Quantitative proteomic analysis of whey proteins in the colostrum
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and mature milk of yak (Bos grunniens)
Yongxin Yang1,2, Xiaowei Zhao2, Shumin Yu1, Suizhong Cao1, *
1
College of Veterinary Medicine, Sichuan Agricultural University, Ya’an 625014,
China 2
Institute of Animal Science and Veterinary Medicine, Anhui Academy of Agricultural
Sciences, Hefei 230031, China
*Corresponding author: Suizhong Cao E-mail address: [email protected] Tel.: +86-835-2885312; Fax: +86-835-2885302
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/jsfa.6791
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Abstract BACKGROUND: Yak (Bos grunniens) is an important natural resource in
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mountainous regions. To date, few studies have addressed the differences in the protein profiles of yak colostrum and milk. We used quantitative proteomics to compare the protein profiles of whey from yak colostrum and milk. Milk samples were collected from 21 yaks after calving (1 and 28 d). Whey protein profiles were generated
through
isobaric
tag
for
relative
and
absolute
quantification
(iTRAQ)-labelled proteomics. RESULTS: We identified 183 proteins in milk whey; of these, the expression levels of 86 proteins differed significantly between the whey from colostrum and milk. Hemoglobin expression showed the greatest change; its levels were significantly higher in the whey from colostrum than in mature milk whey. Functional analysis revealed that many of the differentially expressed proteins were associated with biological regulation and response to stimuli. Further, eight differentially expressed proteins involved in the complement and coagulation cascade pathway were enriched in milk whey. CONCLUSION: These findings add to the general understanding of the protein composition of yak milk, suggest potential functions of the differentially expressed proteins, and provide novel information on the role of colostral components in calf survival.
Keywords: colostrum; milk whey; proteomics; yak
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INTRODUCTION Milk contains many nutrients such as proteins, fat, lactose, vitamins, and minerals,
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and milk protein is considered one of the most important sources of natural bioactive components. Colostrum is secreted by mammary glands in the first several days post-parturition, and it contains immunoglobulins, growth factors, cytokines, and nucleosides in abundance to protect the newborn calf against diseases and to support neonatal growth 1. Colostrum and milk are known to differ in the quantity and composition of their proteins. For instance, immunoglobulin concentrations are much higher in colostrum than in milk
2, 3
. These qualitative and quantitative differences in
protein expression would be clearer if the protein expression profiles of colostrum and milk can be compared. Proteomic characterization can be used to generate qualitative and quantitative profiles of global protein expression in milk
4, 5
. In fact, in recent years, many groups
have characterized the colostrum and milk of humans and other mammals using proteomics techniques. For example, in humans, the developmental changes in the proteomes of milk whey and milk fat globule membrane (MFGM) during a twelve-month lactation period have been studied by liquid chromatography tandem mass spectrometry
6, 7
. Similarly, low-abundance proteins and proteins in the MFGM
of bovine colostrum and milk have also been studied by two-dimensional electrophoresis coupled with mass spectrometry, isobaric tag for relative and absolute quantification (iTRAQ), and ion-exchange approaches 8-12. Furthermore, the proteome of the colostrum and milk of the sow and the marsupial Trichosurus vulpecula have
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also been studied by two-dimensional electrophoresis coupled with mass spectrometry 13, 14
. The results of these studies demonstrated that a unique subset of proteins was
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expressed in the colostrum and that the expression levels of some proteins involved in immune functions changed during the course of lactation. Yak (Bos grunniens) is an important natural resource in the mountainous regions of China, Mongolia, Russia, Nepal, and India; it supplies the population with milk and meat 15. Several groups have studied yak milk to characterize its composition and to determine the functions of the various components
16-18
. The utility of yak milk as a
functional food has also been reviewed 19. However, to date, only a single study has explored the proteome of yak milk whey 20, and the proteome of yak colostrum whey is yet to be studied. Therefore, in the present study, we evaluated the low-abundance proteins in the colostrum and milk of yak by using a quantitative iTRAQ-labeling proteomics approach. We believe that our quantitative proteomics data can contribute substantially to the present understanding of the protein profile of yak colostrum and milk. Moreover, our results may provide useful insights into previously unknown milk components and help indicate associations between milk production and mammary gland development in yaks.
MATERIALS AND METHODS Sample Preparation Milk samples were collected from 21 yaks at 1 and 28 days after calving at the local farm in Qinghai province. Colostrum and mature milk samples were pooled into three fractions each. The pooled samples were centrifuged at 3000 × g at 4°C for 10
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min. The fat layer was removed using a spatula; skimmed milk samples were collected and centrifuged twice at 22 000 × g at 4°C for 1 h to collect the supernatant.
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Protein concentrations were assayed using a modified Bradford method with bovine serum albumin as standard. Tryptic digestion and iTRAQ Labelling Two-hundred micrograms of milk whey proteins were diluted in SDT buffer (40 g L-1 SDS, 100 mmol L-1 Tris-HCl pH 7.6, and 100 mmol L-1 dithiothreitol) and heated at 95°C for 5 min. After cooling to room temperature, the samples were transferred to an ultrafilter (10 kDa cutoff, Sartorius, German), and then 200 μL of UT buffer (8 mol L-1 urea and 150 mmol L-1 Tris-HCl, pH 8.0) was injected into the filter and mixed. The mixture was centrifuged at 14 000 × g for 30 min and washed with UT buffer. Then, 100 μL of 50 mmol L-1 iodoacetamide solutions was added to the filter and mixed, and the mixture was incubated for 30 min at room temperature in the dark. The mixture was then centrifuged at 14 000 × g for 20 min and washed with UT buffer. Subsequently, 100 μL of dissolution buffer (Applied Biosystems, USA) was added to the filter, and the mixture was centrifuged at 14 000 × g for 30 min. This step was repeated once. Finally, the samples were incubated with 2 μg of modified sequencing-grade trypsin (Promega, Madison, WI, USA) in 40 μL of dissolution buffer at 37°C for 16–18 h. Tryptic peptides were eluted by centrifugation at 10 000 × g for 10 min. Samples were desalted using a C18 solid-phase extraction column and dried in a SpeedVac. One-hundred micrograms of peptide mixtures were labelled with iTRAQ tags
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according to the manufacturer’s instructions (Applied Biosystems). Peptide mixtures from colostrum were labelled with reagent 113 and those from milk were labelled
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with reagent 114. Three independent experiments were performed for each group. Chromatography Separation Labelled samples were acidified with 10 mL L-1 trifluoroacetic acid and fractionated
by
strong
cationic-exchange
chromatography
(SCX)
in
a
PolysulfoethylTM column (PolyLC Inc., Columbia, Maryland, USA). Peptides were separated on a linear binary gradient as follows: 100% solvent A (10 mmol L-1 KH2PO4 in 250 mL L-1 acetonitrile, pH 3.0) for 25 min, 0–10% solvent B (solvent A containing 500 mmol L-1 KCl) in A for 5 min, 10–20% solvent B in A for 10 min, 20–45% solvent B in A for 5 min, 45–100% solvent B for 5 min, and 100% solvent B for 8 min at a flow rate of 1000 μL/min. Fractions were collected and pooled into 10 SCX fractions per sample set, and the pooled fractions were desalted on a C18 solid-phase extraction cartridge. Dried SCX fractions were dissolved in solvent C (1 mL L-1 formic acid in pure H2O). Peptide mixtures were separated on a nano-LC system (EASY-nLC1000, Thermo Fisher Scientific, San Jose, CA, USA). Samples were loaded using an autosampler onto the 5-μm-C18 trapped column (20 mm × 100 μm) and separated on an RP column with 3-μm C18 beads (100 mm × 75 μm). For separation, a linearly increasing concentration of solvent D (1 mL L-1 formic acid in 840 mL L-1 acetonitrile) was used, as follows: 0–35% for 100 min, 35–100% for 8 min, and 100% for 12 min at a flow rate of 200 nL/min.
