International Dairy Journal 35 (2014) 15e20

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International Dairy Journal

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Animal species milk identication by comparison of two-dimensional

gel map prole and mass spectrometry approach
Yongxin Yang a, b,1, Nan Zheng a,1, Jinhui Yang a, Dengpan Bu a, Jiaqi Wang a, *, Lu Ma a,
Peng Sun a
a
State Key Laboratory of Animal Nutrition, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China
b
Institute of Animal Science and Veterinary Medicine, Anhui Academy of Agricultural Sciences, Hefei 230031, China

a r t i c l e i n f o a b s t r a c t

Article history: The aim of the current study was to present the primary protein prole of cow, goat, camel, yak and
Received 29 May 2013 buffalo milk, along with binary mixtures of these milks through two-dimensional gel electrophoresis
Received in revised form coupled with mass spectrometry for detection of specic milks in mixtures. Distributions of a-lactal-
21 September 2013
bumin and/or b-lactoglobulin spots on gel maps were used to detect goat, camel, yak and buffalo milk
Accepted 23 September 2013
adulterated with cow milk. Appearance of a-lactalbumin and b-lactoglobulin protein spots were helpful
for detection of camel, yak and buffalo milk adulteration with goat milk. aS1-Casein from cow and goat
milk was also used to determine camel milk adulteration. In particular, b-lactoglobulin from cow, goat,
yak and buffalo milk, and a-lactalbumin from camel milk were useful to detect adulteration of specic
milk mixtures at levels as low as 0.5%. These results highlight applicability of this method for charac-
terisation of milk proteome and detection of specic milk in mixtures.
2013 Elsevier Ltd. All rights reserved.

1. Introduction (Kasemsumran, Thanapase, & Kiatsoonthon, 2007; Nicolaou, Xu, &

Variation in environmental conditions and the natural selection
process have promoted diversication of animal species and
breeds, which has resulted in differing milk composition and pro-
duction (Murgiano, DAlessandro, Zolla, Valentini, & Pariset, 2012).
Milk from minor dairy species, such as goat, buffalo, yak and camel,
has high nutritional and economical value (Medhammar et al.,
2012). For nancial prot, higher priced milk (such as yak and
camel milk) substituted by lower priced milk (such as cow milk)
has been found in dairy products. To protect consumer benets and
product authenticity, it has been necessary to develop analytical
procedures able to detect fraudulent substitution.
The development of analytical techniques, such as capillary
electrophoresis (Herrero-Martnez, Sim-Alfonso, Ramis-Ramos,
Gel, & Righetti, 2000), polymerase chain reaction (Bai et al.,
2009; Guerreiro, Fernandes, & Bardsley, 2012), chromatography
(Bramanti, Sortino, Onor, Beni, & Raspi, 2003; Cordeiro, Bordin,
Rodriguez, & Hart, 2001), enzyme-linked immunosorbent assay
(Hurley, Coleman, Ireland, & Williams, 2004), infrared spectroscopy
* Corresponding author. Tel.: 86 10 62890458.
E-mail address: jqwangcaas@gmail.com (J. Wang).
1
These authors contributed equally to this work.
Goodacre, 2010), as well as nuclear magnetic resonance (Lamanna,
Braca, Di Paolo, & Imparato, 2011), has enabled their use to evaluate
content of mixtures of milk of different species. Recently, mass
spectrometry methods have been developed for investigating milk
adulteration. In particular, liquid chromatographyetandem mass
spectrometry (Chen, Chang, Chung, Lee, & Ling, 2004; MacMahon,
Begley, Diachenko, & Stromgren, 2012) and matrix-assisted laser
desorption/ionisation time-of-ight mass spectrometry (MALDIe
TOF MS) (Calvano, De Ceglie, Monopoli, & Zambonin, 2012;
Cozzolino et al., 2001; Nicolaou, Xu, & Goodacre, 2011) have been
utilised to improve milk product analysis for quantication of
specic proteins.
Two-dimensional gel electrophoresis (2-DE) is a powerful
technique used to acquire protein proles and allow protein spots
to be visualised on gels. This method resolves proteins into spots,
and the map of protein spots can be considered as the protein
prole of a specic sample (Yarmush & Jayaraman, 2002). Thus, 2-
DE maps of milk were constructed and used to characterise the
protein prole of different mammalian milk (DAuria et al., 2005;
Hinz, OConnor, Huppertz, Ross, & Kelly, 2012). Roncada et al.
(2012) reviewed the most recent progress in characterisation of
the milk proteome that allows the milk proteome of farm animal
species to be easily distinguished. However, literature data have
hardly been concerned with changes of protein prole on gel maps

