bolic activity of the mammary gland, such as lactogenesis andinvolution.Proteins in human milk may also be used as markers of in-

fection and inflammation of the mammary gland. Several studieshave reported changes to the levels of immunological andinflammatory proteins during mastitis. For example, lactoferrin,sIgA, secretory leukocyte protease inhibitor, and serum albuminhave all been found to be present at higher levels in human milk



Table 2

name peptidesa unusedb fold differencec

Up-regulated in preterm milkAbundant proteins Serum albumin 57 57.57 2.35

Bile-salt activated lipase 26 29.91 5.19Cysteine desulfurase 1 1.64 1.62Lactotransferrind 204 127.89 2.83Lysozyme-Ce 12 8.28 2.30

Low abundance proteins T-complex protein 1 subunit thetad 3 3.65 1.21Isocitrate dehydrogenase [NADP] cytoplasmic 4 6.39 2.1140S ribosomal protein S19 9 19.09 1.85Phosphoglycerate kinase 1 10 15.64 2.52Alanine aminotransferase 1 2 2.02 1.84Glyceraldehyde-3-phosphate dehydrogenase 9 10.31 2.1140S ribosomal protein S20 4 6.04 1.76Antithrombin-III 2 4 1.67UDP-GlcNAc:betaGalbeta-1,3-N-acetylglucosaminyltransferase 3 2 2.17 2.10Nucleolin 3 2.02 2.00Perilipin-3 7 11.4 2.48Putative elongation factor 1-alpha-like 3 5 11.9 4.91Apolipoprotein B-100 32 63.34 4.20Cytosolic nonspecific dipeptidase 16 27.42 5.27Apolipoprotein A-II 3 5.82 3.91S-acyl fatty acid synthase thioesterase, medium chain 3 4.74 3.45Ras-related protein Rab-1A 5 7.25 6.36Apolipoprotein E 13 23.25 12.01DNA-binding protein A 2 4 53.47Cytoplasmic dynein 1 light intermediate chain 2 2 2 58.65Syntaxin-binding protein 2 2 2.16 87.90Ras-related protein Rab-8A 3 1.83 87.90Elongation factor 1-gamma 2 1.64 56.77

Down-regulated in preterm milkAbundant proteins Polymeric immunoglobulin receptor 59 58.91 −6.67

Clusterin 12 21.97 −2.38Prolactin-inducible protein 2 3.38 −50.0Lactadherin 9 14.71 −1.50Alpha-lactalbumin 22 11.82 −33.3Mucin-4 3 4.31 −1.02Ig heavy chain V−I region HG3d 2 2.35 −6.25Biotinidase 1 1.82 −33.3Gamma-glutamyl transpeptidase 1 1.73 −1.50Complement C3e 12 21.1 −2.44

Low abundance proteins Glutathione S-transferase omega-1 2 2 −79.05C-type lectin domain family 11 member A 2 4 −74.35Tenascin 45 69.8 −26.18Lipoprotein lipase 10 13.6 −7.16Protein disulfide-isomerase 12 23.79 −2.37Proactivator polypeptide 18 22.11 −2.15Beta-1,4-galactosyltransferase 1 7 13.1 −2.93Ceruloplasmin 7 9.79 −2.43Cathepsin B 6 9.69 −2.16Vitronectin 16 25.3 −2.20Lysyl oxidase homologue 4 2 2.6 −1.76L-xylulose reductase 2 2.29 −1.04EGF-containing fibulin-like extracellular matrix protein 1 4 7.78 −1.19Gelsolin 4 2.16 −1.27Fibrinogen gamma chain 2 3.91 −1.33Triosephosphate isomerase 10 19.35 −1.226-phosphogluconate dehydrogenase, decarboxylating 4 2.97 −1.05

aThe number of peptides identified. bUnused ProtScore is a measure of the protein confidence for a detected protein. An Unused ProtScore of 1.3corresponds to 95% confidence, with a higher score representing a higher level of confidence. cThe ratio between the abundance of each protein inthe pooled term and preterm samples, expressed as an average of the duplicate experiments. The value is positive when the protein is more abundantin the preterm milk, and negative when the reverse is true. dProteins that were found to be differentially expressed in one replicate at a confidencelevel >95% and in the other replicate at a confidence level >90%. eProteins that were found to be differentially expressed in both replicates at aconfidence level >90%.