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MS/MS Analysis and Quantification After separation, the peptides were analyzed by Q-Exactive mass spectrometry
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(Thermo Fisher Scientific, San Jose, CA, USA). Data were acquired in positive ion mode for 120 min. MS spectra were acquired at 70 000 resolution, with a mass/charge (m/z) of 200, a scan mass range of 300–1800 m/z, and a maximum ion inject time of 10 ms. The top 10 most abundant and multiple-charged precursor ions from the survey scan were selected for MS/MS analysis. Fragmentation was performed by high-energy collisional dissociation with normalized collision energies of 30 eV. Resolution was set to 17 500 at 200 m/z with a maximum ion inject time of 60 ms. Dynamic exclusion for selected ions was set to 40 s. Proteins were identified from at least two unique peptide identifications. Raw
data
were
processed
using
ProteomicsTools
(Version
3.0.5)
(http://www.proteomics.ac.cn/), and the Mascot search engine (version 2.2; Matrix Science) was used to search the Cetartiodactyla in an in-house UniProt database
21
.
The search parameters were as follows: enzyme trypsin with two miscleavages allowed, iTRAQ labelling with cysteine carbamidomethylation as a fixed modification, methionine oxidation as a variable modification, fragment mass tolerance at 0.1 Da, and peptide mass tolerance at ± 20 ppm. The false discovery rate for peptide and protein identification was set to 0.01. Proteins were identified from at least two unique peptides through database searches. For protein quantification, the match ratios for the identified peptides were calculated from the relative intensities of released iTRAQ reporter ions in labelled samples. Relative ratios were calculated
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from the weighted ratios of uniquely identified peptides. Statistical analysis was performed using t-test. P values less than 0.05 were considered significant.
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ELISA Quantification The α-lactalbumin in the colostrum and milk samples was quantified using a commercial ELISA kit (Bethyl Laboratories, Montgomery, TX, USA) according to the manufacturer’s instructions. The detection limit for α-lactalbumin was 0.78 ng/mL. The dynamic range of the assay was 0.78–50 ng/mL.
RESULTS AND DISCUSSION In this study, 183 proteins were identified in the whey from the colostrum and milk of yak by using the quantitative iTRAQ proteomics method. To date, in studies on animal milk proteome, researchers have mostly focused on Holstein cow milk. In the past few years, interest in comprehensive characterization of bovine whey protein has increased progressively. In addition, information of bovine protein sequences available in databases has also increased substantially. For example, Yamada et al. first compared the low-abundance proteins in bovine colostrum and milk and a total of 15 unique gene products were identified 8. In a later study, the host defense proteins in bovine milk were studied, and 95 gene products were identified in the skim milk, whey, and MFGM fractions. Of these, 55 proteins were found to be expressed only in the MFGM fraction, and 17, only in the whey 22. Recently, for more detailed characterization of whey components, milk whey was treated using combinatorial peptide ligand libraries, and 149 unique proteins were identified
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23
. In
another study, using an ion-exchange approach, 293 unique gene products were identified in colostral and mature whey 12. More recently, 376 unique proteins in two
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replicates were identified through extensive fractionation
24
. The number of proteins
identified in this study seems lower than that obtained in other recent studies. Furthermore, almost all the proteins identified in this study coincided with those reported in Holstein milk, except for several variants of hemoglobin. These factors could be due to the protein identification approach, which was based on the information available in the databases; this information is limited because very few studies have investigated the milk proteome of yak or the differences between the protein profiles of milk of yak and Holstein cows. The expression levels of 86 of the identified proteins significantly differed between colostrum and milk (Table 1). Forty-one proteins were found to be significantly up-regulated in yak colostrum, including immunoglobulin J chain, immunoglobulin light chain, apolipoprotein E, and hemoglobin beta and alpha. Forty-five proteins were significantly down-regulated in colostrum, including osteopontin and folate receptor alpha. The expression of α-lactalbumin in colostrum was 0.41 times that in milk; this result was partially consistent with the iTRAQ proteomic data. Previous proteomics investigations have shown that the colostrum of Holstein cows is rich in immunoglobulins as well as other proteins associated with the immune response, such as lactoferrin, beta-2-microglobulin, and polymeric immunoglobulin receptors
8-10,12
.
Consistent with these findings, our results showed that yak colostrum too is rich in several proteins involved in immune functions, including immunoglobulins,
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beta-2-microglobulin, complement factor B, and C-X-C motif chemokine 6. We also identified variants of hemoglobin, such as hemoglobin subunit alpha-1 and
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alpha-2, subunit beta, and beta-A, in yak milk. Hemoglobin beta and alpha, which are involved in oxygen binding and transport, were significantly up-regulated in yak colostrum. In particular, hemoglobin beta expression differed the most, with the expression in colostrum whey being 20 times that in milk whey. Yak hemoglobin contains two types of alpha chain and beta
25
. Expression of multiple hemoglobin
isoforms with graded oxygen affinities is thought to provide regulatory reserve oxygen transport capacity
26
. This combined with the high hemoglobin content in
colostrum may provide sufficient oxygen to the new-born calf to survive in extreme low-oxygen environments. These proteins were not identified previously in Holstein colostrum using 2-DE coupled with mass spectrometry
8-10
, and although hemoglobin
beta was identified in bovine colostrum and milk using an ion-exchange approach, its relative proportion was not defined 12. In addition, it is well known that hemoglobin is found in red blood cells and their progenitor lines. However, high levels of hemoglobin were detected in yak colostrum; whether these originated from the red blood cells in the blood or from outside red blood cells needs to be confirmed by analyzing hemoglobin expression in yak mammary epithelial cells. Gene Ontology (GO) annotation was used for functional analysis of the differentially expressed proteins. In the biological process category, most of the proteins were assigned to the biological regulation, followed by response to stimuli. Other functions included localization, multicellular organismal processes, and
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immune system processes (Fig. 1A). In the molecular function analysis, a large number of proteins were found to belong to the binding category (66%), which
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included protein, ion, pattern, heparin, and vitamin binding. A small number of proteins were assigned to other functional categories based on their properties, such as antioxidant activity, enzyme regulator activity, and transporter activity (Fig. 1B). The proteins found to be cellular components were assigned to the extracellular region and cytoplasm, and a small number of these proteins were assigned to the chylomicron, cytosol, and the plasma membrane (Fig. 1C). In a previous GO annotation-based study, many proteins were associated with the binding category, including protein, ion, lipid, nucleotide, carbohydrate and vitamin binding, and a small number of proteins were
assigned
to
other
categories,
including
hydrolase,
transferase,
and
oxidoreductase activity 23. In another study, whey proteins of several animals species (Holstein cow, buffalo, yak, goat, and camel) were analyzed, and the most common molecular function was found to binding activity, including ion, protein, carbohydrate, pattern, cell surface, and lipid binding; the other major functional category was enzyme regulation 20. Our results were consistent with these previous findings. Pathway analysis of differentially expressed proteins was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Six up-regulated proteins, namely, alpha-2-macroglobulin, prothrombin plasminogen, fibrinogen alpha chain, fibrinogen gamma chain, and complement factor B, and two down-regulated proteins, namely, complement C3 and factor XIIa inhibitor, in colostrum were assigned to the complement and coagulation cascade pathway (P = 1.91E-08; Table 2).
