Available online at http://journal-of-agroalimentary.ro
Journal of Agroalimentary Processes and
Technologies 2010, 16 (2), 169-176
Journal of
Agroalimentary Processes and
Technologies
Two-Dimensional gel electrophoresis of peptides as lap in
bovine milk
Ramona Suharoschi, Bartalici Corina-Irina*, Petrut Raul, Semeniuc A. Cristina,
Rotar M. Ancuţa
University of Agricultural Sciences and Veterinary Medicine of Cluj-Napoca, Faculty of Agriculture,
400372, Manastur Street, no.3-5, Cluj-Napoca, Romania
Received: 1 April 2010; Accepted: 10 May 2010
______________________________________________________________________________________
Abstract
SDS-PAGE (sodium dodecyl sulphate – polyacrylamide gel electrophoresis) was used as preliminary
analysis of whey proteins of raw, commercial pasteurized and commercial ultra-high temperature (UHT)
milks. Four whey proteins bands, including β-lactoglobulin variants (β-LG A and B), could be distinctively
separated in the gel. The results showed that levels of the major whey proteins were reduced in the
pasteurized milks and UHT milks as compared with raw milk. Peptides of bovine milk were separated
further on by two-dimensional gel electrophoresis (2-D electrophoresis, 2-DE). The peptides were
separated in the pH range 3-10 in the first dimension, while 12%T homogenous gels were used in the
second dimension. 2-D electrophoresis analysis of UHT milk sample showed a decrease in the number of
proteins and peptides.
Keywords: cow milk, heat treatment, SDS-PAGE, whey proteins, 2-D electrophoresis (two dimensional gel
electrophoresis)
______________________________________________________________________________________
1. Introduction
The Milk proteins have been studied continuously
for over five decades. Knowledge of this complex
protein system has evolved incrementally in recent
years, largely coinciding with advances in
technology. Heat treatments are usually used to
obtain bacteriological safe milk and to ensure a
long shelf life. Pasteurized and UHT treatments of
whey proteins has been explored by many groups
using a wide range of techniques. Proteomics and
associated technologies have the potential to
facilitate further advances in our knowledge of
milk proteins.
Proteomics allows for the detection, identification
and characterization of milk proteins. More
importantly, proteomics facilitates the analysis of
large numbers of milk proteins simultaneously.
Milk proteins have been studied in depth for well
over 50 years.
_______________________________________________
Corresponding author: e-mail: corina_bac@yahoo.com
Despite a plethora of in-depth studies analysing the
various milk protein components, many questions
concerning milk protein expression, structure and
modifications remain unanswered. The details of
protein interactions and protein modifications are
also poorly understood. Given high levels of
evolutionary divergence [1,2]. the presence of
numerous genetic variants and post-translational
modifications (PTMs) make the milk proteome
extremely complex. PTMs such as glycosylation,
phosphorylation, disulphide bond formation and
proteolysis have the potential to create a large
number of different protein variants from a single
gene product. Nowadays, various systems and
technologies associated with different time–
temperature profiles have been developed to obtain
pasteurized and Ultra-High Temperature (UHT) milk,
and the effect on the nutritional and sensory quality
of commercial milk samples may vary substantially
depending on the process [3,4].
Ramona Suharoschi et. al. / Journal of Agroalimentary Processes and Technologies 2010, 16(2)
For regulatory purposes, attention should be paid
to evaluate the authentication of heat-treated milks
and milk integrity since heat treatments change the
biochemical composition of milk. Denaturation of
whey proteins in milks is regarded as a good
indicator of thermal damage.
Proteins can be separated by many techniques of
which the most common are various types of
chromatography and electrophoresis. As a classic
separation method, with the help of advanced
scanner and image analysis patterns, the
electrophoresis method has resulted in precise
quantification, good separations and high
resolutions, leading to accurate and reproducible
results. However, little attention has been paid to
the application of SDS-PAGE polyacrylamide gel
electrophoresis in protein quantification, by which
proteins are separated under non-reducing
conditions[5-8].
Two-dimensional gel electrophoresis (2-DE) was
first described by O’Farrell in 1975 [9]. Research
on proteins using 2-DE has developed during
recent years. Some studies have been performed to
evaluate the use of 2-DE in proteomics. Ong and
Pandey (2001) and Rabilloud (2002) [10,11]
outlined the areas where 2-DE can best be utilised
as a separation method in proteomic strategies.