during episodes of mastitis.35,37 A recent study in bovine milkalso identified a number of immunological proteins as being up-regulated during Escherichia coli infection.38 Of the proteins weidentified, 120 (24% of total) were associated with immune andinflammatory pathways and may have the potential to act assensitive biomarkers of mammary gland inflammation. Promisingcandidates include eight heat shock proteins that we identified inhuman milk for the first time (Table 1), which are known torespond to cellular insults.39

In addition to yielding information regarding mammarygland function, the expansion of the human milk proteome inthe present study is also of potential interest from an infantcentered perspective, in that many of the proteins may beinvolved in providing nutritional, protective, and developmentaladvantages to breastfed infants. With respect to infant growthand development, we identified 33 proteins (7%) as being in-volved in tissue development, including a number of proteinsidentified for the first time, such as granulin (GRN), cysteine-rich motor neuron protein (CRIM1), gremlin-2 (GREM2),nephronectin (NPNT), and bone morphogenetic protein 1(BMP1). GRN is a growth factor with marked functionalsimilarities to the epidermal growth factor family and has beenshown to regulate cellular proliferation, particularly in thehematopoietic and reproductive systems.40 Similarly, GREM2also regulates cellular differentiation and is involved in the Wntsignaling pathways.41 NPNT, BMP1, and CRIM1 have beenshown to interact with several growth factors and to play im-portant roles in the differentiation of osteoclasts, chondrocytes,and motor neurons, respectively, although more general roles indevelopment have also been proposed.42−44 While furtherresearch is required to assess whether these proteins retain theiractivity upon digestion, it is known that human-milk-fed infantsexperience developmental advantages over formula-fed in-fants,45−47 and it is possible that proteins within this groupmay 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 protectionrather than the provision of nutrition that was the original func-tion of the mammary gland in premammals. Although there isan abundance of literature describing the protective advantagesconferred upon infants through breastfeeding, the mechanismsinvolved are far from delineated.49 Individual proteins havebeen shown to exert protective effects through multiple path-ways, even after proteolytic digestion.50,51 Furthermore, there is agreat deal of interaction between different proteins involved inthe immune response, as well as with the infant’s own defenses.49

The identification of additional potential immune proteins pre-sent in human milk (Table 1) may be of use in further elucidat-ing these complex protective mechanisms. Immune responseproteins that we identified in human milk for the first timeinclude C1q and tumor necrosis factor related protein-1(CTRP1), peroxiredoxins-1, -2, and -6 (PRDX1, PRDX2,PRDX6), glutathione peroxidase 3 (GPX3), and HLA class Iand II histocompatibility antigens (HLA-A, HLA-DRA). CTRP1is a cytokine that possesses both immunomodulatory and meta-bolic functionality, inhibiting common pro-inflammatory path-ways, as well as being involved in glucose and insulinregulation.52,53 PRDX1, PRDX2, PRDX6, and GPX3 are allinvolved in systems of oxidative stress regulation, protecting cells,enzymes, and other proteins from oxidative damage,54 whereasHLA-A and HLA-DRA are both membrane proteins, involved inpresenting foreign antigens to the immune system.55 While thepresence of these proteins in human milk does not indicate thatthey are functionally active in the infant gut, they nonethelessprovide useful targets for further investigation.To investigate whether the human milk proteome under-

goes detectable changes at different stages of mammary glanddevelopment, a pilot study involving iTRAQ analysis was usedto compare the protein composition of term and preterm milk.Although iTRAQ experiments are designed to detect differ-ences in the fractional composition of protein samples, in thepresent study the original protein concentration of each pooled