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This pathway plays crucial roles in host defence against pathogens and other invaders 27
. Activated complement and coagulation cascades have previously been observed in
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bovine mammary glands by transcriptome analysis 28. Interestingly, our results show that the differentially expressed proteins are associated with the complement and coagulation cascade pathway in the milk whey; although further investigation is needed to confirm this result, it may help elucidate proteins associated with physiological function. In conclusion, in this study, 183 proteins were identified in whey from yak milk and colostrum by using a quantitative iTRAQ-labeling proteomics approach, and of these, 86 proteins showed differential expression in colostrum and milk. The differentially expressed proteins were predominantly associated with biological regulation and response to stimuli in the biological process category, and with binding activity in the molecular function category. In addition, eight differentially expressed proteins were assigned to the complement and coagulation cascade pathway. Collectively, these results may contribute to the current understanding of milk synthesis in mammary glands of yaks, which live in mountainous regions with low oxygen levels.
ACKNOWLEDGEMENT This project was supported by the Key Technology R & D Program of Sichuan Province (2011NZ0060).
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10. Zhang L, Wang J, Yang Y, Bu D, Li S and Zhou L. Comparative Proteomic Analysis of Changes in the Bovine Whey Proteome during the Transition from
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17. Sun WC, Luo YH and Ma HQ. Preliminary study of metal in yak (Bos grunniens) milk from Qilian of the Qinghai Plateau. Bull Environ Contam Toxicol 86: 653-656
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Table 1 Differentially expressed whey proteins in yak colostrum and mature milk. accession
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No. F1MK50
Protein name Uncharacterized protein
Unique
Coverage
peptide
(%)
2
10.67
MW
PI
19.80
7.15
Fold change1 0.15
P value 2.19E-03
P31096
Osteopontin
6
19.64
30.90
4.49
0.19
1.28E-04
P02702
Folate receptor alpha
5
24.48
27.92
8.12
0.19
4.99E-04
44
38.51
147.10
7.74
0.26
1.01E-06
12
9.01
146.76
7.87
0.26
2.65E-06
3
16.88
17.03
5.27
0.27
1.47E-05
6
15.78
49.15
7.64
0.27
8.41E-05
G1AQP3 F1MUT3 P80195 G3EHG6
Xanthine dehydrogenase/oxidase Uncharacterized protein Glycosylation-dependent cell adhesion molecule-1 Adipose differentiation-related protein
G5E5H7
Uncharacterized protein
14
81.46
19.91
4.93
0.28
6.08E-05
C3W955
Beta-lactoglobulin
14
80.56
20.01
4.93
0.30
3.17E-05
F1S3Y7
Uncharacterized protein
7
4.48
147.01
7.06
0.31
1.04E-04
F2X043
Lactoperoxidase
25
44.80
80.69
8.7
0.31
3.14E-04
E5G7E7
Fatty acid-binding protein
8
49.62
14.78
6.73
0.32
4.91E-05
Q4VS17
FGFBP
2
28.04
11.94
9.16
0.33
4.40E-03
G0Z2N2
L-lactate dehydrogenase
5
13.17
36.72
5.72
0.33
1.03E-03
Q9MZY2
Airway lactoperoxidase
19
31.00
80.49
8.95
0.33
4.00E-04
P02769
Serum albumin
14
17.96
69.69
6.08
0.34
1.93E-04
2
70.00
2.27
8.5
0.35
2.29E-04
7
52.03
14.16
4.61
0.36
1.71E-05
3
24.00
12.93
5.73
0.36
2.35E-04
3
20.80
16.64
8.2
0.37
1.74E-04
Q9TRB9 P00711 Q52RN5 P79345
Enterotoxin-binding glycoprotein PP20K Alpha-lactalbumin Superoxide dismutase [Cu-Zn] Epididymal secretory protein E1
F1RRP2
Uncharacterized protein
5
7.71
81.47
8.82
0.38
5.67E-03
F6R3I5
Uncharacterized protein
7
29.34
27.14
8.29
0.39
1.57E-02
3
7.36
52.58
5.52
0.40
5.77E-03
Q32KV6
Nucleotide exchange factor SIL1
P22226
Cathelicidin-1
2
18.06
17.65
7.54
0.43
3.17E-02
P26779
Proactivator polypeptide
5
12.57
58.05
5.08
0.50
1.78E-02
6
20.25
41.08
4.79
0.51
3.21E-03
2
9.24
20.99
6.39
0.52
8.04E-03
3
32.63
10.60
6.79
0.53
1.65E-03
A5YVD9 P62833 Q307G4
45 kDa calcium-binding protein Ras-related protein Rap-1A Cytosolic NADP-isocitrate dehydrogenase
P50448
Factor XIIa inhibitor
5
7.90
51.72
6.19
0.54
3.30E-02
Q9XSG3
Isocitrate dehydrogenase
10
25.36
46.78
6.13
0.55
3.60E-02
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[NADP] cytoplasmic Q861V5
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P08037
Peptidyl-prolyl cis-trans isomerase Beta-1,4-galactosyltransferase I
5
31.61
16.93
8.46
0.57
1.80E-03
10
30.35
44.76
9.2
0.59
1.99E-03
P11151
Lipoprotein lipase
5
15.48
53.59
8.61
0.59
1.00E-02
Q0IIH5
Nucleobindin 2
8
23.85
49.19
5.12
0.62
3.55E-02
12
18.49
82.43
7.07
0.65
1.03E-03
P81265
Polymeric immunoglobulin receptor
F6PQI6
Uncharacterized protein
20
30.91
82.52
7.68
0.69
6.11E-03
A2I7N2
Serpin A3-6
4
8.00
46.39
5.34
0.70
6.31E-03
G3N1U4
Uncharacterized protein
3
6.81
46.16
6.05
0.72
1.05E-03
G8JKW7
Uncharacterized protein
4
9.95
46.34
6.29
0.72
2.48E-03
A1E0X4
Beta-actin
13
48.90
40.58
5.66
0.73
4.72E-03
D7NJ85
Beta casein
7
25.45
25.10
5.26
0.77
1.23E-02
Q95100
Kappa-casein
3
35.25
13.14
5.86
0.79
1.13E-02
F1MZW0
Uncharacterized protein
7
14.41
67.88
5.91
0.81
3.70E-02
Q0P569
Nucleobindin-1
8
23.84
54.98
5.11
0.84
2.46E-02
Q9GKP1
Complement C3
18
11.56
186.80
6.09
0.84
3.57E-02
F1N5M2
Uncharacterized protein
7
17.30
53.36
5.42
1.24
2.81E-02
Q66RQ0
Vitamin D-binding protein
