ARTICLE pubs.acs.org/jpr
Proteomic Characterization of Human Milk Fat Globule Membrane Proteins during a 12 Month Lactation Period Yalin Liao,† Rudy Alvarado,‡ Brett Phinney,‡ and Bo L€onnerdal*,† †
Department of Nutrition, ‡Genome Center Proteomics Core Facility, University of California, Davis, California 95616, United States ABSTRACT: The milk fat globule membrane (MFGM) contains proteins which have been implicated in a variety of health benefits. Milk fat globule membrane proteins were isolated from human milk during a 12 month lactation period and subjected to in-solution digestion and liquid chromatography tandem mass spectrometry analysis. Data were pooled, and our results showed that 191 proteins were identified. Relative quantification of the identified MFGM proteins during the course of lactation was performed by label free spectral counting and differentiation expression analysis, which showed some proteins decreasing during the course of lactation whereas some increased or remained at a relatively constant level. The human MFGM proteins are distributed between intracellular, extracellular, and membrane-associated proteins, and they are mainly involved in cell communication and signal transduction, immune function, metabolism and energy production. This study provides more insights into the dynamic composition of human MFGM proteins, which in turn will enhance our understanding of the physiological significance of MFGM proteins. KEYWORDS: human milk, milk fat globule membrane, protein, proteomics, LC MS/MS
’ INTRODUCTION The milk fat globule membrane (MFGM) is the membrane surrounding lipid droplets in milk. MFGM consists of a complex organization of lipid bilayer rich in integral and peripheral proteins, which surrounds a triacylglycerol core. MFGM proteins represent 1 4% of total milk protein content and 1% of the total globule mass.1,2 MFGM proteins have been implicated in beneficial bioactivities, such as antimicrobial (bacteria, virus)3 and anticancer effects.4,5 A bovine milk fraction containing MUC1 has been shown to inhibit hemagglutination of Vibrio cholerae and Escherichia coli.6 In addition, purified mucin, a milk fat globule membrane constituent, was demonstrated to decrease the adherence of Yersinia enterolytica to intestinal membranes.7 Human milk mucin components were able to bind to various rotavirus strains and prevent replication, and the ability was correlated to lactadherin.8 Furthermore, the content of lactadherin in breast milk was shown to be negatively correlated to symptomatic rotavirus infection in Mexican infants.9 The MFGM fraction has also been found to reduce rotavirus in vitro.10 Our preliminary results have shown a significant inhibitory effect of camel MFGM against hepatitis C virus (HCV) infection in vitro (unpublished data). Based on these observations, the MFGM fraction has been proposed as a potential nutraceutical, see review.11 A recent study has shown that addition of a bovine MFGM protein fraction to complementary food given daily to 6 12 months old Peruvian infants reduced the prevalence of diarrhea.13 A comprehensive proteomic characterization to unveil the entire profile of MFGM proteins is indispensable to understand the biological significance of this unique milk compartment. Human MFGM proteins have been characterized in the past but only to a limited extent. A recent study by Reinhardt and r 2011 American Chemical Society
Lippolis using quantitative shotgun proteomics identified 138 proteins in the bovine MFGM, with 26 proteins up-regulated and 19 proteins down-regulated at 1 week of lactation compared to colostrum MFGM.13 In another study, six MFGM proteins from buttermilk protein concentrates (BPC50 and BPC60) (fatty acid binding protein, butyrophilin, PAS 6/7, adipophilin, xanthine oxidase, and mucin 1) were quantified using highresolution selected reaction monitoring mass spectrometry.14 Charlwood et al. used human milk from 6 to 373 days postpartum and identified seven major MGFM proteins: R-lactalbumin, lysozyme precursor, β-casein, clusterin, lactoferrin, polymeric immunoglobulin receptor precursor, and human milk fat globule EGF-factor 8 protein.15 Interestingly, by using 2-DE separation and in-gel digestion followed by MALDI-TOF MS, Fortunato et al. were able to derive 107 2-DE spots which correspond to ∼50 proteins from pooled human colostrum.16 The objective of this study was to further identify human MFGM proteins and examine their dynamic expression during the 12 month lactation period using a liquid chromatography tandem mass spectrometry (LC MS/MS) approach.