Rabilloud highlighted the strengths and
weaknesses of proteomics approaches, such as
peptide separation, protein recognition and
selection on dedicated arrays, as well as 2-DE.
When using 2-DE, proteins are separated
according to their isoelectric point along a pH
gradient by isoelectric focusing (IEF) in the first
dimension, and according to their molecular
weight
by
sodium
dodecyl
sulphatepolyacrylamide gel electrophoresis (SDS-PAGE)
in the second dimension [10]. The gel is stained to
visualise the protein pattern, and image analysis
software and amino acid analysis can be used for
evaluation. A comparison between two analysis
software packages has been published by Raman,
Cheung, and Marten (2002) [12].
Proteins and peptides in milk are of major
importance to the dairy industry due to their
technological and nutritional values. Bovine milk
contains 2.3–4.4% (w/w) proteins [13] and
approximately 80% (76–86%) of these proteins
consists of caseins [14]. The rest is mainly whey or
serum proteins, enzymes and other minor proteins
and peptides.
The most common whey proteins are b-lactoglobulin
(b-Lg), a-lactalbumin (a-La), blood serum albumin
(BSA) and immunoglobulins (Igs) [14]. Whey also
contains proteins such as lactoferrin (LF),
lactoperoxidase (LP) and lysozyme, as well as more
than 60 different enzymes [15,16]. In addition, milk
contains peptides that exist naturally in the milk or
are formed from milk proteins, mainly caseins, by
various chemical or biological treatments [17,18].
The proteins and peptides have biological activities,
e.g., ion carriers (caseins, a-La), retinol and fatty acid
carriers (b-Lg and BSA), immunomodulators (a-La,
LF) and immune protection (Igs). Some peptides
influence gastrointestinal function, while others can
stimulate the immune system or affect blood
coagulation or blood pressure [17, 19].
Results of 2-D electrophoresis of bovine milk
proteins have previously been published. Yamada,
Murakami, Wallingford, and Yuki (2002) [20] have
identified low-abundance proteins in bovine
colostrum and mature milk after the removal of bcasein and IgG. Protein identification of the spots on
the gels was performed by either gel comparison,
microsequencing, matrix-assisted laser desorption
ionization–time
of
flight–mass
spectrometry
(MALDI–TOF–MS), peptide mass fingerprinting or
peptide sequencing using tandem MS. They found
significant differences in protein pattern between
colostrum and mature milk. Several low-abundance
proteins, such as fibrinogen b-chain, a-antitrypsin and
apolipoprotein H, were observed only in colostrum.
The present study assessed whether the SDS-PAGE
electrophoresis could separate and analysis
differences between raw milk and commercial heattreated milk samples and correlate the proteins
profile with 2-D GE maps as a technique for studying
the composition and variation of proteins and
peptides with a special focus on LAP (low abundance
proteins) in bovine milk with various kinds of heat
treatment and storage conditions. Compared to
previous 2-D GE studies of milk, we have exploited
the possibilities to study LAP in milk samples.
2. Materials and methods
2.1. Sample preparation
Commercial bovine milk samples from different
types [two pasteurized milks and four Ultra-High
Temperature (UHT) milks] were purchased from a
local super-market. Bulk whole raw bovine milk
(Holstein) was collected from the herd of Cluj.
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Protein standard were obtained from Fermentas
(Unstained Protein Molecular Weight Marker with
MW size range 14.4 to 116 kDa). For evaluating
the influence of pH on thermally-treated whey
proteins, the pH of partly skimmed raw milk was
varied in narrow and wide ranges with 0.1 N HCl
or 0.1 N NaOH.
Table 1. Milk Samples assessed for proteomics profile
The supernatant was run on SDS-PAGE. Molecular
weight standards, specifically SDS-PAGE low-range
standards (Fermentas, Unstained Protein Molecular
Weight Marker) with molecular weights ranging
from 14.4 to 116 kDa were used. The gels were
stained in 0.1% (w/v) R250 Coomassie Brilliant Blue
(BioRad, Hercules, CA, USA) and destained in a
solution of 7.5% (v/v) ethanol and 7.5% (v/v) acetic
acid (fig1).