4
17.89
24.54
5.02
1.26
2.49E-02
P29701
Alpha-2-HS-glycoprotein
8
24.45
38.68
5.17
1.26
1.64E-02
B8R1K3
Transferrin
18
27.56
77.66
6.92
1.26
2.40E-02
P81187
Complement factor B
3
5.65
85.37
7.87
1.37
2.25E-02
P00735
Prothrombin
3
6.08
70.50
5.97
1.38
4.51E-02
E1BI82
Uncharacterized protein
6
10.77
69.15
8.29
1.40
3.96E-02
P12725
Alpha-1-antiproteinase
10
20.43
45.98
5.83
1.54
2.77E-02
G3EHG5
TIP47
4
14.87
47.36
5.74
1.70
7.83E-03
P00978
Protein AMBP
3
10.23
39.23
7.81
1.72
6.61E-03
Q7SIH1
Alpha-2-macroglobulin
17
16.76
167.57
5.71
1.73
5.70E-03
F1MFI4
Uncharacterized protein
2
4.17
69.88
5.1
1.88
2.01E-02
Q3SZZ9
FGG protein
8
21.15
49.17
5.56
1.88
3.18E-02
G3N0V0
Uncharacterized protein
6
15.64
35.95
8.05
2.01
5.34E-03
G5E604
Uncharacterized protein
3
29.90
11.06
8.02
2.15
3.49E-02
F1MAV0
Uncharacterized protein
3
8.48
56.44
8.5
2.17
2.75E-03
P17697
Clusterin
5
14.35
51.11
5.73
2.19
2.57E-03
C0LQK8
Myostatin
8
27.47
42.51
6.05
2.23
1.26E-02
P80221
C-X-C motif chemokine 6
2
22.32
11.59
9.4
2.40
4.43E-03
A4ZVY8
Beta-2-microglobulin
2
16.95
13.61
6.4
2.42
1.02E-04
A7E350
PLG protein
6
9.53
81.58
8.33
2.54
6.33E-04
P02672
Fibrinogen alpha chain
11
18.37
67.01
6.73
2.74
2.17E-02
F1MNN7
Uncharacterized protein
3
6.44
53.69
6.61
2.91
4.42E-03
A6QNJ8
GANAB protein
3
3.53
109.08
5.83
2.97
2.66E-02
G3N2D7
Uncharacterized protein
2
37.93
12.11
4.78
3.36
4.55E-04
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P20757
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Q1RMN8
Angiotensinogen Immunoglobulin light chain, lambda gene cluster
6
11.14
51.30
6.54
3.39
2.14E-03
6
33.33
24.54
7.54
4.53
8.21E-03
A6QM09
Uncharacterized protein
4
15.09
24.73
7.51
4.77
6.72E-03
P01968
Hemoglobin subunit alpha-2
2
17.02
14.98
8.91
5.03
1.25E-02
G5E5T5
Uncharacterized protein
6
15.93
42.36
4.89
5.63
3.90E-07
Q0ZCB4
Apolipoprotein E
5
23.21
27.07
8.87
5.69
5.28E-04
G5E513
Uncharacterized protein
4
10.50
49.97
5.32
6.00
2.53E-05
Q3SYR8
Immunoglobulin J chain
2
14.65
17.86
5.1
6.53
2.21E-03
Q3Y443
Alpha s2 casein
10
36.94
26.13
8.24
6.57
6.00E-03
G3N342
Uncharacterized protein
3
7.28
47.83
7.23
6.94
1.03E-02
Q9TSN7
Hemoglobin subunit alpha-1
2
16.90
15.13
8.08
7.71
4.12E-02
F1MPP2
Uncharacterized protein
8
37.23
29.05
8.25
9.48
1.77E-03
F1MLW8
Uncharacterized protein
3
15.88
24.62
5.58
10.38
3.74E-05
A6QNW7
CD5L protein
6
18.31
50.21
5.24
14.35
8.53E-07
P04346
Hemoglobin subunit beta-A
6
44.83
15.96
6.36
17.14
1.02E-03
P02072
Hemoglobin beta
5
42.76
15.99
7.06
19.77
1.32E-04
1
Relative abundance of whey proteins in colostrum versus mature milk
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Table 2 Pathway categories of differentially expressed proteins in yak colostrum and mature milk: Kyoto Encyclopedia of Genes and Genomes (KEGG).
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Pathway Name
bta04610:Complement and coagulation cascades
Accession NO. P02672, P50448, Q3SZZ9,
Count
Hits
Percentage
P Value
Fold Enrichment
8
71
14.04
1.91E-08
23.19
P81187, Q7SIH1, P06868, Q2UVX4, P00735
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(A)
(B)
(C) Fig. 1 Classification of differentially expressed milk proteins based on gene ontology annotation. (A) Biological process; (B) molecular function; (C) cellular component.
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Accepted Article
and mature milk of yak (Bos grunniens)
Yongxin Yang1,2, Xiaowei Zhao2, Shumin Yu1, Suizhong Cao1, *
1
College of Veterinary Medicine, Sichuan Agricultural University, Ya’an 625014,
China 2
Institute of Animal Science and Veterinary Medicine, Anhui Academy of Agricultural
Sciences, Hefei 230031, China
*Corresponding author: Suizhong Cao E-mail address: [email protected] Tel.: +86-835-2885312; Fax: +86-835-2885302
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/jsfa.6791
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Abstract BACKGROUND: Yak (Bos grunniens) is an important natural resource in
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mountainous regions. To date, few studies have addressed the differences in the protein profiles of yak colostrum and milk. We used quantitative proteomics to compare the protein profiles of whey from yak colostrum and milk. Milk samples were collected from 21 yaks after calving (1 and 28 d). Whey protein profiles were generated
through
isobaric
tag
for
relative
and
absolute
quantification
(iTRAQ)-labelled proteomics. RESULTS: We identified 183 proteins in milk whey; of these, the expression levels of 86 proteins differed significantly between the whey from colostrum and milk. Hemoglobin expression showed the greatest change; its levels were significantly higher in the whey from colostrum than in mature milk whey. Functional analysis revealed that many of the differentially expressed proteins were associated with biological regulation and response to stimuli. Further, eight differentially expressed proteins involved in the complement and coagulation cascade pathway were enriched in milk whey. CONCLUSION: These findings add to the general understanding of the protein composition of yak milk, suggest potential functions of the differentially expressed proteins, and provide novel information on the role of colostral components in calf survival.