0958-6946/$ e see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.idairyj.2013.09.008
16 Y. Yang et al. / International Dairy Journal 35 (2014) 15e20
from different animal species. We hypothesised that milk mixtures
from different animal species would result in alteration of distri-
bution of protein spots on the 2-DE gels. Thus, 2-DE coupled with
MALDIeTOF MS was used to investigate detection markers and
distinguish milk authentication including goat, yak, buffalo and
camel milk adulteration with cow milk, as well as yak, buffalo and
camel milk adulteration with goat milk.
2. Materials and methods
2.1. Sample preparation
Thirty cow milk samples were collected from a dairy farm in
Beijing city; 27 goat milk samples collected from a farm in Shanxi
province; 21 camel milk samples collected from a farm in Urumchi
city in Xinjiang; 30 yak milk samples collected from a farm in
Qinghai province; and 21 buffalo milk samples collected from a
farm in Yunnan province. These raw milk samples were collected
from animals free of disease, as based on veterinarian records.
Milk samples from each farm were pooled for three fractions.
Binary mixtures were prepared as in Table 1. Different milk types
were pooled according to volume ratios and mixed by vortexing for
5 min. Then 2 mL milk samples were centrifuged at 3000  g for
10 min at 4  C to separate the skimmed milk and fat layer. Skimmed
milk, as milk protein, was recovered from each sample and
centrifuged again at 100,000  g for 1 h at 4  C to recover the su-
pernatant. Concentration of protein was determined by the modi-
ed Bradford method with bovine serum albumin as a standard
(Bio-Rad, Hercules, CA, USA).
2.2. Two-dimensional gel electrophoresis separation
Two hundred and 50 mg of protein of skimmed milks or ultra-
centrifugal supernatants were mixed in 350 mL immobilised
pH gradient (IPG) rehydration buffer comprising 8 M urea, 2 M
thiourea, 4% (w/v) 3-[(3-cholamidopropyl) dimethylammonio]-1-
propanesulfonate, 0.5% (v/v) pH 4e7 IPG buffer and trace bromo-
phenol blue. First dimensional isoelectric focussing was carried out
using 17 cm pH 4e7 IPG strips (Bio-Rad) at 20  C. The IPG strips
were rehydrated for 12 h and then taken through a series of
focussing. They were focused at 0e500 V for 1 h, 500e1000 V for
1 h, 1000e9000 V for 5 h, and then 9000 V for 80,000 Vh.
After being focused, IPG strips were equilibrated with 2% (w/v)
dithiothreitol, 50 mM TriseHCl pH 8.8, 6 M urea, 30% (v/v) glycerol,
and 2% (w/v) sodium dodecyl sulphate at room temperature for
12 min followed by incubation with 2.5% (w/v) iodoacetamide,
50 mM TriseHCl pH 8.8, 6 M urea, 30% (v/v) glycerol, and 2% (w/v)
Table 1
List of milk binary mixtures of cow, goat, camel, yak and buffalo milk.
Milk binary mixtures Volume ratio of specic milk mixtures
Cow milk adulterated with goat milk 95:5 98:2
Cow milk adulterated with camel milk 95:5 98:2
Cow milk adulterated with yak milk 95:5 98:2
Cow milk adulterated with buffalo milk 95:5 98:2
Goat milk adulterated with cow milk 95:5 98:2 99:1
Camel milk adulterated with cow milk 95:5 98:2 99:1
Yak milk adulterated with cow milk 95:5 98:2 99:1
Buffalo milk adulterated with cow milk 95:5 98:2 99:1
Goat milk adulterated with camel milk 95:5 98:2 99:1
Goat milk adulterated with yak milk 95:5 98:2 99:1
Goat milk adulterated with buffalo milk 95:5 98:2 99:1
Camel milk adulterated with goat milk 95:5 98:2 99:1
Yak milk adulterated with goat milk 95:5 98:2 99:1
Buffalo milk adulterated with goat milk 95:5 98:2 99:1