2.4. Densitometry
The gels were scanned by a scanner and image
system (Molecular Imager GS-800 Calibrated
Densitometer, BioRad, Hercules, CA, USA), the
electrophoresis patterns and densitometry curves of
the bands were processed with the aid of a specific
software of Bio-Rad Quantity One.
2.5. Two-dimensional gel electrophoresis
2.2. Whey protein preparation
The fat was removed from milk samples by
centrifugation at 3500g for 15 min at 40C. The
samples were preserved at 40C for 30 min and the
fat was removed from all milk samples. The skim
milk samples were frozen at -800C until analysis.
Whey proteins were obtained by precipitation of
milk at pH 4.6 with 1N acetic acid (Sigma-Aldrich,
D). The volume of the samples was not
significantly changed. The caseins were removed
by centrifugation at 6000g for 30 min at 40C and
the supernatant of the whey proteins was obtained
prior to SDS-PAGE and protein content
determination. As a control, the whey proteins of
skimmed raw milk were prepared following the
same steps. The protein concentration was
determined following the method of Bradford
(1976)[22] (fig 1).
2.3. SDS-PAGE
SDS-PAGE of whey proteins was performed
according to Laemmli (1970) [21] with a 12% T
gel (gel size: 60x70x1 mm; 10 lanes, model miniPROTEAN® 3 cell, BioRad, Hercules, CA, USA).
The electrophoresis was run under non-reducing
conditions. Whey proteins samples were mixed
with three times the volume of a sample protein
loading buffer [0.1 M Tris-HCl, pH 6.8; 20% v/v
glycerol, 2% (v/v) SDS, 5% (v/v) βmercaptoethanol and 0.01% bromphenol blue] at a
1:3 ratio (v/v) were heated in boiling water for 5
min, and centrifuged at 6000g for 5 min.
Immobilised pH gradient (IPG) strips, ReadyStripTM
IPG Strips gel (pH 3-11, NL, Bio-Rad, Hercules, CA,
USA) with a length of 7 cm were used for separation
in the first dimension. The IPG strips were
rehydrated before IEF. In the period between the first
and second dimensional electrophoresis, the IPG
strips were equilibrated in a SDS buffer (Bio-Rad,
Hercules, CA, USA). Handcast gels 12%T [3.4 ml
DDI H2O, 4 ml 30% Acrylamide/Bis, 2.5 ml Gel
Buffer (1x Tris/Glycine, pH 8.3), 0.1 ml 10% (w/v)
SDS and immediately prior to pouring the gel, add 50
µl 10% APS and 5 µl TEMED] were used for
electrophoresis in the second dimension.
The IPG strips were rehydrated in an
Rehydration/Equilibration Tray (BioRad, Hercules,
CA, USA) with a rehydration solution consisting of
8mM urea, 2% CHAPS, 50 mM DTT, 0.2% BioLyteTM 3/10 ampholytes, 0.001% Bromphenol Blue
(Bio-Rad, Hercules, CA, USA) suitable for the pH
non-linear range 3-10. The IPG strips were
rehydrated overnight at room temperature for 17-18h.
IEF was carried out using a PROTEAN IEF Cell
(BioRad, Hercules, CA, USA). The equipment was
run at 200C according to a programme comprising of
two linear increases in voltage over varying periods
of time, i.e., (1) 0-300 V over 1 min, (2) 300-3500 V
over 90 min, followed by (3) 3500 V for over 300
min. The total voltage-hours (Vh) was approximately
12 000 and the current was 50 µA per gel.
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Figure 1. Experimental Design – Flow chart of SDS-PAGE and 2-D electrophoresis milk protein analysis
In the period between the samples resolution using
first and second dimensional electrophoresis, the
IPG strips were equilibrated in two equilibration
solutions. Equilibration solution I contained 375
mM Tris-HCl, pH 8.8, 6 M urea, 2% SDS, 2%
DTT, and equilibration solution II contained 375
mM Tris-HCl, pH 8.8, 6 M urea, and 2% SDS
(Bio-Rad, Hercules, CA, USA). The IPG strips
were equilibrated at room temperature in
individual
Rehydration/Equilibration
Trays
(BioRad, Hercules, CA, USA) with 2-2.5 mL of
equilibration solution I followed by equilibration
solution II, for 15 min each. SDS-PAGE was
performed at a temperature of 150C. The whey
proteins were separated on mini-Protean 3 cell
(BioRad, Hercules, CA, USA) on hand cast SDSPAGE Gels homogenous 12% at 600 V and 20 mA
for 28 min followed by 50 mA for 75 min.