Keywords: colostrum; milk whey; proteomics; yak
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INTRODUCTION Milk contains many nutrients such as proteins, fat, lactose, vitamins, and minerals,
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and milk protein is considered one of the most important sources of natural bioactive components. Colostrum is secreted by mammary glands in the first several days post-parturition, and it contains immunoglobulins, growth factors, cytokines, and nucleosides in abundance to protect the newborn calf against diseases and to support neonatal growth 1. Colostrum and milk are known to differ in the quantity and composition of their proteins. For instance, immunoglobulin concentrations are much higher in colostrum than in milk
2, 3
. These qualitative and quantitative differences in
protein expression would be clearer if the protein expression profiles of colostrum and milk can be compared. Proteomic characterization can be used to generate qualitative and quantitative profiles of global protein expression in milk
4, 5
. In fact, in recent years, many groups
have characterized the colostrum and milk of humans and other mammals using proteomics techniques. For example, in humans, the developmental changes in the proteomes of milk whey and milk fat globule membrane (MFGM) during a twelve-month lactation period have been studied by liquid chromatography tandem mass spectrometry
6, 7
. Similarly, low-abundance proteins and proteins in the MFGM
of bovine colostrum and milk have also been studied by two-dimensional electrophoresis coupled with mass spectrometry, isobaric tag for relative and absolute quantification (iTRAQ), and ion-exchange approaches 8-12. Furthermore, the proteome of the colostrum and milk of the sow and the marsupial Trichosurus vulpecula have
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also been studied by two-dimensional electrophoresis coupled with mass spectrometry 13, 14
. The results of these studies demonstrated that a unique subset of proteins was
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expressed in the colostrum and that the expression levels of some proteins involved in immune functions changed during the course of lactation. Yak (Bos grunniens) is an important natural resource in the mountainous regions of China, Mongolia, Russia, Nepal, and India; it supplies the population with milk and meat 15. Several groups have studied yak milk to characterize its composition and to determine the functions of the various components
16-18
. The utility of yak milk as a
functional food has also been reviewed 19. However, to date, only a single study has explored the proteome of yak milk whey 20, and the proteome of yak colostrum whey is yet to be studied. Therefore, in the present study, we evaluated the low-abundance proteins in the colostrum and milk of yak by using a quantitative iTRAQ-labeling proteomics approach. We believe that our quantitative proteomics data can contribute substantially to the present understanding of the protein profile of yak colostrum and milk. Moreover, our results may provide useful insights into previously unknown milk components and help indicate associations between milk production and mammary gland development in yaks.
MATERIALS AND METHODS Sample Preparation Milk samples were collected from 21 yaks at 1 and 28 days after calving at the local farm in Qinghai province. Colostrum and mature milk samples were pooled into three fractions each. The pooled samples were centrifuged at 3000 × g at 4°C for 10
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min. The fat layer was removed using a spatula; skimmed milk samples were collected and centrifuged twice at 22 000 × g at 4°C for 1 h to collect the supernatant.
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Protein concentrations were assayed using a modified Bradford method with bovine serum albumin as standard. Tryptic digestion and iTRAQ Labelling Two-hundred micrograms of milk whey proteins were diluted in SDT buffer (40 g L-1 SDS, 100 mmol L-1 Tris-HCl pH 7.6, and 100 mmol L-1 dithiothreitol) and heated at 95°C for 5 min. After cooling to room temperature, the samples were transferred to an ultrafilter (10 kDa cutoff, Sartorius, German), and then 200 μL of UT buffer (8 mol L-1 urea and 150 mmol L-1 Tris-HCl, pH 8.0) was injected into the filter and mixed. The mixture was centrifuged at 14 000 × g for 30 min and washed with UT buffer. Then, 100 μL of 50 mmol L-1 iodoacetamide solutions was added to the filter and mixed, and the mixture was incubated for 30 min at room temperature in the dark. The mixture was then centrifuged at 14 000 × g for 20 min and washed with UT buffer. Subsequently, 100 μL of dissolution buffer (Applied Biosystems, USA) was added to the filter, and the mixture was centrifuged at 14 000 × g for 30 min. This step was repeated once. Finally, the samples were incubated with 2 μg of modified sequencing-grade trypsin (Promega, Madison, WI, USA) in 40 μL of dissolution buffer at 37°C for 16–18 h. Tryptic peptides were eluted by centrifugation at 10 000 × g for 10 min. Samples were desalted using a C18 solid-phase extraction column and dried in a SpeedVac. One-hundred micrograms of peptide mixtures were labelled with iTRAQ tags
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according to the manufacturer’s instructions (Applied Biosystems). Peptide mixtures from colostrum were labelled with reagent 113 and those from milk were labelled
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with reagent 114. Three independent experiments were performed for each group. Chromatography Separation Labelled samples were acidified with 10 mL L-1 trifluoroacetic acid and fractionated
by
strong
cationic-exchange
chromatography
(SCX)
in
a
PolysulfoethylTM column (PolyLC Inc., Columbia, Maryland, USA). Peptides were separated on a linear binary gradient as follows: 100% solvent A (10 mmol L-1 KH2PO4 in 250 mL L-1 acetonitrile, pH 3.0) for 25 min, 0–10% solvent B (solvent A containing 500 mmol L-1 KCl) in A for 5 min, 10–20% solvent B in A for 10 min, 20–45% solvent B in A for 5 min, 45–100% solvent B for 5 min, and 100% solvent B for 8 min at a flow rate of 1000 μL/min. Fractions were collected and pooled into 10 SCX fractions per sample set, and the pooled fractions were desalted on a C18 solid-phase extraction cartridge. Dried SCX fractions were dissolved in solvent C (1 mL L-1 formic acid in pure H2O). Peptide mixtures were separated on a nano-LC system (EASY-nLC1000, Thermo Fisher Scientific, San Jose, CA, USA). Samples were loaded using an autosampler onto the 5-μm-C18 trapped column (20 mm × 100 μm) and separated on an RP column with 3-μm C18 beads (100 mm × 75 μm). For separation, a linearly increasing concentration of solvent D (1 mL L-1 formic acid in 840 mL L-1 acetonitrile) was used, as follows: 0–35% for 100 min, 35–100% for 8 min, and 100% for 12 min at a flow rate of 200 nL/min.