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



sample was equivalent. This means that the differences infractional composition also directly correspond to differences inthe concentration of individual proteins between term and pre-term milk.In order to detect differentially expressed proteins of both

high and low abundance in term and preterm milk we adopteda workflow including parallel iTRAQ experiments. In a similarmanner to a recent publication,20 iTRAQ comparisons of pool-ed term and preterm milk samples were conducted both withand without prior ProteoMiner treatment. This enabled us todistinguish between high abundance proteins, those identifiedwithout ProteoMiner treatment, and low abundance proteins,those identified only when ProteoMiner treatment was usedprior to iTRAQ analysis. We found that ProteoMiner treat-ment did significantly alter the relative quantitation of the highabundance proteins, and therefore in the iTRAQ analysis ofProteoMiner-treated samples, only the relative abundance ratiosof 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 directionof the differences in expression of five proteins, bile-salt-stimu-lated lipase, lactoferrin, β-casein, sIgA, and serum albumin, interm and preterm milk were consistent between the twoanalytical methods; however, the iTRAQ experiment over-estimated the magnitude of the difference. Alpha-lactalbuminwas found to be differentially expressed in the iTRAQ experi-ment but not by SDS-PAGE. It is likely that these differencesbetween the two methods are due to an overestimation of thelevel of background contamination of the iTRAQ spectra bythe analysis software.56 Although previous studies report thequantitative accuracy of iTRAQ analysis of low abundanceproteins after ProteoMiner-treated samples,20 these results werenot verified in the present study. Despite these limitations,these results provide support for the use of an iTRAQ approachto identify differentially expressed proteins in human milk andillustrate how ProteoMiner treatment can be incorporatedwithin a quantitative analysis.We found 28 proteins that had significantly higher expression

levels in preterm milk compared to term milk and 27 proteinsthat had significantly lower levels of expression (Table 2). Anumber of these differentially expressed proteins such as lacto-ferrin, lysozyme, polymeric immunoglobulin receptor, lactadher-in, prolactin inducible protein, Ig heavy chain, mucin-4, vitro-nectin, and complement C3 are associated with the immuneresponse, protecting infants against infection.57 A number oftargeted studies have found higher levels of specific immunologicfactors in preterm milk and have accounted for these differenceson a teleological basis, arguing that the composition of pretermmilk differs from term milk to render it more suited to theprotection of vulnerable preterm infants.10,58 Our data contra-dicts this assertion, in that we found no concerted difference inthe expression of immunological proteins between term and pre-term milk, with some present at higher levels in preterm milkand vice versa (Table 2). This suggests that differences in proteincomposition between term and preterm milk are due to phys-iological differences in the mammary gland affecting proteinsynthetic and transport pathways, rather than being the result ofdiffering infant requirements, and supports the idea that aspreterm infants have only been able to survive in recent years,there has not been any selection pressure to encourage an in-crease in protective proteins in preterm milk.4

Similarly, three digestive enzymes, biotinidase (BTD), lipo-protein lipase (LPL), and bile-salt-stimulated lipase (BSSL)

were differentially expressed in term and preterm milk, withBSSL present at a higher level in preterm milk and both LPLand BTD found at a higher level in term milk. The presence ofthese enzymes in human milk is thought to compensate for alack of endogenous enzymes in the immature pancreatic juiceof newborns, enabling the efficient digestion of triacylglycerolsand biotin, respectively.59−61 Given the importance of theabsorption of fat and biotin to an infant’s growth and develop-ment, it is likely that the delivery of these enzymes is ofparticular importance to preterm infants. Again, their pattern ofexpression defies teleological explanation, in that while thehigher levels of BSSL in preterm milk may promote additionalgrowth in preterm infants, the lower levels of BTD and LPLmay be of detriment.Identifying proteins that are differentially expressed in term