The gels were Coomassie Blue stained using
Coomassie Brilliant Blue R-250 Staining Solutions
Kit (Bio-Rad, Hercules, CA, USA). The stained
gels were distained with Destain Solution (40%
methanol, 10% acetic acid) scanned using
Molecular
Imager
GS-800
Calibrated
Densitometer (BioRad, Hercules, CA, USA).
The evaluation was performed with the image
analysis software for 2-D GE, PDQuest 8.0
(Bio-Rad, Hercules, CA, USA).
172
2.6. Protein content
The protein contents in milk, and whey were
estimated by a protein assay kit from Bio-Rad
(Hercules, CA, USA) using BSA (Bio-Rad, Hercules,
CA, USA) as a standard (fig. 1).
2.7. Statistical analysis
All experiments were performed in triplicate.
Statistical analyses were performed by using SPSS
computer software (version 16) and GraphPad Prism
version 3.00 for Windows, GraphPad Software, (San
Diego California, USA, www.graphpad.com).
3. Results and Discussion
3.1. Application of SDS-PAGE
SDS-PAGE of proteins of commercial milks and
whey proteins of commercial milks and individual
bovine milks
The proteins in six commercial milk samples and
whey proteins from same milk samples compared
with raw milk proteins and whey proteins were
characterized and quantified by SDS-PAGE.
Individual proteins and whey protein contents were
calculated based on densitometric curve of the
standard proteins. As shown in figure 2, the major
proteins content.
Ramona Suharoschi et. al. / Journal of Agroalimentary Processes and Technologies 2010, 16(2)
As shown in Figures 2 and 3, the major whey
protein content (sum of BSA, α-LA, β-LGA and βLGB) of four UHT milks was significantly
reduced (over 85%) when compared with raw
milk, while mostly all of the native BSA and β-LG
disappeared in the gel, and only a small amount of
native α-LA milk products was reduced (under
27%). The native BSA was obviously reduced and
the α-LA mostly remained in its native state.
account, the square correlation coefficient (R2) of two
methods was 0.9815 (fig.1). The samples of raw milk
contain many other whey proteins (such as
immunoglobulins, lactoferrin, lactoperoxidase and
lysozyme) are mostly denaturated in commercial
bovine milks (the top part of lane in figure 2 and 3).
Concerning individual bovine milk, the SDS-PAGE
of milk and whey proteins focused on the
identification of two variants of bovine β-LG, which
are the most common genetic variants differing by
only two residues: variant A has Val118, Asp64, and
variant B has Ala118, Gly64 [23]. From this point of
view SDS-PAGE would be a meaningful method in
the field of cow breeding with its advantage of
simple operation and lower cost.
3.2. SDS-PAGE of whey proteins from different
thermally-treated milks
Figure 2. 12% T SDS-PAGE of milk proteins from
different heat-treated bovine milks: raw bovine milk as
control; lane 1, protein marker Fermentas; lane 2, hightemperature pasteurised, PET; lane 3, UHT, TetraPack;
lane 4 UHT, Eco, TetraPack; lane 5, high-temperature
pasteurised, TetraPack, lane 6 empty, lane 7, UHT,
TetraPack, lane 8, UHT, TetraPack, lane 9, raw bovine
milk
To get more information on whey proteins influenced
by heating, different heat-treated commercial milk
samples were assessed by SDS-PAGE. As shown in
Fig. 3, BSA, α -LA, β -LGB and β -LGA exhibited
different thermal tolerances. These results on the
sensitivities of whey proteins to heat are in
accordance with other studies [24,25]. The heat
tolerance of whey proteins could be visualized
directly and precisely using SDS-PAGE, excluding
the effect of other milk composites. The R2 of the
quantification of the total major whey proteins by
native-PAGE and the classic protein determination
method was 0.9815 (data shown in fig.1), indicating
that SDS-PAGE can be a credible method of protein
quantification.