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MS/MS Analysis and Quantification After separation, the peptides were analyzed by Q-Exactive mass spectrometry
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(Thermo Fisher Scientific, San Jose, CA, USA). Data were acquired in positive ion mode for 120 min. MS spectra were acquired at 70 000 resolution, with a mass/charge (m/z) of 200, a scan mass range of 300–1800 m/z, and a maximum ion inject time of 10 ms. The top 10 most abundant and multiple-charged precursor ions from the survey scan were selected for MS/MS analysis. Fragmentation was performed by high-energy collisional dissociation with normalized collision energies of 30 eV. Resolution was set to 17 500 at 200 m/z with a maximum ion inject time of 60 ms. Dynamic exclusion for selected ions was set to 40 s. Proteins were identified from at least two unique peptide identifications. Raw
data
were
processed
using
ProteomicsTools
(Version
3.0.5)
(http://www.proteomics.ac.cn/), and the Mascot search engine (version 2.2; Matrix Science) was used to search the Cetartiodactyla in an in-house UniProt database
21
.
The search parameters were as follows: enzyme trypsin with two miscleavages allowed, iTRAQ labelling with cysteine carbamidomethylation as a fixed modification, methionine oxidation as a variable modification, fragment mass tolerance at 0.1 Da, and peptide mass tolerance at ± 20 ppm. The false discovery rate for peptide and protein identification was set to 0.01. Proteins were identified from at least two unique peptides through database searches. For protein quantification, the match ratios for the identified peptides were calculated from the relative intensities of released iTRAQ reporter ions in labelled samples. Relative ratios were calculated
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from the weighted ratios of uniquely identified peptides. Statistical analysis was performed using t-test. P values less than 0.05 were considered significant.
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ELISA Quantification The α-lactalbumin in the colostrum and milk samples was quantified using a commercial ELISA kit (Bethyl Laboratories, Montgomery, TX, USA) according to the manufacturer’s instructions. The detection limit for α-lactalbumin was 0.78 ng/mL. The dynamic range of the assay was 0.78–50 ng/mL.
RESULTS AND DISCUSSION In this study, 183 proteins were identified in the whey from the colostrum and milk of yak by using the quantitative iTRAQ proteomics method. To date, in studies on animal milk proteome, researchers have mostly focused on Holstein cow milk. In the past few years, interest in comprehensive characterization of bovine whey protein has increased progressively. In addition, information of bovine protein sequences available in databases has also increased substantially. For example, Yamada et al. first compared the low-abundance proteins in bovine colostrum and milk and a total of 15 unique gene products were identified 8. In a later study, the host defense proteins in bovine milk were studied, and 95 gene products were identified in the skim milk, whey, and MFGM fractions. Of these, 55 proteins were found to be expressed only in the MFGM fraction, and 17, only in the whey 22. Recently, for more detailed characterization of whey components, milk whey was treated using combinatorial peptide ligand libraries, and 149 unique proteins were identified
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23
. In
another study, using an ion-exchange approach, 293 unique gene products were identified in colostral and mature whey 12. More recently, 376 unique proteins in two
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replicates were identified through extensive fractionation
24
. The number of proteins
identified in this study seems lower than that obtained in other recent studies. Furthermore, almost all the proteins identified in this study coincided with those reported in Holstein milk, except for several variants of hemoglobin. These factors could be due to the protein identification approach, which was based on the information available in the databases; this information is limited because very few studies have investigated the milk proteome of yak or the differences between the protein profiles of milk of yak and Holstein cows. The expression levels of 86 of the identified proteins significantly differed between colostrum and milk (Table 1). Forty-one proteins were found to be significantly up-regulated in yak colostrum, including immunoglobulin J chain, immunoglobulin light chain, apolipoprotein E, and hemoglobin beta and alpha. Forty-five proteins were significantly down-regulated in colostrum, including osteopontin and folate receptor alpha. The expression of α-lactalbumin in colostrum was 0.41 times that in milk; this result was partially consistent with the iTRAQ proteomic data. Previous proteomics investigations have shown that the colostrum of Holstein cows is rich in immunoglobulins as well as other proteins associated with the immune response, such as lactoferrin, beta-2-microglobulin, and polymeric immunoglobulin receptors
8-10,12
.
Consistent with these findings, our results showed that yak colostrum too is rich in several proteins involved in immune functions, including immunoglobulins,
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beta-2-microglobulin, complement factor B, and C-X-C motif chemokine 6. We also identified variants of hemoglobin, such as hemoglobin subunit alpha-1 and
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alpha-2, subunit beta, and beta-A, in yak milk. Hemoglobin beta and alpha, which are involved in oxygen binding and transport, were significantly up-regulated in yak colostrum. In particular, hemoglobin beta expression differed the most, with the expression in colostrum whey being 20 times that in milk whey. Yak hemoglobin contains two types of alpha chain and beta
25
. Expression of multiple hemoglobin
isoforms with graded oxygen affinities is thought to provide regulatory reserve oxygen transport capacity
26
. This combined with the high hemoglobin content in
colostrum may provide sufficient oxygen to the new-born calf to survive in extreme low-oxygen environments. These proteins were not identified previously in Holstein colostrum using 2-DE coupled with mass spectrometry
8-10
, and although hemoglobin
beta was identified in bovine colostrum and milk using an ion-exchange approach, its relative proportion was not defined 12. In addition, it is well known that hemoglobin is found in red blood cells and their progenitor lines. However, high levels of hemoglobin were detected in yak colostrum; whether these originated from the red blood cells in the blood or from outside red blood cells needs to be confirmed by analyzing hemoglobin expression in yak mammary epithelial cells. Gene Ontology (GO) annotation was used for functional analysis of the differentially expressed proteins. In the biological process category, most of the proteins were assigned to the biological regulation, followed by response to stimuli. Other functions included localization, multicellular organismal processes, and
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immune system processes (Fig. 1A). In the molecular function analysis, a large number of proteins were found to belong to the binding category (66%), which
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included protein, ion, pattern, heparin, and vitamin binding. A small number of proteins were assigned to other functional categories based on their properties, such as antioxidant activity, enzyme regulator activity, and transporter activity (Fig. 1B). The proteins found to be cellular components were assigned to the extracellular region and cytoplasm, and a small number of these proteins were assigned to the chylomicron, cytosol, and the plasma membrane (Fig. 1C). In a previous GO annotation-based study, many proteins were associated with the binding category, including protein, ion, lipid, nucleotide, carbohydrate and vitamin binding, and a small number of proteins were
assigned
to
other
categories,
including
hydrolase,
transferase,
and
oxidoreductase activity 23. In another study, whey proteins of several animals species (Holstein cow, buffalo, yak, goat, and camel) were analyzed, and the most common molecular function was found to binding activity, including ion, protein, carbohydrate, pattern, cell surface, and lipid binding; the other major functional category was enzyme regulation 20. Our results were consistent with these previous findings. Pathway analysis of differentially expressed proteins was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Six up-regulated proteins, namely, alpha-2-macroglobulin, prothrombin plasminogen, fibrinogen alpha chain, fibrinogen gamma chain, and complement factor B, and two down-regulated proteins, namely, complement C3 and factor XIIa inhibitor, in colostrum were assigned to the complement and coagulation cascade pathway (P = 1.91E-08; Table 2).