sodium dodecyl sulphate at room temperature for another 12 min.
Subsequently, strips were transferred onto gels and sealed with
0.5% (w/v) low melting point agarose and separated in a second
dimension with 12% polyacrylamide gels.
After electrophoresis, gels were stained with Coomassie Brilliant
Blue G-250 solution, as described by Candiano et al. (2004). Gel
images were analysed using ImageMaster 5.0 software (GE
Healthcare, Piscataway, NJ, USA). Each pooled sample was repeated
3 times. For comparison of relative abundance of proteins among
gels, protein spots were automatically detected and manually
conrmed with all or none as the determining criterion that
protein spot was only detected in milk binary mixtures compared
with control milk.
2.3. In-gel digestion
Selected protein spots were excised manually from the gels and
washed twice with MilliQ water for 15 min. Then, gel piece was
washed with 50% (v/v) acetonitrile and destained with 50% (v/v)
acetonitrile in 0.1 M ammonium bicarbonate for 15 min and then
dried to completeness in pure acetonitrile. Subsequently, dried gel
pieces were rehydrated with a small volume of digestion buffer
(50 mM ammonium bicarbonate containing 0.01% (w/v) sequence-
grade trypsin) and then 20 mL 10 ng mL1 digestion buffer was
added to cover the gel piece. Finally, the gel piece was incubated at
37  C overnight and then digestion was terminated by addition of
2 mL of 5% (v/v) triuoroacetic acid.
2.4. Protein identication and database search
Extracted peptide mixtures were loaded onto one spot of a
MALDI target plate. Mass spectra and tandem mass spectra (MS/
MS) were obtained by using 4800 Plus MALDI TOF/TOF Analyzer
(Applied Biosystems, Foster City, CA, USA) with 355 nm Nd:YAG
(Neodymium-doped Yttrium Aluminium Garnet) laser, and at an
acceleration voltage of 20 kV. Positively charged ions were obtained
in the reector mode by delayed extraction. Peptide mass nger-
printing was gained in the mass range of 800e4000 Da. Eight
highest precursor ions with a signal-to-noise ratio of at least 50
from each sample were analysed using MS/MS mode with 2500
laser shots, and at collision energy of 20 keV.
Protein identication was performed with MASCOT
(Matrix Science) to search the NCBI nonredundant database (http://
www.ncbi.nlm.nih.gov). All peaks with a signal-to-noise ratio of at
least 15 were included in the search. Peptide mass ngerprinting
search parameters were set as follows: monoisotopic mass
accuracy < 100 ppm, allowance for one missed cleavage, carba-
midomethylation of cysteine as a xed modication, and methio-
nine oxidation as a variable modication. Fragment mass tolerance
and peptide mass tolerance were set to a 0.4 Da and 100 ppm,
respectively.
3. Results
In the present study, 2-DE gels were repeated 3 times for each
99.5:0.5 sample to detect reproducibility. Image analysis showed that these
99.5:0.5 2-DE maps were reproducible. To construct a 2-DE map, it was
99.5:0.5
important to have a representative sample. Thus, master gel maps
99.5:0.5
99.5:0.5 were constructed by comparing the 2-DE maps from the same
99.5:0.5 samples with ImageMaster 2-DE analysis software, and then mas-
99.5:0.5 ter gel maps were used to perform differential expression analysis.
99.5:0.5 Protein proles on the gel maps of cow, yak and buffalo milk were
99.5:0.5
99.5:0.5
similar; however, protein proles of goat and camel milk presented
obvious differences.
 Y. Yang et al. / International Dairy Journal 35 (2014) 15e20 17

