ARTICLE IN PRESS

International Dairy Journal 18 (2008) 2346

www.elsevier.com/locate/idairyj

Fractionation of bovine whey proteins and characterisation by

proteomic techniques
Bertram Y. Fong, Carmen S. Norris, Kate P. Palmano
LactoPharma, Fonterra Research Centre, Dairy Farm Road, Private Bag 11029, Palmerston North, New Zealand
Received 30 January 2007; accepted 19 June 2007

Abstract

To further elucidate and understand bovine milk protein composition and its relation to bioactivity, we have investigated the bovine
whey proteome by gel-based proteomic methods, after rst fractionating whey from late-lactation milk into acidic, basic and non-bound
fractions by semi-coupled anion and cation exchange chromatography. Characteristic two-dimensional gel ngerprints were obtained for
each fraction, with protein isoform patterns clearly apparent. A large number of minor whey proteins were identied, several of which
have not been reported previously in bovine milk. Notably, a cluster of osteopontin peptides not priorly described in milk was
consistently observed in the acidic protein fraction, presupposing novel bioactivities.
r 2007 Elsevier Ltd. All rights reserved.

Keywords: Bovine milk proteins; Whey proteins; Whey fractionation; Proteome; Bioactivity

1. Introduction incidence of the numerous less abundant proteins and

Whey is now generally regarded as a functional food,
which has measurable effects on health outcomes, and the
bioactive properties of whey proteins and whey protein
fractions are becoming increasingly recognised (Kruger
et al., 2005; Marshall, 2004; Michaelidou & Steijns, 2006;
Toba, Takada, & Yamamura, 2000). Although the major
bovine whey proteins, b-lactoglobulin (b-Lg) and
a-lactalbumin (a-Lac), and several of the predominant minor
proteins, such as lactoferrin and lactoperoxidase, have been
extensively studied (Floris, Recio, Berkhout, & Visser,
2003; Fox, 2003; Severin & Wenshui, 2005) and several
approaches to proteomic analysis of milk or whey protein
have been reported (Galvani, Hamdan, & Righetti, 2001;
Goldfarb, Savadove, & Inman, 1989; Lindmark-Mansson,
Timgren, Aden, & Paulsson, 2005; Manso, Leonil, Jan, &
Gagnaire, 2005; Murakami, Lagarde, & Yuki, 1998;
Yamada, Murakami, Wallingford, & Yuki, 2002), the
bovine whey proteome has yet to be fully elucidated.
Further, relatively little is known about the expression or
Corresponding author. Tel.: +64 6 350 4600; fax: +64 6 350 4607.
E-mail address: kate.palmano@fonterra.com (K.P. Palmano).
peptides in whey, some of which are derived by proteolytic
cleavage from the major milk proteins, extrusion or
leakage from the plasma or mammary epithelia (Hogarth
et al., 2004; Larsen et al., 2006), or are products of the
somatic cells (leukocytes and mammary epithelial cells)
present in milk (Kelly & McSweeney, 2003; Paape,
Bannerman, Zhao, & Lee, 2003). Many of these proteins
and peptides may have interesting properties that might
contribute to the health benets of whey. Understanding
the regulation of biosynthesis and expression, origin
and incidence of milk proteins is essential to the under-
standing of milk composition and characteristics and how
environmental, nutritional, genotypical, seasonal and other
factors might impact upon these. In particular, increased
understanding of the whey proteome might have conse-
quence in terms of manipulating the bioactive properties
of milk.
Like many complex matrices, the whey protein fraction
is dominated by a small number of so-called major
proteins, which collectively constitute over 80% of the
whey protein (Tremblay, Laporte, Leonil, Dupont, &
Paquin, 2003). In particular, b-Lg alone comprises 50%
of the whey protein. This dominance poses problems with

0958-6946/$ - see front matter r 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.idairyj.2007.06.005
 ARTICLE IN PRESS
24 B.Y. Fong et al. / International Dairy Journal 18 (2008) 2346

respect to application of proteomic techniques and 2.3. Fractionation of whey protein

visualisation and identication of less abundant proteins.
Generally, some kind of fractionation or partitioning is
required to remove or deplete dominant proteins and
increase resolution of the minor proteins. Approaches such
as immunoabsorption (Murakami et al., 1998; Palmer
et al., 2006; Yamada et al., 2002), afnity tagging (Holland,
Deeth, & Alewood, 2006) and solution isoelectric focusing
(IEF) (Zuo & Speicher, 2002) have been utilised. In this
paper, we describe fractionation of bovine whey using
semi-coupled anion and cation exchange chromatography,
preparatory to gel-based proteomic analysis. The strategy
of fractionation was designed to optimise the removal of
the hugely dominant whey protein b-Lg and also a-Lac
into a single non-bound fraction, while partitioning the
remaining proteins into characteristic acidic and basic
fractions in a minimum number of steps. The advantage of
such strategy is that it can be applied easily to scale-up for
production of fractions in which bioactives of interest can
be amplied or enriched. Samples of late lactation milk
were used for initial study as the bioactive protein
lactoferrin is known to be upregulated in late lactation
(Turner, Thomson, & Auldist, 2007), raising the possibility
that other potentially bioactive components of low
abundance might also be amplied and therefore visible
by proteomics techniques.
2. Materials and methods
2.1. Materials
Complete mini ethylenediamine tetraacetic acid-free
protease inhibitor tablets were from Roche Diagnostics
(Mannheim, Germany). The dialysis membrane, 3500 Da
molecular mass cut-off, was from Spectrum Laboratories
Inc. (Rancho Dominguez, CA, USA). Mercaptoethanesul-
phonic acid (MES) was from Merck (Darmstadt, Germany).
Trypsin was sequence grade (Promega Corporation, Madison,
WI, USA). All other reagents were electrophoresis grade
and sourced from BioRad Laboratories (Hercules, CA,
USA).
To obtain sufcient sample for fractionation, whey from
six consecutive milkings was pooled for each cow prior to
fractionation, giving a representative whey sample for each
cow. The whey samples were fractionated using an
integrated fast protein liquid chromatography system
(Amersham Biosciences, Uppsala, Sweden) with UV
monitoring at 214 and 280 nm.
The mineral acid whey was diluted 1:1 with MilliQ
water, the pH was readjusted to 4.6 using hydrochloric
acid (0.1 M) and approximately 75 mL (conductivity
5.25.4 mS cm 1) was loaded at 2 mL min 1 onto an
anion HiTrapTM Q HP column (5 mL, Amersham Bios-
ciences), which had been pre-equilibrated with 40 mM
sodium chloride (NaCl), pH 4.6 at 2 mL min 1. The
column was sufciently washed with 40 mM NaCl, pH 4.6
to remove all anion breakthrough (non-bound) material.
The breakthrough and the wash were collected as a single
fraction (Fig. 1). Once the UV absorbance had returned to
baseline, the bound proteins (collectively termed the 1 M
acidic fraction) were eluted off the column using 1 M NaCl
at 2 mL/min. The anion breakthrough fraction was
adjusted to pH 6.5 using sodium hydroxide (0.1 M), claried
by centrifugation (15 000  g, 15 min, 10 1C) and then
passed at 2 mL min 1 through a cation exchange
HiTrapTM S HP column (5 mL, Amersham Biosciences),
equilibrated with 20 mM MES buffer pH 6.5. The column
was sufciently washed with 20 mM MES buffer pH 6.5 to
remove all unbound material and the breakthrough and the
wash were collected as one fraction (cation breakthrough
fraction CX-BT). Once the UV absorbance had returned
to baseline (Fig. 1), the bound proteins were eluted