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This pathway plays crucial roles in host defence against pathogens and other invaders 27
. Activated complement and coagulation cascades have previously been observed in
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bovine mammary glands by transcriptome analysis 28. Interestingly, our results show that the differentially expressed proteins are associated with the complement and coagulation cascade pathway in the milk whey; although further investigation is needed to confirm this result, it may help elucidate proteins associated with physiological function. In conclusion, in this study, 183 proteins were identified in whey from yak milk and colostrum by using a quantitative iTRAQ-labeling proteomics approach, and of these, 86 proteins showed differential expression in colostrum and milk. The differentially expressed proteins were predominantly associated with biological regulation and response to stimuli in the biological process category, and with binding activity in the molecular function category. In addition, eight differentially expressed proteins were assigned to the complement and coagulation cascade pathway. Collectively, these results may contribute to the current understanding of milk synthesis in mammary glands of yaks, which live in mountainous regions with low oxygen levels.
ACKNOWLEDGEMENT This project was supported by the Key Technology R & D Program of Sichuan Province (2011NZ0060).
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10. Zhang L, Wang J, Yang Y, Bu D, Li S and Zhou L. Comparative Proteomic Analysis of Changes in the Bovine Whey Proteome during the Transition from
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17. Sun WC, Luo YH and Ma HQ. Preliminary study of metal in yak (Bos grunniens) milk from Qilian of the Qinghai Plateau. Bull Environ Contam Toxicol 86: 653-656
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2011. 18. Dorji T, Namikawa T, Mannen H and Kawamoto Y. Milk protein polymorphisms in cattle (Bos indicus), mithun (Bos frontalis) and yak (Bos grunniens) breeds and their hybrids indigenous to Bhutan. Anim Sci J 81: 523-529 (2010). 19. Nikkhah A. Equidae, Camel, and Yak Milks as Functional Foods: A Review. J Nutr Food Sci 1: 2 (2011). 20. Yang Y, Bu D, Zhao X, Sun P, Wang J and Zhou L. Proteomic Analysis of Cow, Yak, Buffalo, Goat and Camel Milk Whey Proteins: Quantitative Differential Expression Patterns. J Proteome Res 12: 1660-1667 (2013). 21. Sheng Q, Dai J, Wu Y, Tang H and Zeng R. BuildSummary: using a group-based approach to improve the sensitivity of peptide/protein identification in shotgun proteomics. J Proteome Res 11: 1494-1502 (2012). 22. Smolenski G, Haines S, Kwan FY, Bond J, Farr V, Davis SR, Stelwagen K and Wheeler TT. Characterisation of host defence proteins in milk using a proteomic approach. J Proteome Res 6: 207-215 (2007). 23. D'Amato A, Bachi A, Fasoli E, Boschetti E, Peltre G, Senechal H and Righetti PG. In-depth exploration of cow's whey proteome via combinatorial peptide ligand libraries. J Proteome Res 8: 3925-3936 (2009). 24. Nissen A, Bendixen E, Ingvartsen K L and Røntved CM. Expanding the bovine milk proteome through extensive fractionation. J Dairy Sci 96: 7854-7866 (2013).
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Table 1 Differentially expressed whey proteins in yak colostrum and mature milk. accession
Accepted Article
No. F1MK50
Protein name Uncharacterized protein
Unique
Coverage
peptide
(%)
2
10.67
MW
PI
19.80
7.15
Fold change1 0.15
P value 2.19E-03
P31096
Osteopontin
6
19.64
30.90
4.49
0.19
1.28E-04
P02702
Folate receptor alpha
5
24.48
27.92
8.12
0.19
4.99E-04
44
38.51
147.10
7.74
0.26
1.01E-06
12
9.01
146.76
7.87
0.26
2.65E-06
3
16.88
17.03
5.27
0.27
1.47E-05
6
15.78
49.15
7.64
0.27
8.41E-05
G1AQP3 F1MUT3 P80195 G3EHG6
Xanthine dehydrogenase/oxidase Uncharacterized protein Glycosylation-dependent cell adhesion molecule-1 Adipose differentiation-related protein
G5E5H7
Uncharacterized protein
14
81.46
19.91
4.93
0.28
6.08E-05
C3W955
Beta-lactoglobulin
14
80.56
20.01
4.93
0.30
3.17E-05
F1S3Y7
Uncharacterized protein
7
4.48
147.01
7.06
0.31
1.04E-04
F2X043
Lactoperoxidase
25
44.80
80.69
8.7
0.31
3.14E-04
E5G7E7
Fatty acid-binding protein
8
49.62
14.78
6.73
0.32
4.91E-05
Q4VS17
FGFBP
2
28.04
11.94
9.16
0.33
4.40E-03
G0Z2N2
L-lactate dehydrogenase
5
13.17
36.72
5.72
0.33
1.03E-03
Q9MZY2
Airway lactoperoxidase
19
31.00
80.49
8.95
0.33
4.00E-04
P02769
Serum albumin
14
17.96
69.69
6.08
0.34
1.93E-04
2
70.00
2.27
8.5
0.35
2.29E-04
7
52.03
14.16
4.61
0.36
1.71E-05
3
24.00
12.93
5.73
0.36
2.35E-04
3
20.80
16.64
8.2
0.37
1.74E-04
Q9TRB9 P00711 Q52RN5 P79345
Enterotoxin-binding glycoprotein PP20K Alpha-lactalbumin Superoxide dismutase [Cu-Zn] Epididymal secretory protein E1
F1RRP2
Uncharacterized protein
5
7.71
81.47
8.82
0.38
5.67E-03
F6R3I5
Uncharacterized protein
7
29.34
27.14