Fig. 1. 2-DE maps and densitometric values of protein spots of milk proteins and milk binary mixtures. 2-DE maps are of (A) cow, goat, camel, yak and buffalo milk protein and of
binary mixtures including: (B) goat, camel, yak, and buffalo milk adulterated with cow milk; (D) cow milk adulterated with goat, camel, yak, and buffalo milk; (F) camel, yak, and
buffalo milk adulterated with goat milk; (H) binary mixtures including goat milk adulterated with camel, yak, and buffalo milk. Protein spots labelled with an arrow only detected in
milk binary mixtures compared with control milk. Densitometric values are of protein spots only detected in milk binary mixtures compared with: (C) goat, camel, yak, and buffalo
milk; (E) cow milk; (G) camel, yak and buffalo milk; (I) goat milk. Values are means  standard error (n 3).

The well-resolved and reproducible 2-DE maps of control milk
and minimum adulterated milk mixtures are displayed in Figs. 1
and 2. Protein spots were labelled that only were detected in
milk binary mixtures compared with control milk. Three pairs of
protein spots (co1 and co4, b1 and b2, g3 and g4) in gels were
identied as three different proteins. Data for all identied protein
spots are shown in Table 2, and in Figs. 1 and 2.
Goat and camel milk were adulterated with 2% (v/v) cow milk.
Protein spots of a-lactalbumin and b-lactoglobulin from cow milk
were only detected in gels of binary mixtures of above species. The
spot of aS1-casein from cow milk was also used to detect camel milk
adulterated with cow milk. Yak and buffalo milk were adulterated
with 2% (v/v) cow milk and then increased to 5% (v/v) cow milk for
sure detection. This resulted in protein spots of a-lactalbumin and/
or b-lactoglobulin from cow milk being detected in gels and these
served as molecular markers to detect yak and buffalo milk
adulteration.
In addition, cow milk was adulterated with 2% (v/v) goat, camel,
yak and buffalo milk, and protein spots from goat, camel, yak and
buffalo milk were detected on gels of binary mixtures. Distribution
of a-lactalbumin, b-lactoglobulin and two isoforms of aS2-casein
spots from goat milk was used to detect cow milk adulterated with
goat milk. Protein spots of aS1-casein and a-lactalbumin from camel
milk were used to detect cow milk adulterated with camel milk.
Protein spot of b-lactoglobulin isoform from yak milk was used to
detect cow milk adulterated with yak milk. Finally, distribution of
k-casein isoform spot from buffalo milk was used to detect cow
milk adulterated with buffalo milk.
For camel, yak and buffalo milk adulterated with 2% (v/v) goat
milk, protein spots of a-lactalbumin and b-lactoglobulin from goat
milk were present on gels of binary mixtures and served as mo-
lecular markers to detect camel, yak and buffalo milk adulteration
with goat milk. In addition, the protein spot of aS1-casein from goat
milk was used to detect camel milk adulterated with goat milk.
Spots of two isoforms of aS2-casein from goat milk were used to
detect yak and buffalo milk adulterated with goat milk. In goat milk
adulterated with 2% (v/v) camel, yak and buffalo milk, protein spots
from camel, yak and buffalo milk were detected in gels of binary
mixtures of above species. Protein spots of aS1-casein and a-lact-
albumin from camel milk were scattered on gel maps and used to
detect goat milk adulterated with camel milk. Distribution of a-
lactalbumin and b-lactoglobulin spots of yak milk were used to
detect goat milk adulterated with yak milk. In addition, the exis-
tence of a-lactalbumin, b-lactoglobulin and two isoforms of k-
casein spots from buffalo milk were used to detect goat milk
adulterated with buffalo milk.
Control milk and specic binary milk samples were ultra-
centrifuged and then separated by 2-DE. Goat and camel milk were
adulterated with 0.5% (v/v) cow milk; protein spots of b-lacto-
globulin from cow milk were detected on gels of binary mixtures of
above species and served as molecular markers to detect goat and
camel milk adulteration. Yak and buffalo milk were adulterated
18 Y. Yang et al. / International Dairy Journal 35 (2014) 15e20