2.2. Mineral acid whey preparation

Individual milk samples were collected from each of six

Friesian cows and from several milkings over a short
period in late lactation. Immediately after each milking, a
2530 mL sample of the collected milk was withdrawn into
a plastic container and protease inhibitor added. The milk
samples were maintained at 4 1C and processed within 48 h.
Whey was prepared from each sample as follows. The milk
was defatted by centrifugation (15 000  g, 15 min, 10 1C)
and the pH was then adjusted to 4.6 using hydrochloric
acid (1 M). The precipitated casein was removed by Fig. 1. Fractionation of bovine whey by (A) anion exchange chromato-
centrifugation (15 000  g, 15 min, 10 1C) and the resulting graphy followed by (B) cation exchange chromatography. Fractions were
mineral acid whey was stored at 80 1C. collected as indicated.
 ARTICLE IN PRESS
B.Y. Fong et al. / International Dairy Journal 18 (2008) 2346
consecutively with 0.4 M NaCl and 1.5 M NaCl to give the
0.4 M basic fraction and the 1.5 M basic fraction, respec-
tively. The completeness of elution at each salt step was
monitored by UV absorbance. The recovered fractions
were dialysed against MilliQ water and then freeze dried
and stored at 30 1C until analysed using proteomic
techniques.
2.4. Protein analysis
The major whey proteins and lactoferrin were analysed
in whey samples and fractions using ResourceTM reverse
phase (RP)-high-performance liquid chromatography
(HPLC) (Elgar et al., 2000; Palmano & Elgar, 2002). The
total protein in fractions was assayed using a micro-
Kjeldahl technique.
2.5. Two-dimensional gel electrophoresis (2-DE)
IEF was carried out using an IPGphor system (Amer-
sham Biosciences) according to the manufacturers instruc-
tions. Briey, the freeze-dried protein fractions (approxi-
mately 2 mg of protein) were dissolved in 1 mL of IEF
sample buffer (5 M urea, 2 M thiourea, 2% 3[(3-cholamido-
propyl)dimethylammonio]-propanesulphonic acid (CHAPS),
0.5% dithiothreitol (DTT) and 0.5% pH 310 Biolyte) and
incubated for 1 h with continuous sonication in a sonicator
bath (Astrason, Farmingdale, NY, USA). Samples
(350 mL) were loaded on to immobilised pH gradient
(IPG) strips (pH 310, 18 cm, Amersham Biosciences),
which were then actively rehydrated overnight (18 h, 50 V,
20 1C) in IPGphor ceramic cartridges. The IPG strips were
then focused at 20 1C using the following conditions:
gradient to 200 V (30 min); gradient to 500 V (30 min);
gradient to 1000 V (30 min); gradient to 3000 V (1 h);
gradient to 8000 V (1 h). Focusing was continued at 8000 V
for approximately 8 h with overnight focusing at
15003000 V to achieve a total of 100 000 Vh. After
focusing, the IPG strips were stored immediately at
80 1C until required for second dimension sodium
dodecyl sulphate polyacrylamide gel electrophoresis
(SDS-PAGE). SDS-PAGE was carried out using 816%
Tris-HCl Protean II Ready Precast gels (18 cm) and a
Protean II gel electrophoresis system (BioRad) with Tris
(25 mM)/glycine (192 mM)/SDS (0.01%), pH 8.3 as elec-
trode buffer. Prior to SDS-PAGE, the focused IPG strips
were equilibrated with 2% SDS/1% DTT/30% glycerol in
0.375 M Tris base for 15 min, followed by a further 15 min
in 2% SDS/2.5% iodoacetamide/30% glycerol in 0.375 M
Tris base. The equilibrated strips were then embedded on
the SDS gels in 0.5% w/v agarose in electrode buffer. The
gels were run at 16 mA/gel for the rst 40 min, and then at
24 mA/gel until the bromophenol blue dye was at the
bottom edge of the gel. The entire electrophoresis unit was
maintained at 12 1C using a circulating water bath. For
visualisation of the protein spots, the gels were stained with
25
Coomassie Brilliant Blue R-250 (CBB) and then over-
stained with silver (Rabilloud, 1999).
2.6. In-gel digestion
The protein spots were excised from the gels and
destained for silver using potassium ferricyanide/sodium
thiosulphate solution according to Gharahdaghi, Wein-
berg, Meagher, Imai, and Mische (1999), followed by CBB
destaining using 50% acetonitrile (ACN) in 0.2 M ammo-
nium hydrogen carbonate. The destained gel pieces were
dried at 40 1C under a stream of nitrogen.
In-gel protein digestion was carried out as described by
Hellman, Wernstedt, Gonez, and Heldin (1995) using
0.10.5 mg of trypsin. The peptides were extracted from
the gel pieces twice using 250 mL of 60% ACN in 0.1%
triuoroacetic acid. The extracted peptides were dried
under a stream of nitrogen at 40 1C. The dried peptide
extracts were rehydrated in 5% ACN/0.2% formic acid
(1020 mL) and centrifuged (10 000  g, 5 min, 20 1C) before
nano-RP-liquid chromatography (LC)-mass spectrometry
(MS/MS) analysis.
2.7. Nano-RP-LC-MS/MS
Nano-reversed phase-liquid chromatography mass spec-
trometry (RP-LC-MS/MS) was performed on an LC
Packings system (LC Packings, Amsterdam, The Nether-
lands). The samples were loaded on to the nano-column
(PepMap C18, 5, 75 mm id, 15 cm; LC Packings) using a
pre-concentration/desalting step on the nano-precolumn
cartridge (300 mm id, 1 mm; LC Packings). Five microlitres
of the sample was loaded on to the nano-precolumn at
20 mL min 1 (0.1% formic acid). After 5 min, the pre-
column was switched in-line with the nano-column. The
peptides were separated with an ACN gradient (4.5% for
7 min, 4.536% over 33 min, 3681% over 5 min) in 0.2%
formic acid at 300 nL min 1. The nano-RP-LC system was
coupled to a QSTAR XL mass spectrometer (PE SCIEX,
Foster City, CA, USA), using a nano-electrospray-ionisa-
tion interface. This consisted of a housing unit (Protana
Inc., Toronto, Canada) that was modied to hold a micro
ion-spray unit (PE SCIEX). The micro ion-spray tips
(uncoated silica tip, 150 mm od  20 mm id, 10 mm tip id)
were from New Objective Inc. (Woburn, MA, USA). Ions
were generated and focused using a positive ion-spray
voltage of 1900 V, a focusing potential of 295 V and a
declustering potential of 80 V. Information-dependent
acquisition experiments were performed according to the
following parameters: 1 s survey scan and 3 s MS/MS scan
on the most intense ion as determined from the survey
scan. Former target ions were excluded for 10 s.
2.8. Protein identification
Protein identication was carried out using the MS/MS
spectral data matching search engine Mascot (Version
 ARTICLE IN PRESS
26 B.Y. Fong et al. / International Dairy Journal 18 (2008) 2346
2.0.0.4, Matrix Science, London, UK) as described in
Perkins, Pappin, Creasy, and Cottrell (1999). The ltered
MS/MS data (spectra were rejected if less than 10 peaks)
were searched against the National Center for Biotechnol-
ogy Information (NCBI) Bos Taurus genomic database
(NCBI build 2.1, based on Btau_2.0, www.ncbi.nlm.nih.
gov), a reversed NCBI Bos Taurus genomic database
(where the amino acid sequence of each Bos Taurus entry
was reversed), the NCBI Bos Taurus database (extracted
from NCBI nr database) and the Swiss-Prot database
(other mammalia only, Version 25-07-06, www.expasy.org/
sprot).
The following search parameters were used: MS error
70.1 Da; MS/MS error 70.1 Da; xed modications:
carbamidomethyl cysteine; variable modications: phos-
phorylation of serine and threonine residues, oxidation of
methionine. A maximum of two missed tryptic cleavages
was allowed. Protein matches were considered to be valid if
there were at least two valid matched peptides with Mascot
scores summing to at least 60. Beyond this score, additional
unique peptides matching the protein were not necessarily
validated. Valid peptides were required to have all of the
major peaks in the spectra assigned.
Protein functions alluded to without reference in the text
were obtained from the Swiss-Prot database.
3. Results and discussion
Fractionation of bovine whey using semi-coupled anion
and cation exchange under selected conditions of pH and
ionic strength resulted in effective isolation of the major
whey proteins b-Lg and a-Lac into a single (non-bound)
breakthrough fraction (CX-BT), allowing proteomic ana-
lysis from 2-DE gels of sub-dominant and minor proteins,
which were largely partitioned into the acidic and basic
fractions. The elution of basic proteins from the cation
exchange resin was performed in two steps so as to isolate
lactoferrin, a predominant basic protein, away from other
basic proteins such as lactoperoxidase (Yoshida & Xiuyun,
1991). The 2-DE ngerprints obtained were characteristic
for each whey fraction and showed a high degree of
similarity between whey samples.
3.1. 1 M acidic fraction
A proteomic map of the 1 M acidic fraction from bovine
whey is shown in Fig. 2, with accompanying data on
protein spot identication given in Table 1. The CBB-
stained gel (Fig. 2A) gave a pattern of spots that was
characteristic for the acidic fraction, with clearly dominant
proteins/peptides in the lower mass (Mr), low isoelectric
point (pI) region of the gel. The b-casein N-terminal
phosphorylated fragment proteose peptone 5 (PP5)
(f 1105/107, spots 4446), occurring as several isoforms
due to variable cleavage and genetic variance (Swaisgood,
2003), contributed a large proportion of the protein in this
fraction. Assignment of this peptide was made on the basis
of observed Mr (1820 kDa (LeRoux, Girardet, Hum-
bert, Laurent, & Linden, 1995), previous characterisation
of isolated material by Edman sequencing and mass
analysis (Elgar et al., 2000; Reid, Cornish, Haggarty, &
Palmano, 2000) and compositional data (not shown). In
this study, MS sequence coverage was too limited to
indicate N-terminal bias and, in any case, small C-terminal
peptides were identied in spot 45, suggesting that some
non-PP5 b-casein proteolytic product was also present in
this spot. PP5 is universally present in bovine whey as a
major component of proteose peptone, the heat-stable
acid-soluble protein component of whey (Swaisgood,
2003), and occurs due to the activity of the indigenous
milk enzyme plasmin on b-casein. The proteose peptone
lactophorin, a phosphorylated glycoprotein also known as
glycosylation-dependent cell adhesion molecule 1, PP3 and
28 kDa milk protein, was also present as a series of spots
(17, 18, 2334, 37, 38) in the 2530 kDa region of the gel.
Lactophorin is highly and specically expressed in the
lactating mammary gland (Sorensen & Petersen, 1993a;
Groenen, Dijkhof, & der Poel, 1995) and appears as a
distinct series of isoforms on 2-D gels due to post-
translational heterogeneity (Kanno, 1989). The pattern of
isoforms observed in the 1 M acidic fraction was very
similar to that observed for lactophorin derived from milk
fat globule membrane (MFGM) (Fong, Norris, & Mac-
Gibbon, 2007; Girardet, Mati, Sanogo, Etienne, & Linden,
1994) suggesting a common derivation but differential
location in milk. Although lactophorin has a nominal pI of
6, the presence of multiple phosphate groups confers a
stronger acidic nature to the protein; hence, its capture into
the acidic fraction. Several lower Mr forms (1720 kDa)
of lactophorin were also observed in the neutral to basic pI
range (spots 5457) although at low levels. It is possible
that these are related to the so-called 17 kDa fragment
(residues 54135) of lactophorin, which arises from limited
proteolysis in the milk, most likely by plasmin (Sorensen &
Petersen, 1993a, b; Swaisgood, 2003). This fragment is
considerably basic (pI9) and contains no phosphoryla-
tion sites. Presumably, variable glycosylation or, alterna-
tively, further minor proteolytic or chemical modication
accounts for fragment heterogeneity. The occurrence of
basic peptides in the acidic fraction can probably be
accounted for by proteinprotein interactions and, indeed,
the 17 kDa fragment is reported to associate closely with its
parent (Sorensen & Petersen, 1993b). A clear pattern of
spots (27, 4043, 44a) in the 2025 kDa range was identied
as secreted phosphoprotein 1, more commonly known as
osteopontin (OPN). OPN is a highly phosphorylated,
highly acidic glycoprotein that is closely associated with
bone development and maintenance (Denhardt, Noda,
ORegan, Pavlin, & Berman, 2001), although it is known to
have roles in other aspects of physiology such as cell
signalling, adhesion and migration (Giachelli & Steitz,
2000), and has been linked to immunological development
in breast-fed infants (Nagatomo et al., 2004). In bovine
milk, it was originally isolated from the proteose peptone
 ARTICLE IN PRESS
B.Y. Fong et al. / International Dairy Journal 18 (2008) 2346 27

Fig. 2. Representative 2-D gel of the 1 M acidic fraction from late-lactation bovine whey. Proteins (0.7 mg) were separated by rst dimension IEF
(18 cm, pH 310 IPG strip) followed by second dimension SDS-PAGE (816% precast Tris-HCl gel). The gel was stained with CBB (A) and overstained
with silver (B, top half of gel illustrated). Spots or bands indicated numerically were excised and components were identied by RP-LC-MS/MS following
in-gel tryptic digestion.

fraction and shown to migrate on SDS gels as a single band
at 60 kDa (Sorensen & Petersen, 1993a), much higher
than its actual polypeptide mass of 29 kDa due to
extensive post-translational heterogeneity (Sorensen,
Hoejrup, & Petersen, 1995). Although limited cleavage
products in the Mr range 5060 kDa have been observed
for bovine milk OPN (Azuma, Maeta, Fukuchi, & Kanno,
2006), there have been no prior reports on the presence of
low Mr OPN isoforms or peptides in bovine milk. The
apparent mass of the OPN peptides observed in this study
would indicate that they were cleavage products of the
parent molecule and, in fact, only C-terminal fragments
were identied by MS. Interestingly, 2025 kDa C-terminal
species of OPN have also been reported in kidney cell lines
(Ullrich, Mann, Haase, & Koch-Brandt, 1991) and extracts
from porcine bone (Zhang, Domenicucci, Goldberg,
Wrana, & Sodek, 1990). OPN has a natural thrombin
cleavage site, which yields N- and C-terminal fragments of
30 and 2023 kDa, respectively (Zhang et al., 1990). The
form and the function of such peptides remain to be
claried as does their presence in whey other than from late
lactation. It should be noted, however, that OPN and
fragments have potential for use in increasing calcium
availability in the gut (Kumara, Minato, & Shimazaki,
 28
Table 1
Proteins identied in the 1 M acidic fraction of late-lactation whey by RP-LC-MS/MS

Spot no.a Accession no.b Name Molecular Theoretical pIc No. of peptides Protein sequence Total score
weightc identied coverage (%)d