8.29
0.39
1.57E-02
3
7.36
52.58
5.52
0.40
5.77E-03
Q32KV6
Nucleotide exchange factor SIL1
P22226
Cathelicidin-1
2
18.06
17.65
7.54
0.43
3.17E-02
P26779
Proactivator polypeptide
5
12.57
58.05
5.08
0.50
1.78E-02
6
20.25
41.08
4.79
0.51
3.21E-03
2
9.24
20.99
6.39
0.52
8.04E-03
3
32.63
10.60
6.79
0.53
1.65E-03
A5YVD9 P62833 Q307G4
45 kDa calcium-binding protein Ras-related protein Rap-1A Cytosolic NADP-isocitrate dehydrogenase
P50448
Factor XIIa inhibitor
5
7.90
51.72
6.19
0.54
3.30E-02
Q9XSG3
Isocitrate dehydrogenase
10
25.36
46.78
6.13
0.55
3.60E-02
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[NADP] cytoplasmic Q861V5
Accepted Article
P08037
Peptidyl-prolyl cis-trans isomerase Beta-1,4-galactosyltransferase I
5
31.61
16.93
8.46
0.57
1.80E-03
10
30.35
44.76
9.2
0.59
1.99E-03
P11151
Lipoprotein lipase
5
15.48
53.59
8.61
0.59
1.00E-02
Q0IIH5
Nucleobindin 2
8
23.85
49.19
5.12
0.62
3.55E-02
12
18.49
82.43
7.07
0.65
1.03E-03
P81265
Polymeric immunoglobulin receptor
F6PQI6
Uncharacterized protein
20
30.91
82.52
7.68
0.69
6.11E-03
A2I7N2
Serpin A3-6
4
8.00
46.39
5.34
0.70
6.31E-03
G3N1U4
Uncharacterized protein
3
6.81
46.16
6.05
0.72
1.05E-03
G8JKW7
Uncharacterized protein
4
9.95
46.34
6.29
0.72
2.48E-03
A1E0X4
Beta-actin
13
48.90
40.58
5.66
0.73
4.72E-03
D7NJ85
Beta casein
7
25.45
25.10
5.26
0.77
1.23E-02
Q95100
Kappa-casein
3
35.25
13.14
5.86
0.79
1.13E-02
F1MZW0
Uncharacterized protein
7
14.41
67.88
5.91
0.81
3.70E-02
Q0P569
Nucleobindin-1
8
23.84
54.98
5.11
0.84
2.46E-02
Q9GKP1
Complement C3
18
11.56
186.80
6.09
0.84
3.57E-02
F1N5M2
Uncharacterized protein
7
17.30
53.36
5.42
1.24
2.81E-02
Q66RQ0
Vitamin D-binding protein
4
17.89
24.54
5.02
1.26
2.49E-02
P29701
Alpha-2-HS-glycoprotein
8
24.45
38.68
5.17
1.26
1.64E-02
B8R1K3
Transferrin
18
27.56
77.66
6.92
1.26
2.40E-02
P81187
Complement factor B
3
5.65
85.37
7.87
1.37
2.25E-02
P00735
Prothrombin
3
6.08
70.50
5.97
1.38
4.51E-02
E1BI82
Uncharacterized protein
6
10.77
69.15
8.29
1.40
3.96E-02
P12725
Alpha-1-antiproteinase
10
20.43
45.98
5.83
1.54
2.77E-02
G3EHG5
TIP47
4
14.87
47.36
5.74
1.70
7.83E-03
P00978
Protein AMBP
3
10.23
39.23
7.81
1.72
6.61E-03
Q7SIH1
Alpha-2-macroglobulin
17
16.76
167.57
5.71
1.73
5.70E-03
F1MFI4
Uncharacterized protein
2
4.17
69.88
5.1
1.88
2.01E-02
Q3SZZ9
FGG protein
8
21.15
49.17
5.56
1.88
3.18E-02
G3N0V0
Uncharacterized protein
6
15.64
35.95
8.05
2.01
5.34E-03
G5E604
Uncharacterized protein
3
29.90
11.06
8.02
2.15
3.49E-02
F1MAV0
Uncharacterized protein
3
8.48
56.44
8.5
2.17
2.75E-03
P17697
Clusterin
5
14.35
51.11
5.73
2.19
2.57E-03
C0LQK8
Myostatin
8
27.47
42.51
6.05
2.23
1.26E-02
P80221
C-X-C motif chemokine 6
2
22.32
11.59
9.4
2.40
4.43E-03
A4ZVY8
Beta-2-microglobulin
2
16.95
13.61
6.4
2.42
1.02E-04
A7E350
PLG protein
6
9.53
81.58
8.33
2.54
6.33E-04
P02672
Fibrinogen alpha chain
11
18.37
67.01
6.73
2.74
2.17E-02
F1MNN7
Uncharacterized protein
3
6.44
53.69
6.61
2.91
4.42E-03
A6QNJ8
GANAB protein
3
3.53
109.08
5.83
2.97
2.66E-02
G3N2D7
Uncharacterized protein
2
37.93
12.11
4.78
3.36
4.55E-04
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P20757
Accepted Article
Q1RMN8
Angiotensinogen Immunoglobulin light chain, lambda gene cluster
6
11.14
51.30
6.54
3.39
2.14E-03
6
33.33
24.54
7.54
4.53
8.21E-03
A6QM09
Uncharacterized protein
4
15.09
24.73
7.51
4.77
6.72E-03
P01968
Hemoglobin subunit alpha-2
2
17.02
14.98
8.91
5.03
1.25E-02
G5E5T5
Uncharacterized protein
6
15.93
42.36
4.89
5.63
3.90E-07
Q0ZCB4
Apolipoprotein E
5
23.21
27.07
8.87
5.69
5.28E-04
G5E513
Uncharacterized protein
4
10.50
49.97
5.32
6.00
2.53E-05
Q3SYR8
Immunoglobulin J chain
2
14.65
17.86
5.1
6.53
2.21E-03
Q3Y443
Alpha s2 casein
10
36.94
26.13
8.24
6.57
6.00E-03
G3N342
Uncharacterized protein
3
7.28
47.83
7.23
6.94
1.03E-02
Q9TSN7
Hemoglobin subunit alpha-1
2
16.90
15.13
8.08
7.71
4.12E-02
F1MPP2
Uncharacterized protein
8
37.23
29.05
8.25
9.48
1.77E-03
F1MLW8
Uncharacterized protein
3
15.88
24.62
5.58
10.38
3.74E-05
A6QNW7
CD5L protein
6
18.31
50.21
5.24
14.35
8.53E-07
P04346
Hemoglobin subunit beta-A
6
44.83
15.96
6.36
17.14
1.02E-03
P02072
Hemoglobin beta
5
42.76
15.99
7.06
19.77
1.32E-04
1
Relative abundance of whey proteins in colostrum versus mature milk
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Table 2 Pathway categories of differentially expressed proteins in yak colostrum and mature milk: Kyoto Encyclopedia of Genes and Genomes (KEGG).
Accepted Article
Pathway Name
bta04610:Complement and coagulation cascades
Accession NO. P02672, P50448, Q3SZZ9,
Count
Hits
Percentage
P Value
Fold Enrichment
8
71
14.04
1.91E-08
23.19
P81187, Q7SIH1, P06868, Q2UVX4, P00735
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Accepted Article
(A)
(B)
(C) Fig. 1 Classification of differentially expressed milk proteins based on gene ontology annotation. (A) Biological process; (B) molecular function; (C) cellular component.
This article is protected by copyright. All rights reserved.