Fig. 2. 2-DE maps and densitometric values of protein spots of whey from cow, goat, camel, yak, buffalo and milk binary mixtures with ultracentrifugation. 2-DE maps are of milk
whey from (A) cow, goat, camel, yak and buffalo milk with ultracentrifugation and from binary mixtures including: (B) goat, camel, yak, and buffalo milk adulterated with cow milk;
(D) camel, yak, and buffalo milk adulterated with goat milk; (F) goat milk adulterated with camel, yak, and buffalo milk. Protein spots labelled with an arrow only detected in milk
whey of binary mixtures compared with control milk whey. Densitometric values are of protein spots only detected in milk whey of binary mixtures compared with: (C) goat, camel,
yak, and buffalo milk whey; (E) goat milk whey; (G) camel, yak and buffalo milk whey. Values are means  standard error (n 3).

with 0.5% (v/v) and 1% (v/v) cow milk, protein spots from cow milk
were not detected. In addition, camel, yak and buffalo milk were
adulterated with 0.5% (v/v) goat milk; protein spots of b-lacto-
globulin from goat milk were detected in gels of binary mixtures of
above species. For the contrary scenario, goat milk was adulterated
with 0.5% (v/v) camel, yak and buffalo milk; protein spots of a-
lactalbumin from camel milk and b-lactoglobulin from yak and
buffalo milk were also detected in gels of binary mixtures of above
species.
4. Discussion
In the present study, we used a 2-DE approach to create protein
proles for cow, yak, buffalo, goat and camel milk and found that
distributions of protein spots of some caseins, a-lactalbumin, and/
or b-lactoglobulin on gel maps could be used to detect specic
milk adulteration. In particular, b-lactoglobulin from cow, goat,
yak and buffalo milk, and a-lactalbumin from camel milk allowed
detection of the adulteration of specic milk mixtures as low as
0.5%.
Previously, based on mass difference of a-lactalbumin and/or
b-lactoglobulin, MALDIeTOF MS was used to detect ewe and
buffalo milk adulterated with cow milk with detection limits
below 10% and 5%, respectively (Cozzolino et al., 2001). Accord-
ing to retention time and molecular mass of b-lactoglobulin from
cow milk, high-performance liquid chromatography with elec-
trospray ionisation mass spectrometry was used to detect goat
milk adulterated with cow milk at levels as low as 5% (Chen et al.,
2004). Also, b-lactoglobulin as a marker for detection of buffalo
milk adulterated with cow milk was demonstrated by liquid
chromatographyetandem mass spectrometry (Czerwenka,
Mu} ller, & Lindner, 2010). Subsequently, the ratio of the sum of
non-bovine b-lactoglobulins to total b-lactoglobulins in milk
mixtures were used to detect either ovine or caprine milk adul-
terated with bovine milk, and ovine or caprine milk added into
bovine milk by capillary electrophoresisemass spectrometry
(Mu} ller et al., 2008). Similarly, distributions of a-lactalbumin and
b-lactoglobulin on the native-polyacrylamide gels were linear
relationships with amounts of bovine milk that were used to
detect caprine and ovine milk adulterated with bovine milk
(Pesic et al., 2011). In agreement with previous studies, our re-
sults indicated that protein spots of b-lactoglobulin from cow
milk on 2-DE gels could serve as a molecular marker to detect
goat milk adulterated with cow milk at level of 0.5%.
However, few studies have investigated camel or yak milk
adulterated with cow and goat milk in regards to important
nutritional and health implications (Nikkhah, 2011). More inter-
estingly, Hinz et al. (2012) separated the primary proteins of cow,
caprine, buffalo, equine and camel milk by 2-DE and major proteins
were identied by mass spectrometry. Camel milk showed quite a
different protein pattern with the least number of protein spots in
the area of caseins (Hinz et al., 2012) and an absence of b-lacto-
globulin (Merin et al., 2001). a-Lactalbumin was the most abundant
whey protein in camel milk and, having 123 residues with a mo-
lecular mass of 14.6 kDa, exhibited a comparatively large difference
from other dairy animal milk (Hinz et al., 2012). Thus, the distri-
bution of a-lactalbumin and b-lactoglobulin of cow and goat milk
on gels served as the molecular markers to detect camel milk
adulteration with cow or goat milk. In addition, aS1-casein of cow
and goat milk on gels was also used to detect camel milk
 Y. Yang et al. / International Dairy Journal 35 (2014) 15e20 19

Table 2
Identication of protein spots only detected in milk binary mixtures compared with control milk on the gels by MALDIeTOF MS.