1 gi|31340900 Serine (or cysteine) proteinase inhibitor, clade A (alpha-1- 46,289 5.57 18 32 950
antiproteinase, antitrypsin), member 3e
gi|76632120 PREDICTED: similar to inter-alpha globulin inhibitor H2 103,277 8.44 2 2 144
polypeptide
2 gi|76622034 PREDICTED: similar to complement component C3 188,675 6.41 50 27 2693
precursor isoform 1
gi|30794292 Lactoferrin 80,002 8.69 3 4 170
3 gi|27807349 Serine (or cysteine) proteinase inhibitor, clade G (C1 51,919 6.19 16 26 956
inhibitor), member 1f
gi|76670704 PREDICTED: similar to CD5 antigen-like 44,001 5.84 13 30 789
gi|31340900 Serine (or cysteine) proteinase inhibitor, clade A (alpha-1- 46,289 5.57 6 19 302
antiproteinase, antitrypsin), member 3e
4 gi|30794280 Albumin 71,274 5.82 35 55 1936
5 gi|76646499 PREDICTED: similar to complement component C9 61,387 5.66 10 21 572
precursor
gi|27806233 Butyrophilin, subfamily 1, member A1 59,909 5.10 9 22 398
gi|27807349 Serine (or cysteine) proteinase inhibitor, clade G (C1 51,919 6.19 8 20 378
inhibitor), member 1f
gi|27806447 Prosaposin 59,837 5.08 4 11 166
gi|76631371 PREDICTED: similar to L-plastin (lymphocyte cytosolic 70,752 5.17 2 4 104
protein 1) (LCP-1) isoform 1
6 gi|76668190 PREDICTED: similar to alpha-1B-glycoprotein 54,091 5.29 14 30 735
gi|27806233 Butyrophilin, subfamily 1, member A1 59,909 5.10 11 26 515
gi|76681914 PREDICTED: similar to immunoglobulin alpha-2 chain C 40,847 5.00 5 12 278
ARTICLE IN PRESS

region, partial
gi|76641927 PREDICTED: similar to nucleobindin 1 isoform 1 53,248 5.08 5 12 228
gi|76678688 PREDICTED: similar to immunoglobulin gamma-1 chain 45,936 4.98 4 13 246
C region, membrane-bound form
7 gi|31340900 Serine (or cysteine) proteinase inhibitor, clade A (alpha-1- 46,289 5.57 34 35 1729
antiproteinase, antitrypsin), member 3e
B.Y. Fong et al. / International Dairy Journal 18 (2008) 2346

gi|27806751 Alpha-2-HS-glycoprotein 39,193 5.26 14 30 740

gi|28461175 Kininogen 1 49,510 5.92 10 25 588
gi|59676562 Endopin 2B 47,200 5.82 11 27 675
gi|76641927 PREDICTED: similar to nucleobindin 1 isoform 1 53,248 5.08 12 27 655
gi|27806233 Butyrophilin, subfamily 1, member A1 59,909 5.10 7 16 388
8 gi|28461175 Kininogen 1 49,510 5.92 10 22 539
gi|76647789 PREDICTED: similar to alpha-1-antichymotrypsin 46,429 6.29 12 22 639
precursor (ACT)
gi|27806751 Alpha-2-HS-glycoprotein 39,193 5.26 11 24 658
gi|59676562 Endopin 2B 47,200 5.82 8 19 448
gi|27806233 Butyrophilin, subfamily 1, member A1 59,909 5.10 7 13 347
gi|76641927 PREDICTED: similar to nucleobindin 1 isoform 1 53,248 5.00 3 6 157
gi|76613612 PREDICTED: similar to vitamin D binding protein 50,360 5.61 11 23 537
precursor (DBP) (group-specic component)
gi|76647977 PREDICTED: similar to antithrombin III precursor 52,920 6.38 6 13 297
(ATIII) isoform 1
9a, 9b gi|27806941 Serine (or cysteine) proteinase inhibitor, clade A (alpha-1- 46,417 6.05 313 729 138765
antiproteinase, antitrypsin), member 1
gi|76613612 PREDICTED: similar to vitamin D binding protein 50,360 5.61 818 1542 383842
precursor (DBP) (group-specic component)
gi|76647977 PREDICTED: similar to antithrombin III precursor 52,920 6.38 79 1617 300417
(ATIII) isoform 1
gi|28461175 Kininogen 1 49,510 5.92 24 49 143242
9c gi|76647977 PREDICTED: similar to antithrombin III precursor 52,920 6.38 7 18 398
(ATIII) isoform 1
gi|76613612 PREDICTED: similar to vitamin D binding protein 50,360 5.61 5 11 245
precursor (DBP) (group-specic component)
gi|27806941 Serine (or cysteine) proteinase inhibitor, clade A (alpha-1- 46,417 6.05 6 16 239
antiproteinase, antitrypsin), member 1e
gi|27806233 Butyrophilin, subfamily 1, member A1 59,909 5.10 2 3 91
gi|76678688 PREDICTED: similar to immunoglobulin gamma-1 chain 45,936 5.25 13 36 586
C region, membrane-bound form
gi|76681914 PREDICTED: similar to immunoglobulin alpha-2 chain C 40,847 5.00 2 6 132
region, partial
10a gi|76678688 PREDICTED: similar to immunoglobulin gamma-1 chain 45,936 5.25 12 25 635
C region, membrane-bound form
gi|76681914 PREDICTED: similar to immunoglobulin alpha-2 chain C 40,847 5.00 2 6 143
region, partial
gi|41386719 Milk fat globule-EGF factor 8 protein 48,611 6.81 5 13 246
10b gi|76678688 PREDICTED: similar to immunoglobulin gamma-1 chain 45,936 5.25 6 11 281
C region, membrane-bound form
10c gi|41386719 Milk fat globule-EGF factor 8 protein 48,611 6.81 7 20 362
gi|76678688 PREDICTED: similar to immunoglobulin gamma-1 chain 45,936 5.25 5 12 183
C region, membrane-bound form
11 gi|41386719 Milk fat globule-EGF factor 8 protein 48,611 6.81 33 60 1815
12 gi|76622034 PREDICTED: similar to complement component C3 18,8675 6.41 23 12 1367
precursor isoform 1
13a gi|30794292 Lactoferrin 80,002 8.69 62 60 3268
gi|76693216 PREDICTED: similar to immunoglobulin mu chain C 23,723 8.29 7 35 395
ARTICLE IN PRESS

region, partial
13b & 13c gi|30794292 Lactoferrin 80,002 8.69 410 717 348485
14 gi|30794348 Casein alpha-S1 24,570 4.98 11 24 555
15 & 16 gi|30794348 Casein alpha-S1 24,570 4.98 311 1223 119576
gi|27881412 Casein kappa 21,370 6.30 24 1420 124183
B.Y. Fong et al. / International Dairy Journal 18 (2008) 2346

17 & 18 gi|30794348 Casein alpha-S1 24,570 4.98 14 42 805

gi|27807339 Glycosylation-dependent cell adhesion molecule 1g 17,198 6.21 18 38 801
gi|27806963 Casein alpha-S2 26,173 8.54 8 22 428
gi|30794310 Casein beta 25,139 5.14 6 18 423
gi|27881412 Casein kappa 21,370 6.30 4 20 269
19, 20 & 21 gi|27806963 Casein alpha-S2 26,173 8.54 23 715 64124
gi|30794310 Casein beta 25,139 5.14 38 320 81493
22 gi|30794310 Casein beta 25,139 5.14 2 10 177
gi|76639444 PREDICTED: similar to immunoglobulin lambda-like 21,338 8.89 5 21 434
polypeptide 1 precursor
23, 24 & 26 gi|30794348 Casein alpha-S1 24,570 4.98 411 2429 217574
gi|27807339 Glycosylation-dependent cell adhesion molecule 1g 17,198 6.21 78 31 383393
gi|27881412 Casein kappa 21,370 6.30 9 32 436
gi|32401410 Immunoglobulin J chain 18,359 5.10 3 21 154155
25 gi|27807339 Glycosylation-dependent cell adhesion molecule 1g 17,198 6.21 9 31 416
gi|27881412 Casein kappa 21,370 6.30 6 32 312
29
 30
Table 1 (continued )

Spot no.a Accession no.b Name Molecular Theoretical pIc No. of peptides Protein sequence Total score
weightc identied coverage (%)d

27 gi|27806401 Secreted phosphoprotein 1h 31,042 4.49 9 20 506

gi|27807339 Glycosylation-dependent cell adhesion molecule 1g 17,198 6.21 6 20 273
28 gi|27807339 Glycosylation-dependent cell adhesion molecule 1g 17,198 6.21 13 31 584
gi|27881412 Casein kappa 21,370 6.30 6 32 306
gi|30794310 Casein beta 25,139 5.14 2 15 145
gi|30794348 Casein alpha-S1 24,570 4.98 2 12 84
29 gi|30794348 Casein alpha-S1 24,570 4.98 7 22 323
gi|30794310 Casein beta 25,139 5.14 8 23 429
gi|27807339 Glycosylation-dependent cell adhesion molecule 1g 17,198 6.21 5 20 218
gi|27881412 Casein kappa 21,370 6.30 4 22 232
gi|27806963 Casein alpha-S2 26,173 8.54 2 7 68
30 & 31 gi|27807339 Glycosylation-dependent cell adhesion molecule 1g 17,198 6.21 3 12 134
gi|27881412 Casein kappa 21,370 6.30 3 14 147
32, 33 & 34 gi|27807339 Glycosylation-dependent cell adhesion molecule 1g 17,198 6.21 1114 35 557643
35 gi|27881412 Casein kappa 21,370 6.30 2 5 66
36 gi|27881412 Casein kappa 21,370 6.30 3 18 171
gi|30794348 Casein alpha-S1 24,570 4.98 2 10 104
gi|27806963 Casein alpha-S2 26,173 8.54 3 8 114
37 & 38 gi|27881412 Casein kappa 21,370 6.30 68 2832 268404
gi|27807339 Glycosylation-dependent cell adhesion molecule 1g 17,198 6.21 24 719 96184
39 gi|30794348 Casein alpha-S1 24,570 4.98 15 48 694
gi|27806963 Casein alpha-S2 26,173 8.54 6 18 285
gi|76678688 PREDICTED: similar to immunoglobulin gamma-1 chain 45,936 5.25 6 20 247
C region, membrane-bound form
gi|27881412 Casein kappa 21,370 6.30 3 14 218
gi|30794310 Casein beta 25,139 5.14 4 14 148
40, 41, 42, 43, gi|27806401 Secreted phosphoprotein 1h (C-terminal aa170278) 31,042 4.49 314 930 152735
44a
ARTICLE IN PRESS

44 gi|30794310 Casein beta (N-terminal aa4563) 25,139 5.14 3 8 220

45 gi|30794310 Casein beta (N-terminal aa4563, C-terminal aa199198, 25,139 5.14 6 16 373
199217 and 218224)
46 gi|30794310 Casein beta (N-terminal aa4563) 25,139 5.14 3 8 228
47, 48, 53 gi|27806861 Lactoglobulin, beta 20,307 4.85 728 3062 3731565
B.Y. Fong et al. / International Dairy Journal 18 (2008) 2346