and preterm milk may also be useful diagnostically. There havebeen a number of studies investigating the differences in mam-mary gland physiology and metabolism after preterm birth;however, the link between these physiological differences andpreterm milk composition largely remain unknown.4,34,62 Thedifferential expression of proteins in preterm milk may highlightpotential regulatory and metabolic pathways that are disruptedafter preterm delivery. For example, the higher levels of serumalbumin that we found in preterm milk may be due to the per-sistence of an open paracellular pathway after delivery in pre-term mothers, allowing the flow of serum proteins into themammary alveoli. Indeed, levels of serum albumin in milk havebeen used previously as a marker of an open paracellularpathway.63,64 Similarly, the low level of prolactin-inducible pro-tein 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 likelyto have lower levels of serum prolactin, which may be re-sponsible in part for lower levels of milk production in pretermmothers.65 Tenascin is another protein that was found to bepresent at much lower levels in preterm milk compared to termmilk (Table 2). Having been previously implicated in mammarygland development and differentiation,66,67 its concentration inmilk may also potentially be useful as a marker of mammarygland development after preterm delivery.It is also of interest that distinct sets of proteins were identi-

fied in the iTRAQ analysis of pooled term milk collected 7−14days postpartum and in the 2D-LC analysis of pooled term milkcollected 15−28 days postpartum, with over 100 proteinsunique to each sample (Figure 4C). Although no quantitativecomparison was conducted comparing these samples, it is likelythat many of these proteins change in relative abundance interm milk over this time period and therefore may also re-present proteins of interest with regard to mammary glanddevelopment throughout lactation.In summary, the present study represents the most com-

prehensive study to date of the human milk proteome, iden-tifying 261 novel proteins, as well as documenting changes inthe relative abundance of proteins in term and preterm milk.Knowledge obtained from this characterization of the proteinsin human milk will provide insights into the regulatory mech-anisms involved in the synthesis and secretion of human milk,particularly after preterm delivery, as well as identifying poten-tial proteins in human milk responsible for the nutritional,immunological, and developmental advantages conferred ontothe breastfed infant.



■ CONCLUSIONS

We investigated the effectiveness of a number of different an-alytical techniques to analyze the human milk proteome. Dynamicrange compression of human skim milk by the depletion of caseinand ProteoMiner treatment followed by 2D LC−MS/MS was themost successful approach. In total, 415 proteins were identified,over half of which had not been found before in human milk. Inaddition, iTRAQ analysis was used to identify differentiallyexpressed proteins between term and preterm milk, providinginsights into metabolic differences in the mammary gland afterpreterm birth in comparison to term birth.

■ ASSOCIATED CONTENT

*S Supporting Information

Supporting Information Data File 1 contains individualannotated MS/MS spectra of each of the proteins for whichonly one peptide was identified. Supporting Information DataFile 2 displays the preparative 2DE gel of the skim milk aftercasein depletion and ProteoMiner treatment and each of thespots that was analyzed by MS. Supporting Information DataFile 3 contains tables displaying all of the peptides used foridentifying proteins in the 1D-LC, 2D-LC, iTRAQ, 1D SDS-PAGE, and 2DE experiments and their correspondingidentification strengths. Supporting Information File 4 is atable displaying the classification of the proteins according totheir function and location. This material is available free ofcharge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATION

Corresponding Author

*Tel.: +61 6488 4428. Fax: +61 8 6488 7086. E-mail: [email protected].

■ ACKNOWLEDGMENTS

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

■ ABBREVIATIONS

1D-LC, one-dimensional liquid chromatography; 2DE, two-dimensional gel electrophoresis; 2D-LC, two-dimensionalliquid chromatography; iTRAQ, isobaric tags for relative andabsolute quantitation; MALDI, matrix-assisted laser desorp-tion/ionization; MS, mass spectrometry; PAGE, polyacrylamidegel electrophoresis; sIgA, secretory IgA; SDS, sodium dodecylsulfate; TOF, time-of-flight

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proteome mapping of human skim milk proteins in term and preterm milk

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