Protein spot Protein name Accession no. Molecular mass (Da)a Isoelectric pointa Score Number of matched peptides Coverage (%)

co1 b-Lactoglobulin gi: 520 20,307 4.85 228 4 37.64

co2 a-Lactalbumin gi: 68 14,603 4.80 193 6 46.34
co3 aS1-Casein gi: 30794348 24,570 4.98 238 6 35.05
co4 b-Lactoglobulin gi: 520 20,307 4.85 228 4 37.64
g1 b-lactoglobulin gi: 147744630 19,098 5.54 268 4 47.93
g2 a-Lactalbumin gi:125998 16,700 5.06 247 4 28.87
g3 aS2-Casein gi: 416751 26,543 8.24 246 4 18.83
g4 aS2-Casein gi: 416751 26,543 8.24 159 2 9.87
g5 aS1-Casein gi: 311943 24,215 5.19 121 7 41.12
ca1 a-Lactalbumin gi: 125997 14,877 5.00 122 2 14.63
ca2 aS1-Casein gi: 3860335 25,884 5.00 57 2 12.16
y1 b-Lactoglobulin gi: 373159006 18,598 6.20 74 4 28.92
y2 a-Lactalbumin gi: 354832173 16,646 4.93 179 5 27.46
b1 k-Casein gi: 158148601 21,510 6.84 106 3 22.11
b2 k-Casein gi: 158148601 21,510 6.84 106 3 22.11
b3 b-Lactoglobulin gi: 20178290 20,409 4.93 410 7 56.67
b4 a-Lactalbumin gi: 6289063 16,750 4.80 179 5 27.46
a
Molecular mass and isoelectric point derived from the database.

adulteration with cow or goat milk due to molecular weight and
isoelectric point of aS1-casein for both cow and goat being different
from camel milk.
Yak, classied as Bos grunniens or Poephagus grunniens, belong to
the Bovinae subfamily. Previous studies compared the relative per-
centage of milk proteins between yak and cow by sodium dodecyl
sulphate-polyacrylamide gels. Abundance of casein, b-lactoglobulin,
immunoglobulins and lactoferrin was not signicantly different,
while abundance of a-lactalbumin was signicantly lower in yak
milk compared with bovine milk (Sheng, Li, Alam, Fang, & Guo,
2008). Other research also found that the relative percentage of a-
lactalbumin was lower in yak milk, while b-lactoglobulin in yak milk
was higher than in Chinese Holstein cow milk (Mao et al., 2004). In
particular, previous studies demonstrated that b-lactoglobulin
variant was absent in yak milk (Dorji, Namikawa, Mannen, &
Kawamoto, 2010; Xuebin & Jianlin, 2002). Thus, the distribution of
b-lactoglobulin variant spot on gel maps may be used to detect yak
milk adulterated with cow milk. Similarly, distribution of a-lactal-
bumin and b-lactoglobulin of goat milk on gels was used to detect
yak milk adulterated with goat milk. For the reverse, a-lactalbumin
and b-lactoglobulin of yak milk on gels was used to detect goat milk
adulterated with yak milk. Our results demonstrated that 2-DE
combined with MALDIeTOF MS may help to detect the binary
mixtures of different animal species milk.
5. Conclusions
The current study presented the primary protein proles of cow,
goat, camel, yak and buffalo milk, along with binary mixtures of
milk from the above species by 2-DE maps. Gel maps also high-
lighted obvious interspecies differences useful for detection of a
specic milk species in a milk mixture. Distribution of one or more
isoform protein spots, including a-lactalbumin, b-lactoglobulin,
aS1-casein, aS2-casein and k-casein, could serve as molecular
markers to detect adulteration of specic milk samples. In partic-
ular, b-lactoglobulin from cow, goat, yak and buffalo milk, and a-
lactalbumin from camel milk would be useful as molecular markers
for detection of adulteration in some specic milk mixtures as low
as 0.5%.
Acknowledgements
The project was supported by the National Key Basic Research
Program of China (Project No. 2011CB100805).
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