49, 50, 51 & 52 No identication

54, 55, 56, 57 gi|27807339 Glycosylation-dependent cell adhesion molecule 1g 17,198 6.21 34 1112 114204
58 gi|76622034 PREDICTED: similar to complement component C3 188,675 6.41 18 12 951
precursor isoform 1
a
Refer to Fig. 2 for spot numbers.
b
Unless specied, the accession numbers of the identied proteins were obtained from the NBCI bovine genomic database.
c
The molecular weight and theoretical pI of the proteins as identied from the NCBI genomic database. These values do not necessarily reect those observed on the 2-D gel.
d
The protein sequence coverage determined from the sequences identied from the NBCI genomic database.
e
Serine (or cysteine) proteinase inhibitor, clade A (alpha-1-antiproteinase, antitrypsin), member 3; also identied as endopin-1 precursor (muscle endopin-1a, mEndopin 1) (Q9TTE1) in the Swiss-Prot
database.
f
Serine (or cysteine) proteinase inhibitor, clade G (C1 inhibitor), member 1; also identied as factor XIIa inhibitor precursor (P50448) in the Swiss-Prot database.
g
Glycosylation-dependent cell adhesion molecule 1; also identied as lactophorin precursor (28 kDa milk glycoprotein, PP3) (P80195) in the Swiss-Prot database.
h
Secreted phosphoprotein 1; also identied as OPN precursor (bone sialoprotein-1) (P31096) in the Swiss-Prot database.
 ARTICLE IN PRESS
B.Y. Fong et al. / International Dairy Journal 18 (2008) 2346
2006), and putative OPN peptides from whey have been
implicated in the oral bone bioactivity of whey acidic
protein fractions (Kruger et al., 2005). No other forms of
OPN were identied in this study. We have isolated the
parent (intact) protein from similar fractions in our
laboratory (Reid et al., 2000) but have found it very
difcult to consistently visualise using conventional stains
(unpublished data); hence, its presence in the 1 M acidic
fraction in this study cannot be excluded.
Caseins were also identied in the 1 M acidic fraction,
occurring as clusters in the 2530 kDa region of the gel and
co-migrating in some cases with lactophorin. Caseins have
observed pI 4.95.9 (Trieu-Cout & Gripon, 1981), and all
four species (aS1-, aS2-, b-, k-) were present in various
forms, reecting the extensive genetic and post-transla-
tional variation of this group of proteins (Swaisgood,
2003). k-Casein alone has been shown by 2-DE to consist
of multiple isoforms with pI ranging between 4.47 and 5.81
(Holland, Deeth, & Alewood, 2004; Holland et al., 2006).
Whey generally contains residual casein that is not
precipitated in the acid coagulate, but, in late-lactation
milk, the whey proved to be difcult to clarify, indicating a
higher content of non-micellar soluble casein. Minor
amounts of b-Lg and fragment (spots 47, 48, 53) were also
observed on the gel. Silver staining revealed little else in the
lower Mr region of the gel, but revealed more detail on the
proteins in low abundance in the higher Mr region
(Fig. 2B). Butyrophilin and milk fat globule-EGF factor
8 (PAS6/7), proteins derived from the MFGM (Mather,
2000), were identied in clusters with other proteins.
Occurrence of the hydrophobic, limited-solubility MFGM
proteins in the whey fraction probably arises from MFGM
damage and solubilisation of the exposed proteins by
association with other serum proteins or caseins (Cano-
Ruiz & Richter, 1997). The aggregative nature of these
proteins and their association with other proteins would
also explain their presence in the acidic fraction. Alter-
natively, it is possible that these MFGM proteins were
associated with phospholipids, which also occur in the
serum phase of milk (MacGibbon & Taylor, 2006) and
have been shown by us to occur in the acidic fraction
(unpublished data). The highly basic protein lactoferrin,
which has a nominal pI of 8.6 and a molecular mass of
80 kDa, was surprisingly but uniformly identied as a
broad ribbon across a wide pI range (band 13a13c), albeit
in relatively minor amount. It has been demonstrated
recently that lactoferrin can associate electrostatically with
OPN and bind to OPN immobilised on a column (Azuma
et al., 2006) and this interaction might account for its
presence in the acidic fraction. Other proteins observed in
residual amounts in the high Mr region were those
normally associated with vascular functions, i.e. blood
coagulation, blood pressure regulation or complement
activation (antithrombin III; factor XIIa inhibitor/serine
or cysteine proteinase inhibitor clade G; complement
components C3 and C9; kininogen 1), tissue development
(a2-HS-glycoprotein), metabolite binding or transport
31
(inter-a globulin inhibitor H2 polypeptide; vitamin D
binding protein; nucleobindin 1), enzyme inhibition/activa-
tion (serine or cysteine proteinase inhibitor clade A/alpha-
1-antiproteinase; alpha-1-antichymotrypsin; endopin 2B;
prosaposin) or immunity (immunoglobulin (Ig) alpha-2,
gamma-1, J, mu and lambda-like polypeptide chains;
alpha-1B-glycoprotein; CD5 antigen-like). A small amount
of BSA was also present as a typical isoform cluster (spot 4;
Yamada et al., 2002). L-plastin, a protein usually
associated with the lymphocyte cytosol, was also identied
in the 1 M acidic fraction (spot 5).
3.2. 0.4 M basic fraction
While the acidic fraction was dominated by phosphory-
lated proteose peptones, the 0.4 M basic fraction (Fig. 3 and
Table 2) was dominated by a number of Ig and Ig-related
proteins. IgG, the major class of Ig in the cow (Farrell
et al., 2004), was prevalent (Ig gamma-1 chain C region,
spots 13, 2528), with IgA (Ig alpha-2 chain C region; J
chain) and IgM (Ig mu chain C region) also being identied
in the high Mr region of the gel. Polymeric Ig receptor, as
secretory component (Bjorkman et al., 2005; Farrell et al.,
2004) (spots 1213a), also contributed a large proportion
of the protein in this fraction. The Fc fragment of IgG
binding protein was present as a small string of spots at
high Mr (spot 6, and also spots 14, 20) while the Ig lambda
and kappa light chains were also observed along with Ig
lambda-like polypeptide 1 in the mid-Mr region of the gel
(spots 59, 73, 74). As expected, lactoperoxidase, a
glycosylated haem protein integral to the natural anti-
microbial defence system in milk (Seifu, Buys, & Donkin,
2005), was also present in this fraction, being identied
primarily as presumed intact protein in a band at 80 kDa
in the basic region of the gel (spot 7a). As with the 1 M
acidic fraction, MFGM-derived proteins were present in
trace amounts, in this case butyrophilin, adipose differ-
entiation-related protein (adipophilin) and milk fat glo-
bule-EGF factor 8 protein. Lactoferrin was also present in
trace amounts at loci of different pI and Mr. Complement
C3 was identied in greater amount in this fraction,
principally as a cluster of spots at 40 kDa (spots 18, 23,
3036), indicating variable chain cleavage due either to
physiological processing or to milk indigenous enzyme
activity. This protein has also been reported to occur in
human colostrum as multiple fragments (Palmer et al.,
2006). Other members of the complement system, C4, C7,
C8, C9 and factor I, were also identied in the higher Mr
region of the gel. Complement proteins, of which C3 is
predominant, originate from the blood serum globulin
fraction, where they form a cascade of reactions which
protects against pathogens (Bjorkman et al., 2005). Other
proteins apparent in the fraction at lower abundance and
with diverse physiological roles were gelsolin (an actin-
modulating protein), hemopexin (a haem transport pro-
tein), serine (or cysteine) proteinase inhibitor clade
G, cysteine-rich secretory protein 2, tripeptidyl peptidase
 ARTICLE IN PRESS
32 B.Y. Fong et al. / International Dairy Journal 18 (2008) 2346

Fig. 3. Representative 2-D gel of the 0.4 M basic fraction from late-lactation bovine whey. Proteins (0.7 mg) were separated by rst dimension IEF
(18 cm, pH 310 IPG strip) followed by second dimension SDS-PAGE (816% precast Tris-HCl gel). The gel was stained with CBB. Spots or bands
indicated numerically were excised and components were identied by RP-LC-MS/MS following in-gel tryptic digestion.

(a lysosomal proteinase), cartilage acidic protein 1, CD5
antigen-like protein, brinogen gamma polypeptide, stro-
mal cell-derived factor 4 (Cab45 protein), cellular suppres-
sor of E1A-stimulated genes (CREG, involved in
transcriptional control of cell growth and differentiation),
apolipoprotein H and folate receptor 1 (folate binding
protein). Folate binding protein has been identied
previously in human colostral MFGM preparations by
2-DE, as three isoforms migrating at 24.6 kDa (Fortunato
et al., 2003). In this study, it was identied in two spots of
distinctly different pI and Mr (spots 45b, 90).
The remainder of the protein in the 0.4 M basic fraction
consisted mainly of caseins and peptides. A myriad of
casein peptides were identied in the lower Mr region of the
gel, spanning the acidic to the basic range. Such peptides
result from limited proteolysis either in the mammary
gland or in the secreted milk and are likely to vary
according to origin of sample, season and enzyme prole
(e.g., plasmin) (Wheeler, Broadhurst, Rajan, & Wilkins,
1997). Some of these casein peptides may have a role in the
mammary gland, especially at involution (Aslam & Hurley,
1998) but importantly may confer health benets to the
secreted milk as casein peptides have been shown to have a
variety of therapeutic properties (Shah, 2000). Small
amounts of lactophorin as the intact protein were also
found co-migrating with caseins (spots 6266), and the
presumed 17 kDa forms of lactophorin were also present
(spots 7579) but at greater amplitude than in the acidic
fraction, suggesting natural capture into this basic fraction.
3.3. 1.5 M basic fraction
The 1.5 M basic fraction (Fig. 4 and Table 3) was hugely
dominated by lactoferrin, which was as expected given that
the classic isolation protocol for lactoferrin from milk is as
the high ionic strength salt eluate from cation exchange
(Yoshida & Xiuyun, 1991). Lactoferrin is commercially
isolated from bovine milk and is an intriguing, pluripotent
protein with antimicrobial, immune regulatory, anti-
inammatory and mitogenic properties (Cornish et al.,
2006; Wakabayashi, Yamauchi, & Takase, 2006). Analysis
by RP-HPLC (not shown) revealed this protein to be in
excess of 90% of the total protein present in the 1.5 M basic
fraction. Extreme loads of single proteins are generally
unfavourable for 2-DE formats. However, due to both its
relatively high Mr and high pI, lactoferrin was almost
exclusively localised in one corner of the 2-D gel, either as a
monomer (spots 47) or as large Mr complexes that barely
penetrated the gel (spots 13, 5a), thus allowing display of
other proteins captured in this fraction. The vertical streak
of lactoferrin at the basic margin of the gel (spots 10a, 10c,
10d) was no doubt due to overabundance of this protein
Table 2
Proteins identied in the 0.4 M basic fraction of late-lactation whey by RP-LC-MS/MS

Spot no.a Accession no.b Name Molecular Theoretical pIc No. of peptides Protein Total score
weightc identied sequence
coverage (%)d

1 gi|76678688 PREDICTED: similar to immunoglobulin gamma-1 chain 45,936 5.25 10 31 521