These results suggest that the qualitative
determination of whey proteins, which has been a
key tracer in relation to the authentication of heattreated milks [26], could become directly perceived
and convenient by analysis with SDS-PAGE.
Figure 3. 12% T SDS-PAGE of whey proteins from
different heat-treated bovine milks: raw bovine milk as
control; lane 1, protein marker Fermentas; lane 2,
empty, lane 3, UHT, PET; lane 4 high-temperature
pasteurised; TetraPack; lane 5, UHT, Eco, TetraPack;
lane 6 high-temperature pasteurised, TetraPack, lane 7,
UHT, TetraPack, lane 8, UHT, TetraPack, lane 9, raw
bovine milk
The quantification of proteins by SDS-PAGE with
a scanner and image system was assessed by the
method of Bradford (1976), using BSA as a
standard [22]. Taking the total content of the major
whey proteins of six commercial bovine milks into
Figure 4. 2D electropherograms of total proteins of raw
milk (red) and high-temperature pasteurised milk (HTST)
(green)
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The temperatures at which thermal unfolding occurs
for the main proteins in whey are as follows: 770 C, β
-Lg; 670 C, α -La; 65–930 C, depending on the iron
content, LF; and 640 C, BSA [28, 29, 30]. Among the
individual Ig classes, IgG1 was found to unfold at
79.40 C, IgG2 at 76.70 C and IgM at 80.3ºC[28].
However, the unfolding temperature is dependent on
a number of factors including concentration, pH,
solvent and the temperature–time combination.
Figure 5. 2D electropherograms of total proteins of raw
milk (red) and ultra-high temperature milk (UHT)
(green)
Figure 8. 2-D GE of whey proteins in UHT milk samples.
The gel show molecular weights (Mr) and isoelectric
points (pI) of whey samples. The samples were separated
using IPG strip pH 3-10 (non-linear pH gradient) in the
first dimension and SDS-PAGE 2-D Homogenous 12%T
in the second dimension. Apparent molecular weight (Mr,
logarithmic scale) and pI are indicated on the vertical and
horizontal axes, respectively.
Figure 6. 2D electropherograms of whey proteins of
raw milk (red) and high-temperature pasteurised milk
(HTST) (green): more proteins fragments (7) identified
in raw milk compared with HTST milk (4)
4. Conclusion
Figure 7. 2D electropherograms of whey proteins of
raw milk (red) and ultra-high temperature milk (UHT)
(green): more protein fragments (9) identified in raw
milk compared with UHT milk (3)
3.3. Influence of heat treatment on electrophoretic
pattern
The observed content and number of proteins in
whey after heat treatment of milk decreased with
increasing temperature when separating the
samples in the pI range from 3 to 10 (Fig. 8). The
decrease was more pronounced in the whey from
skim milk heated for 1350C for 1 sec (UHT – ultrahigh temperature milk) than whey heated at 710C
for 15-20 min (High Temperature/Short Time –
HTST). This corresponds to findings presented in
the literature, which state that the globular whey
proteins unfold at temperatures above 600 C and
form aggregates [27].
From the results obtained, it can be concluded that:
(a) The described native-PAGE procedure is suitable
for the routine separation and quantification of four
main whey proteins in raw, heattreated and
commercial bovine milks. The native-PAGE of whey
proteins provided a directly perceived, convenient
and lower cost method for the discrimination of whey
proteins of different heattreated bovine milks; (b)
Using native-PAGE, changes in the concentration of
major whey proteins were modeled as a function of
temperature, time, and the pH of bovine milk under
laboratory conditions; (c) With the map of native
whey proteins described in this study, it becomes
possible to model the thermal history of a bovine
milk sample, though better standardization and
interlaboratory validation are necessary for improved
reproducibility.
Our results show that 2-D electrophoresis is a useful
tool for studying the proteins and peptides in milk.
Variations in protein and peptide composition were
detectable in milk with various heat-treatment.
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This technique could be used to study the effects
of breeding, feeding, animal health and milk
processing treatments on the composition of milk
proteins and peptides, as well as bioactive proteins
and peptides and potential allergenic peptides
found in bovine milk.
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