C region, membrane-bound form
2 gi|76678688 PREDICTED: similar to immunoglobulin gamma-1 chain 45,936 5.25 3 13 135
C region, membrane-bound form
gi|27806233 Butyrophilin, subfamily 1, member A1 59,909 5.10 3 4 170
gi|27806759 Adipose differentiation-related protein 49,622 8.72 2 5 111
3 gi|30794292 Lactoferrin 80,002 8.69 8 11 425
gi|76678688 PREDICTED: similar to immunoglobulin gamma-1 chain 45,936 5.25 3 11 145
C region, membrane-bound form
4 gi|76646469 PREDICTED: similar to complement component C7 96,123 7.09 19 28 1083
precursor isoform 1
gi|30794292 Lactoferrin 80,002 8.69 6 8 260
gi|27806851 Lactoperoxidase 81,504 8.83 3 4 145
5 gi|76625353 PREDICTED: similar to gelsolin precursor (actin- 78,154 6.18 24 30 1373
depolymerising factor) (ADF)
6 gi|76677740 PREDICTED: similar to Fc fragment of IgG binding 253,100 4.87 4 1 297
protein
7 gi|27806851 lactoperoxidase 81,504 8.83 11 20 564
gi|76693216 PREDICTED: similar to immunoglobulin mu chain C 23,723 8.29 5 20 287
region, partial
gi|30794292 Lactoferrin 80,002 8.69 2 3 81
7a gi|27806851 Lactoperoxidase 81,504 8.83 1628 1737 7561447
gi|76622034 PREDICTED: similar to complement component C3 188,675 6.41 22 15 1190
ARTICLE IN PRESS

precursor isoform 1
gi|32189338 Polymeric immunoglobulin receptor [Bos taurus] 83,711 7.07 3 3 124163
gi|76650940 PREDICTED: similar to complement component C4 161,421 5.58 5 3 238
precursor [Bos taurus]
7b gi|27806851 Lactoperoxidase [Bos taurus] 81,504 8.83 18 37 773
B.Y. Fong et al. / International Dairy Journal 18 (2008) 2346

gi|76622034 PREDICTED: similar to complement component C3 188,675 6.41 20 13 1091

precursor isoform 1
gi|76625353 PREDICTED: similar to gelsolin precursor (actin- 78,154 6.18 7 13 418
depolymerising factor) (ADF)
7c gi|76622034 PREDICTED: similar to complement component C3 188,675 6.41 22 13 1191
precursor isoform 1
gi|76678688 PREDICTED: similar to immunoglobulin gamma-1 chain 45,936 4.98 2 6 74
C region, membrane-bound form
8 gi|27807349 Serine (or cysteine) proteinase inhibitor, clade G (C1 51,919 6.19 14 19 787
inhibitor), member 1e
gi|76654689 PREDICTED: similar to cartilage acidic protein 1 71,343 5.04 9 16 507
gi|76670704 PREDICTED: similar to CD5 antigen-like 44,001 5.84 10 24 587
gi|32189338 Polymeric immunoglobulin receptor 83,711 7.07 3 5 157
gi|76693216 PREDICTED: similar to immunoglobulin mu chain C 23,723 8.29 3 20 129
region, partial
33
 34
Table 2 (continued )

Spot no.a Accession no.b Name Molecular Theoretical pIc No. of peptides Protein Total score
weightc identied sequence
coverage (%)d

9 gi|30794292 Lactoferrin 80,002 8.69 7 11 402

gi|76646499 PREDICTED: similar to complement component C9 61,387 5.66 4 7 156
precursor
10 gi|30794280 Albumin 71,274 5.82 8 16 450
gi|76635726 PREDICTED: similar to hemopexin isoform 1 (also 52,931 7.59 7 17 351
hypothetical protein LOC534509 gi|77736171)
11 gi|76635726 PREDICTED: similar to hemopexin isoform 1 (also 52,931 7.59 14 29 694
hypothetical protein LOC534509 gi|77736171)
gi|76613793 PREDICTED: similar to complement component C8, 85,266 8.04 8 14 448
alpha polypeptide precursor
gi|32189338 Polymeric immunoglobulin receptor 83,711 7.07 4 7 267
gi|76635772 PREDICTED: similar to tripeptidyl peptidase I precursor 9536 9.42 3 45 166
12, 13, 13a gi|32189338 Polymeric immunoglobulin receptor 83,711 7.07 2336 1332 13782271
14 gi|30794292 Lactoferrin 80,002 8.69 4 7 185
gi|76677740 PREDICTED: similar to Fc fragment of IgG binding 253,100 4.87 6 0 156
protein
15 & 16 gi|76681914 PREDICTED: similar to immunoglobulin alpha-2 chain 40,847 5.00 68 1931 357412
C region, partial
17 gi|27806893 Fibrinogen, gamma polypeptide 50,839 5.54 18 41 1031
18 gi|76622034 PREDICTED: similar to complement component C3 188,675 6.41 11 8 633
precursor isoform 1
20 gi|76677740 PREDICTED: similar to Fc fragment of IgG binding 253,100 4.87 11 6 463
protein
21 gi|30794292 Lactoferrin 80,002 8.69 4 5 209
22 gi|76637700 PREDICTED: similar to stromal cell-derived factor 4 32,600 4.73 7 20 355
ARTICLE IN PRESS

(also hypothetical protein LOC528783, gi|73586919)g

23 gi|76622034 PREDICTED: similar to complement component C3 188,675 6.41 21 9 1092
precursor isoform 1
24 gi|30794280 Albumin 71,274 5.82 11 21 693
gi|76619404 PREDICTED: similar to complement component factor I 42,027 6.56 3 11 179
B.Y. Fong et al. / International Dairy Journal 18 (2008) 2346

(also protein for MGC:128240, gi|81294303)g

25 gi|76678688 PREDICTED: similar to immunoglobulin gamma-1 chain 45,936 4.98 6 16 283
C region, membrane-bound form
gi|76681914 PREDICTED: similar to immunoglobulin alpha-2 chain 40,847 5.00 4 10 281
C region, partial
26 gi|76678688 PREDICTED: similar to immunoglobulin gamma-1 chain 45,936 4.98 5 7 215
C region, membrane-bound form
gi|27806233 Butyrophilin, subfamily 1, member A1 59,909 5.10 2 4 114
27 gi|76678688 PREDICTED: similar to immunoglobulin gamma-1 chain 45,936 4.98 3 7 148
C region, membrane-bound form
gi|76681914 PREDICTED: similar to immunoglobulin alpha-2 chain 40,847 5.00 2 6 120
C region, partial
gi|27806741 Apolipoprotein H 39,450 8.71 2 4 80
28 gi|41386719 Milk fat globule-EGF factor 8 protein 48,611 6.81 4 9 194
gi|27806233 Butyrophilin, subfamily 1, member A1 59,909 5.10 3 5 168
gi|76678688 PREDICTED: similar to immunoglobulin gamma-1 chain 45,936 4.98 5 7 198
C region, membrane-bound form
30, 30a, 30b, gi|76622034 PREDICTED: similar to complement component C3 188,675 6.41 416 112 172821
3136 precursor isoform 1
37, 43, 46, 50, gi|30794348 Casein alpha-S1 24,570 4.98 214 1047 69714
54, 57, 86 & 92
39, 40, gi|30794348 Casein alpha-S1 24,570 4.98 310 941 219535
42, 87, 88 & 89 gi|27806963 Casein alpha-S2 26,173 8.54 2-12 1444 75474
38, 41 gi|27881412 Casein kappa 21,370 6.30 2-5 1020 96204
&66 gi|30794348 Casein alpha-S1 24,570 4.98 29 939 200463
44 gi|27806861 Lactoglobulin, beta 20,307 4.85 3 11 181
gi|30794348 Casein alpha-S1 24,570 4.98 3 19 166
45 gi|30794310 Casein beta 25,139 5.14 3 14 135
45b gi|61868368 PREDICTED: similar to folate receptor 1 precursor 28,815 8.12 11 39 561
isoform 1
46 gi|30794348 Casein alpha-S1 24,570 4.98 7 26 285
47 gi|30794310 Casein beta 25,139 5.14 2 15 145
gi|30794348 Casein alpha-S1 24,570 4.98 5 24 242
48 gi|30794310 Casein beta 25,139 5.14 2 10 107
5152 gi|27806861 Lactoglobulin, beta 20,307 4.85 25 17 92313
gi|30794348 Casein alpha-S1 24,570 4.98 35 1011 164277
53 gi|27806861 Lactoglobulin, beta 20,307 4.85 7 43 425
57 gi|30794348 Casein alpha-S1 24,570 4.98 2 10 93
59 gi|76690231 PREDICTED: similar to immunoglobulin lambda chain 25,302 8.49 2 12 150
V-I region BL2 precursor
gi|27806963 Casein alpha-S2 26,173 8.54 3 15 128
gi|30794348 Casein alpha-S1 24,570 4.98 2 10 113
gi|76681830 PREDICTED: similar to immunoglobulin kappa chain C 26,317 4.69 2 10 96
region isoform 2
60 gi|27806963 Casein alpha-S2 26,173 8.54 2 9 87
gi|76678688 PREDICTED: similar to immunoglobulin gamma-1 chain 45,936 4.98 2 6 72
C region, membrane-bound form
61 gi|30794310 Casein beta 25,139 5.14 6 18 288
ARTICLE IN PRESS

gi|30794348 Casein alpha-S1 24,570 4.98 4 17 165

62 gi|30794348 Casein alpha-S1 24,570 4.98 11 46 626
gi|27807339 Glycosylation-dependent cell adhesion molecule 1f 17,198 6.21 9 31 415
gi|30794310 Casein beta 25,139 5.14 5 21 266
gi|27881412 Casein kappa 21,370 6.30 6 20 342
gi|32401410 Immunoglobulin J chain 18,359 5.10 4 29 165
B.Y. Fong et al. / International Dairy Journal 18 (2008) 2346

gi|76611682 PREDICTED: similar to cellular repressor of E1A- 27,264 6.58 2 15 119

stimulated genes
6365 gi|76611682 PREDICTED: similar to cellular repressor of E1A- 27,264 6.58 25 1821 156212
stimulated genes
gi|27807339 Glycosylation-dependent cell adhesion molecule 1f 17,198 6.21 47 2131 158289
gi|27881412 Casein kappa 21,370 6.30 4 1820 190236
67 gi|27881412 Casein kappa 21,370 6.30 4 20 227
gi|30794348 Casein alpha-S1 24,570 4.98 4 18 169
gi|76611682 PREDICTED: similar to cellular repressor of E1A- 27,264 6.58 2 15 76
stimulated genes
68, 69 gi|27881412 Casein kappa 21,370 6.30 5 20 275
gi|30794310 Casein beta 25,139 5.14 3 14 138
70 gi|30794310 Casein beta 25,139 5.14 3 14 116
73 gi|76611682 PREDICTED: similar to cellular repressor of E1A- 27,264 6.58 4 26 286
stimulated genes
35
 36
Table 2 (continued )

Spot no.a Accession no.b Name Molecular Theoretical pIc No. of peptides Protein Total score
weightc identied sequence
coverage (%)d

gi|76639444 PREDICTED: similar to immunoglobulin lambda-like 21,338 8.89 2 15 179

polypeptide 1 precursor
74 gi|76639444 PREDICTED: similar to immunoglobulin lambda-like 21,338 8.89 2 15 92
polypeptide 1 precursor
gi|76690231 PREDICTED: similar to immunoglobulin lambda chain 25,302 8.49 3 19 166
V-I region BL2 precursor
gi|27806963 Casein alpha-S2 26,173 8.54 3 14 136
7579 gi|27807339 Glycosylation-dependent cell adhesion molecule 1f 17,198 6.21 69 1624 253404
81 gi|27806851 Lactoperoxidase [Bos taurus] 81,504 8.83 9 13 368
gi|76676126 PREDICTED: similar to brinogen alpha chain precursor 92,841 5.74 4 5 223
[Bos taurus]
gi|30794292 Lactoferrin [lactotransferrin] [Bos taurus] 80,002 8.69 21 27 1152
gi|76690514 PREDICTED: similar to cysteine-rich secretory protein 2 17,817 6.35 2 17 118
precursor (CRISP-2)
gi|76622034 PREDICTED: similar to complement component C3 188,675 6.41 20 13 1172
precursor isoform 1
82 gi|76622034 PREDICTED: similar to complement component C3 188,675 6.41 12 8 602
precursor isoform 1 [Bos taurus]
gi|30794292 Lactoferrin [lactotransferrin] [Bos taurus] 80,002 8.69 10 14 511
83 gi|27881412 Casein kappa 21,370 6.30 8 24 419
gi|76622034 PREDICTED: similar to complement component C3 188,675 6.41 7 4 336
precursor isoform 1 [Bos taurus]
gi|30794292 Lactoferrin [lactotransferrin] [Bos taurus] 80,002 8.69 3 4 139
84 gi|27881412 Casein kappa 21,370 6.30 3 17 108
gi|27806963 Casein alpha-S2 26,173 8.54 2 6 75
ARTICLE IN PRESS

85 gi|27806861 Lactoglobulin, beta 20,307 4.85 14 35 700

90 gi|61868368 PREDICTED: similar to folate receptor 1 precursor 28,815 8.12 5 19 256
isoform 1
gi|27806963 Casein alpha-S2 16,173 8.54 2 9 102
gi|30794348 Casein alpha-S1 24,570 4.98 3 10 125
B.Y. Fong et al. / International Dairy Journal 18 (2008) 2346

91 gi|30794310 Casein beta 25,139 5.14 4 14 195

19, 29, 49, 55, No identications
56, 58, 71, 72,
80
a
Refer to Fig. 3 for spot numbers.
b
Unless specied, the accession numbers of the identied proteins were obtained from the NBCI bovine genomic database.
c
The molecular weight and theoretical pI of the proteins as identied from the NCBI genomic database. These values do not necessarily reect those observed on the 2-D gel.
d
The protein sequence coverage determined from the sequences identied from the NBCI genomic database.
e
Serine (or cysteine) proteinase inhibitor, clade G (C1 inhibitor), member 1; also identied as factor XIIa inhibitor precursor (P50448) in the Swiss-Prot database.
f
Glycosylation-dependent cell adhesion molecule 1; also identied as lactophorin precursor (28 kDa milk glycoprotein, PP3) (P80195) in the Swiss-Prot database.
g
NCBI expressed protein+EST bovine database.
 ARTICLE IN PRESS
B.Y. Fong et al. / International Dairy Journal 18 (2008) 2346 37

Fig. 4. Representative 2-D gel of the 1.5 M basic fraction from late-lactation bovine whey. Proteins (0.7 mg) were separated by rst dimension IEF
(18 cm, pH 310 IPG strip) followed by second dimension SDS-PAGE (816% precast Tris-HCl gel). The gel was stained with CBB. Spots or bands
indicated numerically were excised and components were identied by RP-LC-MS/MS following in-gel tryptic digestion.

while, typically, a band of lactoferrin at the acidic margin
of the gel (spot 27) represented protein that remained
unfocused during IEF. Serine (cysteine) proteinase inhi-
bitor clade G and Ig mu chain were also present as minor
constituents in this band. Lactoferrin also occurred as
lower Mr cleavage forms, albeit in minor amount, e.g.,
strings 27b, 27c; 810, possibly corresponding to the
50 kDa N-lobe fragment (Mata, Castillo, Sanchez, Puyol,
& Calvo, 1994). Not surprisingly lactoperoxidase, which is
a common contaminant of lactoferrin preparations, was
also found associated with lactoferrin at higher Mr. Highly
basic proteins at the next level of magnitude were the
ribonucleases PDZ domain containing 6 (secreted protein
ribonuclease 4) and angiogenin 1 (spots 11, 12), both of
which have been isolated chromatographically from the
lactoferrin fraction of bovine milk (Kawakami et al., 2006).
Several other proteins of high pI and mid-high Mr were
identied in spots/regions on the gel where lactoferrin was
identied as the major species. These were quiescin Q6, a
thioredoxin (sulphydryl oxidase); chitinase 3-like 1, which
is known to be expressed in non-lactating bovine mammary
gland and involved in tissue remodelling (Hakala, White, &
Recklies, 1993); heparanase (also involved in tissue
remodelling); lipopolysaccharide binding protein; the
transport protein neutrophil gelatinase-associated lipocalin
(lipocalin 2); and the proteinase inhibitor inter-a-trypsin
inhibitor. Fibroblast growth factor binding protein was
present as two series of presumed isoforms at distinct
molecular masses (25 kDa, spots 21, 23, 24; 30 kDa,
spots 11b, 26, 30), possibly representing alternative post-
translational processing of the protein (Lametsch et al.,
2000). Fibroblast growth factor binding protein has
been isolated from bovine prepartum mammary gland
(Lametsch et al., 2000) and more recently from the
lactoferrin fraction of milk, and is thought not only to be
tightly associated with lactoferrin but also to be physiolo-
gically correlated with this protein (Kawakami et al., 2006).
Signicantly, broblast growth factor binding protein is
involved in recruitment and activation of broblast growth
factors which are, like lactoferrin, mitogenic to certain cell
lines. It is an interesting possibility that the different
isoforms might be functionally different. Tetranectin, a
plasminogen binding C-type lectin implicated in osteogen-
esis and myogenesis, also occurred as a cluster of spots in
the lower mid-Mr region of the gel (spots 1920c).
Although somewhat acidic pI (5.8), this protein binds
strongly to complex sulphated polysaccharides such as
heparan (Clemmensen, 1989), which might explain its
retention on the sulphonated cation exchanger. A trace of
complement component C3 was identied at the basic
margin of the gel in the lactoferrin zone while a further
complement component, factor D, also known as adipsin,
was also clearly identied in this fraction (spot 35). Other
spots throughout the gel were identied as largely caseins
 38

Table 3
Proteins identied in the 1.5 M basic fraction of late-lactation whey by RP-LC-MS/MS

Spot no.a Accession no.b Name Molecular Theoretical pIc No. of peptides Protein sequence Total score
weightc identied coverage (%)d

1, 2, 3, 4, 5, 5a, gi|30794292 Lactoferrin 80,002 8.69 4590 5468 27065669

6, 7
gi|27806851 Lactoperoxidase 81,504 8.83 417 725 180884
8 gi|30794292 Lactoferrin 80,002 8.69 45 54 2307
9, 10 gi|30794292 Lactoferrin 80,002 8.69 3268 5062 17403743
gi|76701028 PREDICTED: similar to quiescin Q6 isoform 27,845 6.68 2 12 99
10a gi|30794292 Lactoferrin 80,002 8.69 67 63 3642
gi|27806583 Heparanase 61,437 9.25 9 17 447
gi|27806851 Lactoperoxidase 81,504 8.83 4 6 201
gi|76701028 PREDICTED: similar to quiescin Q6 isoform a 27,845 6.68 2 12 96
gi|76622034 PREDICTED: similar to complement component 18,8675 6.41 2 1 90
C3 precursor isoform 1
10c gi|30794292 Lactoferrin 80,002 8.69 28 40 1533
gi|76636770 PREDICTED: similar to chitinase 3-like 1 isoform 44,080 8.87 18 39 901
2
10d gi|76630593 PREDICTED: similar to neutrophil gelatinase- 42658 9.64 5 22 345
associated lipocalin precursor (NGAL)
gi|30794292 Lactoferrin 80,002 8.69 3 6 144
ARTICLE IN PRESS

11 gi|30794292 Lactoferrin 80,002 8.69 17 18 858

gi|76652266 PREDICTED: hypothetical protein XP_869612 17,017 8.71 4 43 220
gi|76627221 PREDICTED: similar to PDZ domaine 53,855 9.75 5 5 249
gi|12043712 Angiogenin precursor 17,301 9.10 9 25 426
11b gi|27806963 Casein alpha-S2 26,173 8.54 6 23 329
B.Y. Fong et al. / International Dairy Journal 18 (2008) 2346

gi|30794292 Lactoferrin 80,002 8.69 9 15 399

gi|30794310 Casein beta 25,139 5.14 3 18 179
gi|30794348 Casein alpha-S1 24,570 4.98 3 16 151
gi|27805911 Fibroblast growth factor binding protein (FGF- 26,742 9.14 2 11 77
BP)
12 gi|76627221 PREDICTED: similar to PDZ domaine 53,855 9.75 8 9 424
13 gi|30794292 Lactoferrin 80,002 8.69 8 15 450
14 gi|76615313 PREDICTED: similar to DnaJ (Hsp40) 25,870 7.79 3 7 162
homologue, subfamily B, member 9
15 gi|27806963 Casein alpha-S2 26,173 8.54 2 8 95
16, 17 & 31 No identication
18 gi|30794348 Casein alpha-S1 24,570 4.98 4 22 117
gi|27806963 Casein alpha-S2 26,173 8.54 2 10 92
19, 20, 20b, 20c gi|61819239 PREDICTED: similar to tetranectin precursor 22,586 5.47 58 3540 273504
(TN) (plasminogen kringle 4 binding protein)
21 gi|76639444 PREDICTED: similar to immunoglobulin 21,338 8.89 2 15 135
lambda-like polypeptide 1 precursor
gi|27805911 Fibroblast growth factor binding protein 26,742 9.14 3 7 129
22 gi|27806743 Inter-alpha-trypsin inhibitor (protein HC), light 40,122 7.81 2 5 116
23, 24 gi|27805911 Fibroblast growth factor binding protein 26,742 9.14 2 12 9.14
gi|27807339 Glycosylation-dependent cell adhesion molecule 1f 17,198 6.21 2 11 293
25, 25b gi|27806963 Casein alpha-S2 26,173 8.54 6 22 294
26 gi|27805911 Fibroblast growth factor binding protein 26,742 9.14 4 23 204
gi|30794348 Casein alpha-S1 24,570 4.98 2 10 97
27 gi|30794292 Lactoferrin 80002 8.69 70 59 3809
gi|27807349 Serine (or cysteine) proteinase inhibitor, clade G 51,919 6.19 4 10 307
(C1 inhibitor), member 1g
gi|76693216 PREDICTED: similar to immunoglobulin mu 23,723 8.29 2 9 99
chain C region
27b gi|30794292 Lactoferrin 80,002 8.69 44 46 2512
gi|32189338 Polymeric immunoglobulin receptor 83,711 7.07 3 4 190
27c gi|30794292 Lactoferrin 80,002 8.69 23 36 1387
gi|76633538 PREDICTED: similar to lipopolysaccharide 53,889 6.61 4 10 211
binding protein precursor isoform 1
gi|76678688 PREDICTED: similar to immunoglobulin gamma- 45,936 5.25 2 6 75
1 chain C region, membrane-bound form
30 gi|30794348 Casein alpha-S1 24,570 4.98 7 29 377
gi|27806963 Casein alpha-S2 26,173 8.54 4 18 173
gi|27805911 Fibroblast growth factor binding protein (FGF- 26,742 9.14 3 20 156
BP)
gi|30794310 Casein beta 25,139 5.14 3 15 179
32 gi|27881412 Casein kappa 21,370 6.30 3 14 125
gi|30794348 Casein alpha-S1 24,570 4.98 2 10 114
33 gi|30794348 Casein alpha-S1 24,570 4.98 9 29 496
gi|27881412 Casein kappa 21,370 6.30 4 20 240
gi|27807339 Glycosylation-dependent cell adhesion molecule 1f 17,198 6.21 3 19 116
ARTICLE IN PRESS

34, 34b gi|30794348 Casein alpha-S1 24,570 4.98 1718 52 888978

35 gi|61882363 PREDICTED: similar to complement component 28,664 6.55 4 22 274
factor D precursor (C3 convertase activator)
(properdin factor D)
36 gi|30794348 Casein alpha-S1 24,570 4.98 2 12 78
B.Y. Fong et al. / International Dairy Journal 18 (2008) 2346

a
Refer to Fig. 4 for spot numbers.
b
Unless specied, the accession numbers of the identied proteins were obtained from the NBCI bovine genomic database.
c
The molecular weight and theoretical pI of the proteins as identied from the NCBI genomic database. These values do not necessarily reect those observed on the 2-D gel.
d
The protein sequence coverage determined from the sequences identied from the NBCI genomic database.
e
PREDICTED: similar to PDZ domain; also identied as ribonuclease 4 (P15467) in the Swiss-Prot database.
f
Glycosylation-dependent cell adhesion molecule 1; also identied as lactophorin precursor (28 kDa milk glycoprotein, PP3) (P80195) in the Swiss-Prot database.
g
Serine (or cysteine) proteinase inhibitor, clade G (C1 inhibitor), member 1; also identied as factor XIIa inhibitor precursor (P50448) in the Swiss-Prot database.
39
 ARTICLE IN PRESS
40 B.Y. Fong et al. / International Dairy Journal 18 (2008) 2346

or their fragments, with lactophorin, Ig-associated compo-
nents, b-Lg and a-Lac (indicated on gel) also present in
trace amounts Two other proteins, hypothetical protein
XP_869612 (spot 11) and DnaJ (Hsp40) homologue (spot
14), were matched to genomic sequences (NCBI bovine
genomic database) only. The DnaJ/Hsp40 (heat-shock
protein 40) proteins are conserved proteins that interact
with the Hsp70 chaperone proteins (Qui, Shao, Miao, &
Wang, 2006). Interestingly, Hsp 71 kDa has been identied
in both bovine MFGM and human colostrum (Fong et al.,
2007; Fortunato et al., 2003; Palmer et al., 2006).
XP_869612 protein is similar to a SWI/SNF-related
protein, involved in chromatin remodelling (Pazin &
Kadonaga, 1997). It is tempting to speculate that those
proteins associated with a cell nuclear function/location
and occurring in bovine milk are derived from somatic cell
debris. However, a 100 kDa transcription coactivator
nuclear protein has recently been identied in bovine
MFGM (Reinhardt & Lippolis, 2006), suggesting other
origins.

Fig. 5. Representative 2-D gel of the cation exchange break through (CX-BT) from late-lactation bovine whey. Proteins (0.7 mg) were separated by rst
dimension IEF (18 cm, pH 310 IPG strip) followed by second dimension SDS-PAGE (816% precast Tris-HCl gel). The gel was stained with CBB and
overstained with silver (A). The CBB-stained gel is shown in (B). Spots or bands indicated numerically were excised and components were identied by
RP-LC-MS/MS following in-gel tryptic digestion.
Table 4
Proteins identied in the CX-BT of late-lactation whey by RP-LC-MS/MS

Spot no.a Accession no.b Name Molecular weightc Theoretical pIc No. of peptide Protein sequence Total score
identied coverage (%)d

1 gi|76678688 PREDICTED: similar to immunoglobulin gamma-1 chain C region, 45,936 5.25 14 38 692
membrane-bound form
gi|27806775 Xanthine dehydrogenase 148,885 8.06 2 1 77
2, 3 gi|29135265 Transferrin 79,870 6.75 2046 2454 9512702
gi|32189338 Polymeric immunoglobulin receptor 83,711 7.07 727 927 2841687
4 gi|30794280 Albumin 69,278 5.82 40 33 2224
5 gi|76678688 PREDICTED: similar to immunoglobulin gamma-1 chain C region, 45,138 5.25 12 28 567
membrane-bound form
gi|30794280 Albumin 69,278 5.82 8 12 415
gi|76681914 PREDICTED: similar to immunoglobulin alpha-2 chain C region, partial 40,049 5.00 4 15 270
6 gi|27806941 Serine (or cysteine) proteinase inhibitor, clade A (alpha-1-antiproteinase, 46,417 6.05 10 22 587
antitrypsin), member 1f
gi|76613612 PREDICTED: similar to vitamin D binding protein precursor (DBP) (group- 50,360 5.61 11 22 548
specic component)
gi|76681914 PREDICTED: similar to immunoglobulin alpha-2 chain C region, partial 40,049 5.00 8 31 444
gi|76647789 PREDICTED: similar to alpha-1-antichymotrypsin precursor 46,429 6.29 7 18 333
gi|76647977 PREDICTED: similar to antithrombin III precursor (ATIII) isoform 1 52,920 6.38 5 16 267
gi|76635535 PREDICTED: similar to nucleobinding 2 precursor (DNA binding protein 49,498 5.21 4 10 237
NEFA) (gastric cancer antigen Z)
7, 7a gi|41386760 CD14 antigen 40,283 5.37 913 1724 547849
gi|30794280 Albumin 69,278 5.82 923 1734 5061239
8 gi|76678688 PREDICTED: similar to immunoglobulin gamma-1 chain C region, 45,936 5.25 15 43 741
membrane-bound form
8a gi|30794280 Albumin 69,278 5.82 19 25 1025
gi|41386719 Milk fat globule-EGF factor 8 protein 48,611 6.81 18 34 955
gi|76678688 PREDICTED: similar to immunoglobulin gamma-1 chain C region, 45,936 5.25 5 13 228
membrane-bound form
gi|30794318 Isocitrate dehydrogenase 1 (NADP+) 47,098 6.31 2 7 95
9 No spot
ARTICLE IN PRESS

10 gi|7665408 PREDICTED: similar to zinc-alpha-2-glycoprotein precursor (Zn-alpha-2- 34,059 5.13 4 18 166

glycoprotein) (Zn-alpha-2-GP)g
11 gi|27806907 Clusterin 51,651 5.73 6 13 306
12 gi|30794280 Albumin 69,278 5.82 15 23 823
13 gi|30794280 Albumin 71,274 5.82 5 9 245
14 gi|30794280 Albumin 71,274 5.82 16 28 903
B.Y. Fong et al. / International Dairy Journal 18 (2008) 2346

gi|76678688 PREDICTED: similar to immunoglobulin gamma-1 chain C region, 45,936 5.25 4 10 169
membrane-bound form
15 gi|27806789 Transthyretin (prealbumin, amyloidosis type I) 15,831 5.91 5 30 274
16, 17 gi|30794280 Albumin 71,274 5.82 46 68 201258
18 & 18a gi|30794348 Casein alpha-S1 24,570 4.98 7 28 332
gi|76693955 PREDICTED: similar to alpha-2u globulin PGCL4, partial 12,109 4.57 4 31 168
19 gi|27881412 Casein kappa 21,370 6.30 5 24 255
gi|30794310 Casein beta 25,139 5.14 2 10 147
gi|27807339 Glycosylation-dependent cell adhesion molecule 1e 17,198 6.21 5 26 222
gi|30794348 Casein alpha-S1 24,570 4.98 3 17 157
20 gi|27881412 Casein kappa 21,370 6.30 5 24 301
gi|27807339 Glycosylation-dependent cell adhesion molecule 1e 17,198 6.21 11 37 470
gi|30794310 Casein beta 25,139 5.14 4 18 220
gi|30794348 Casein alpha-S1 24,570 4.98 3 17 133
21, 22, 23 gi|27807339 Glycosylation-dependent cell adhesion molecule 1e 17,198 6.21 412 1943 135579
gi|27881412 Casein kappa 21,370 6.30 67 2425 304388
gi|30794348 Casein alpha-S1 24,570 4.98 23 1017 109156
41
 42
Table 4 (continued )

Spot no.a Accession no.b Name Molecular weightc Theoretical pIc No. of peptide Protein sequence Total score
identied coverage (%)d

24, 25 gi|27881412 Casein kappa 21,370 6.30 610 2324 332391

gi|30794310 Casein beta 25,139 5.14 47 1718 162300
gi|76639444 PREDICTED: similar to immunoglobulin lambda-like polypeptide 1 21,338 8.89 23 17 91143
precursor (immunoglobulin-related 14)
gi|30794348 Casein alpha-S1 24,570 4.98 24 1015 81180
26 gi|27881412 Casein kappa 21,370 6.30 4 20 242
gi|76611322 PREDICTED: similar to 14-3-3 protein sigma (stratin) (epithelial cell 27,946 4.65 3 12 159
marker protein 1) isoform 1
gi|30794348 Casein alpha-S1 24,570 4.98 3 11 150
gi|30794310 Casein beta 25,139 5.14 2 8 49
gi|27819614 Actin, alpha 1, skeletal muscle 42,451 5.31 2 6 98
28, 29, 31, gi|27881412 Casein kappa 21,370 6.30 23 914 74187
33, 43 & gi|30794348 Casein alpha-S1 24,570 4.98 25 1018 90231
46 gi|30794310 Casein beta 25,139 5.14 25 714 84233
30, 32, 39, 68 & 72 gi|30794348 Casein alpha-S1 24,570 4.98 26 1025 116279
34 gi|27806881 NiemannPick disease, type C2h 16,972 8.20 15 55 686
35 gi|30794348 Casein alpha-S1 24,570 4.98 3 11 162
37 gi|30794348 Casein alpha-S1 24,570 4.98 2 10 39
gi|27881412 Casein kappa 21,370 6.30 2 14 79
gi|27806963 Casein alpha-S2 26,173 8.54 2 8 74
gi|30794310 Casein beta 25,139 5.14 3 8 87
39, 51 & 52 gi|27805809 Fatty acid binding protein (heart) like 14,827 6.73 312 1052 195697
40, 42, 47 gi|30794348 Casein alpha-S1 24,570 4.98 25 1020 100210
gi|27881412 Casein kappa 21,370 6.30 23 14 84173
41, 53, 55 & 58 gi|30794310 Casein beta 25,139 5.14 37 1417 74303
45 gi|27806861 Lactoglobulin, beta 20,307 4.85 5 24 273
gi|30794310 Casein beta 25,139 5.14 4 8 133
50 gi|27806789 Transthyretin (prealbumin, amyloidosis type I) 15,831 5.91 8 62 435
59 gi|27806861 Lactoglobulin, beta 20,307 4.85 3 14 138
gi|30794348 Casein alpha-S1 24,570 4.98 5 16 167
63, 64 gi|27806861 Lactoglobulin, beta 20,307 4.85 24 726 110250
ARTICLE IN PRESS

65 gi|41386683 Beta-2-microglobulin 13,782 7.79 4 18 4

67 gi|61820389 PREDICTED: similar to alpha-2u globulin PGCL4 isoform 1 20,927 4.55 2 8 92
66, 6971, 73 & 74 gi|30794348 Casein alpha-S1 24,570 4.98 312 1139 151609
gi|27806963 Casein alpha-S2 26,173 8.54 24 1019 111198
27, 36, 44, 48, 49, No identication
54, 56, 57, 60, 61,
B.Y. Fong et al. / International Dairy Journal 18 (2008) 2346

62

a
Refer to Fig. 5 for spot numbers.
b
Unless specied, the accession numbers of the identied proteins were obtained from the NBCI bovine genomic database.
c
The molecular weight and theoretical pI of the proteins as identied from the NCBI genomic database. These values do not necessarily reect those observed on the 2-D gel.
d
The protein sequence coverage determined from the sequences identied from the NBCI genomic database.
e
Glycosylation-dependent cell adhesion molecule 1; also identied as lactophorin precursor (28 kDa milk glycoprotein, PP3) (P80195) in the Swiss-Prot database.
f
Serine (or cysteine) proteinase inhibitor, clade A (alpha-1-antiproteinase, antitrypsin), member 3; also identied as endopin-1 precursor (muscle endopin-1a, mEndopin 1) (Q9TTE1) in the Swiss-Prot
database.
g
Spot 10, PREDICTED: similar to zinc-alpha-2-glycoprotein precursor; also identied as hypothetical protein LOC508800 (gi|77735615) from the NCBI expressed+EST bovine database.
h
Spot 34, Niemann-Pick disease, type C2 (gi|27806881); also identied as epididymal secretory protein E1 precursor (P79345) in the Swiss-Prot database.
 ARTICLE IN PRESS
B.Y. Fong et al. / International Dairy Journal 18 (2008) 2346
3.4. CX-BT
This fraction (Fig. 5A, Table 4), which contained the
proteins that were not bound to either ion exchangers, was
composed principally of the major whey proteins b-Lg and
a-Lac (indicated on gel), with BSA (spot 4) and Igs (spot 8)
at the next level of abundance. Both silver-stained (Fig. 5A)
and CBB-stained (Fig. 5B) gels are shown because silver
stain is non-linear (Miller, Crawford, & Gianazza, 2006)
and, in this case, gives a false impression of relative casein
protein abundance. Although virtually all the b-Lg, a-Lac
and BSA present in the original whey was concentrated
into the CX-BT, the Igs were partitioned between the 0.4 M
basic fraction and the CX-BT. This partitioning can be
accounted for, at least in part, by the extremely hetero-
geneous nature of the Igs, not only in terms of types but
also in terms of pIs, which range from 5.5 to 8.3 (Hurley,
2003). Comparison of the Ig bands identied in both the
0.4 M basic fraction and the CX-BT suggests that, allowing
for some overlap, the more basic species were retained on
the cation exchanger whereas the more neutral species
remained unadsorbed and appeared in the CX-BT. Both
Igs and BSA appeared in various other spots/bands at
either higher or lower Mr. Caseins constituted a variable
component of the CX-BT and aS1-casein appeared in
signicant amounts in the CX-BT illustrated, probably due
to a greater level of soluble casein in the original sample.
Casein and fragments were in fact ubiquitous, being
identied in virtually all the minor spots throughout the
gel. Transferrin was identied only in the CX-BT and in a
double string of spots similar to those identied previously
in bovine colostrum and mature milk (Yamada et al., 2002)
and also in wallaby whey and human milk (Goldfarb et al.,
1989; Molloy, Herbert, Yan, Williams, & Gooley, 1997).
Lactoferrin and transferrin both belong to the same family
of iron binding proteins but, whereas lactoferrin is
expressed by the mammary gland, transferrin, which has
a lower pI than lactoferrin, is thought to originate from the
blood where it is in signicant concentration and involved
principally in iron transport (Rainard, Poutrel, & Cafn,
1982). Lactophorin was present in a pattern of spots (spots
1923) co-localised in part with various caseins while b-Lg
fragments were found in several minor spots in the low Mr
region of the gel also co-localised in part with casein
fragments. The MFGM proteins xanthine oxidase and
milk fat globule-EGF factor 8 protein were again identied
in trace amounts, and fatty acid binding protein, a fatty
acid transport protein also usually associated with the
MFGM (Spitsberg & Gorewit, 1997), was found in several
spots (spots 39, 51, 52) at an Mr and a pI similar to those
identied in human colostral and bovine MFGM
(Fortunato et al., 2003; Fong et al., 2007). Serine (cysteine)
proteinase inhibitor clade A, antithrombin III, alpha-1-
antichymotrypsin, vitamin D binding protein (all found in
other fractions) and nucleobindin 2 were identied in a
high Mr cluster (spot 6) along with Ig. Interestingly,
nucleobindin 1, which has a predicted pI slightly lower
43
than that of nucleobindin 2, was identied in the 1 M acidic
fraction. Other proteins found largely or solely in the
CX-BT in this study were actin alpha 1; CD14 antigen, a
protein released from the surface of neutrophils (Paape
et al., 2003); the metabolic enzyme isocitrate dehydrogenase,
expressed by the mammary gland and modulated by
regulators of mammary gland differentiation (Liu, Capuco,
& Romagnolo, 2006); zinc-alpha-2-glycoprotein, involved
in the stimulation of lipid degradation; clusterin, a
ubiquitous protein implicated in programmed cell death
and the expression of which is upregulated at sites
undergoing tissue remodelling in parallel with apoptosis
(Jenne & Tshopp, 1992); the thyroid hormone binding
protein transthyretin; 14-3-3 protein sigma (stratin), a
signal transduction protein; epididymal secretory protein,
expressed in the mammary gland; and alpha-2u globulin,
which has extensive homology with lipid binding proteins.
Both alpha-2u globulin and stratin are associated with
keratinocytes (Mancini, Majumdar, Chatterjee, & Roy,
1989; Dellambra et al., 1995), raising the possibility that
the origin of these bovine proteins in milk might have been
epidermal, much as for keratin. However, 14-3-3 proteins
(gamma and beta alpha) have also been identied recently
in bovine MFGM (Reinhardt & Lippolis, 2006).
Beta-2-microglobulin, a major histocompatibility com-
plex class 1 protein, was identied in an isolated location
(spot 65) that corresponded closely with its nominal pI
(7.96) and mass (11.6 kDa). Given the very basic nature of
this protein, it is interesting that it was voided from the
cation exchanger into the CX-BT. However, it is possible
(as with other proteins) that chromatographic fractiona-
tions, especially of the very minor proteins, were inuenced
by the abundance of other proteins.
3.5. General discussion
For the most part, the 2-DE proles showed separation
of proteins by ion exchange according to nominal pI; thus,
the bulk of the proteins in the four fractions were
partitioned according to their acidic or basic nature and
were located in the expected Mr/pI region of the gel. The
appearance of some proteins in several fractions, or in an
unexpected fraction, can be explained by partial or residual
afnities for the ion exchangers, isoform distribution,
interaction with other proteins and cleavage product
distribution. The dispersal of proteins through several
fractions has implications for the production of discrete
bioactive fractions, especially where high potency compo-
nents are concerned since even residual amounts may
contribute bioactivity. Furthermore, the matrix in which
the bioactive compound occurs may modify the activity.
Additionally, specic bioactivities may be encrypted or
explicit depending on the fraction. For example, kininogen
(observed Mr 6065 kDa) was identied in the 1 M acidic
fraction in this study, whereas a 17-kDa fragment of
kininogen was identied by others in a total milk basic
fraction (Yamamura, Takada, Goto, Kumegawa, & Aoe,
 ARTICLE IN PRESS
44 B.Y. Fong et al. / International Dairy Journal 18 (2008) 2346
2000). The fragment, which occurs systemically as a
kallikrein cleavage product, has osteoblastic proliferative
activity whereas kininogen is apparently inactive in this
respect (Yamamura et al., 2000).
The milk samples used in this study were fresh samples
from cows in late lactation. It can be expected that some
variations in proteomic proles and abundances will occur
due to phenotype, stage of lactation and degree of
proteolysis by indigenous milk enzymes (Wheeler et al.,
1997). The last especially applies to caseins and the
appearance of casein peptides in the milk In this respect,
the levels of b-casein fragments in milk have been reported
to increase signicantly during the lactation period from
colostrum to mature milk (Yamada et al., 2002). It might
be expected also that proteins involved in tissue remodel-
ling, such as heparanase, chitinase and osteopontin might
become more prevalent as the mammary gland prepares for
involution. The condition of the mammary gland can also
inuence the protein composition; an increase in proteins
of blood serum origin, including serotransferrin and BSA,
has been observed in the whey from cows with clinical
mastitis (Hogarth et al., 2004). As noted earlier, proteins
can also be derived from the somatic cells present in milk.
Determination of mammary gland protein expression
should allow some differentiation between those proteins
expressed and secreted by mammary tissue and those
derived adventitiously from blood or somatic cells. It may
be that proteins that are manifest in whey at low copy
number in fact represent the somatic (or even epidermal)
cell proteome rather than the mammary expressed pro-
teome. The origin and role of these minor proteins
occurring at such low levels in milk, and with such diverse
functions, remains to be determined but is intriguing.
A number of the minor proteins identied in this study
have been observed previously in both bovine milk and
colostrum by 2-D-gel-based proteomic methods (ODon-
nell, Holland, Deeth, & Alewood, 2004; Yamada et al.,
2002). Generally, the proteins were observed at similar loci
and patterns of distribution. Several proteins, which were
reported to be present only in the colostrum of bovine
milk, gelsolin, chitinase 3-like 1, complement component
C3, brinogen, apolipoprotein H (Yamada et al., 2002),
have now been identied in this study in late-lactation
milk. Additionally, this study documents a number of
proteins that have not been found previously in bovine
milk by proteomic studies, e.g. factor XIIa inhibitor,
tetranectin, CD5 antigen-like, complement components
C4, C8, C9, factors I and D, nucleobindins, among others.
Many of the proteins identied in bovine whey have been
found in human colostrum and mature milk (Murakami
et al., 1998; Palmer et al., 2006), indicating that there might
be commonalities in proles of minor proteins between
species. Interestingly, some of the minor whey proteins
identied in this study, although not typically associated
with the MFGM, have been found in MFGM preparations
from both human milk and bovine milk (Fong et al., 2007;
Fortunato et al., 2003; Quaranta et al., 2001), e.g.,
clusterin, folate binding protein, actin, brinogen. This
might have been due to contamination of the MFGM
preparations with whey proteins (Reinhardt & Lippolis,
2006) or, alternatively, these proteins might be an intrinsic
part of the MFGM and have a dened role within it. Some
proteins may well be dispersed through several phases, as is
the case for OPN, which is associated with both casein
micelles and the whey (Azuma et al., 2006).
As a nal comment, the stringency of the MS protein
validation criteria used in this study precluded some proteins,
known to occur in bovine whey, from assignment in the
tables. For example, transcobalamin-2, a vitamin B12 binding
protein, could only be tentatively assigned on the basis of a
single peptide identication (15 amino acids, top 10 ions
matched) in each of two spots (10a, 10c, 1.5 M basic fraction).
However, it is known to occur in bovine whey (Fedosov,
Petersen, & Nexo, 1996) and, given its high theoretical
basicity and mass (pI 9.22, 46 kDa) combined with reported
cation exchange behaviour, it is likely to occur in this fraction
and to migrate on 2-DE in the region identied.
4. Conclusion
Fractionation of bovine whey using simple ion-ex-
change-based methodology allowed effective display and
identication by 2-DE MS of proteins partitioned into
characteristic acidic and basic fractions, resulting in a more
comprehensive map of the whey proteome than previously
achieved. The merit in such an approach lies in the
relatively rapid visualisation of protein distributions and
isoforms from which some qualitative assessment of the
whey proteome can be made. Such proteomic display may
well be useful in the design of strategies for purication of
select proteins or protein fractions containing targeted
bioactivities. Observations from this study, combined with
data on the bovine whey proteome to date, collectively add
to an emerging inventory of whey proteins, in particular
the minor protein components, and the opportunities for
capture of bioactivities encoded within.
Acknowledgements
This work was funded by the LactoPharma Consortium,
a joint venture between the Foundation for Research,
Science and Technology (NZ) and Fonterra Co-operative
Group Limited. We are grateful to Deborah Husband,
Kerianne Higgs and David Elgar for assistance and advice,
and to SallyAnne Turner (Dexcel Limited, New Zealand)
and Mark Leslie for the provision of milk samples.
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