’ MATERIALS AND METHODS Milk Sample Collection
This study procedure was approved by the Institutional Review Board (IRB) at University of California Davis. Colostrum and milk samples from 1, 2, 3, 6, and 12 months of lactation were Received: February 18, 2011 Published: June 29, 2011 3530
dx.doi.org/10.1021/pr200149t | J. Proteome Res. 2011, 10, 3530–3541
Journal of Proteome Research collected from 30 healthy donors, with samples from 5 mothers collected at each time point. Milk was collected from one breast (at least 2 4 h after prior nursing) by manual expression (colostrum) or manual breast pump into 50 mL polypropylene containers. Mothers who delivered singleton term infants (gestational age 38 42 wks) were recruited, and mothers with maternal illnesses, such as cold, mastitis, and flu were excluded. All mothers were exclusively breast-feeding up to 6 months and partially up to 12 months of lactation. Colostrum was collected within 48 h of lactation initiation.17 All samples were immediately stored at 20 °C until further analysis. Protein Extraction
Milk samples (5 mL) were thawed at 4 °C. CaCl2 was added to adjust the final calcium concentration to 0.06 M, the pH was adjusted to 4.6 and then incubated at room temperature for 1 h.18 This method was used as it also allowed us to obtain the whey proteins for a separate study.19 The samples were subjected to centrifugation at 13,000 g for 30 min twice. The milk cream was collected by a spatula into a clean tube and protease inhibitors (Roche, Complete mini EDTA-free) were added. MFGM proteins were extracted by a procedure adapted from the procedures of Fortunato et al.,16 the procedure of Reinhardt and Lippolis13 and Wilson et al.20 The cream was washed with 10 volumes of cold PBS, and the mixture was incubated at room temperature for 5 min and then vortexed for 15 s prior to centrifugation at 13,000 g for 15 min. Triacylglycerols were removed. The wash step was repeated at least three times until the supernatant was clear. The fat globules were transferred to a clean tube, vortexed for 5 min twice, then incubated at 55 °C for 5 min, followed by centrifugation at 2,000 g for 10 min. The top layer (fat globules) was collected into a clean tube, and buffer (63 mM Tris 3 HCl, pH 6.8, 2% SDS) was added to the fat globules with a fat/buffer ratio of 2:3 (v/v). The mixture was incubated at room temperature for 1 h and then centrifuged at 10,000 g for 10 min. MFGM proteins were collected from the lower phase. Final concentrations of the MFGM proteins were measured by using the Microplate BCA protein assay kit (Fisher Scientific). Sample Processing before LC MS/MS
1. In-Solution Digestion. Soluble proteins were digested insolution using a standard trypsin digestion protocol. Briefly, the proteins were dried in a vacuum centrifuge and then resuspended in 200 μL of 50 mM ammonium bicarbonate. The proteins were then reduced with tris(2-carboxyethyl)phosphine (TCEP) (Pierce), alkylated by iodoacetamide, and treated with trypsin (modified trypsin, sequencing grade, Promega) at a 1:50 enzyme to substrate ratio (w/w) overnight at 37 °C. The trypsin digested samples were then dried. 2. Delipidation. To remove the residual lipids that might affect the performance of the mass spectrometer, the digested samples were further delipidated by the following steps: the samples were resuspended in 200 μL of 50 mM ammonium bicarbonate and sonicated for 10 min. A total of 200 μL of methylene chloride was added. The samples were sonicated for 10 min and then centrifuged for 10 min at 13, 000 g. The top layer, which contains the peptides, was siphoned off and dried by speed-vacuum. The samples were then resuspended in 2% ACN/0.1% TFA for MS analysis. LC MS/MS
A Paradigm MG4 HPLC system (Michrom Bio Resources) coupled with a Thermo Finnigan LTQ ion trap mass spectrometer
ARTICLE
(Thermo Scientific) through a Michrom Advance Captivespray source was used for protein separation and analysis. A total of 15 μg of each digested sample was loaded onto a trap column (Zorbax 300SB-C18, 5 μm, 0.3 mm 5 mm: Agilent Technologies Inc.) and desalted online. Peptides were then eluted from the trap and separated with a reverse-phase Michrom Magic C18AQ (200 μm 150 mm) capillary column (Michrom Bioresources, Inc.) at a flow rate of 2 μL/min. Peptides were eluted using a 90 min gradient of 2% B to 35% B over 60 min, 35% B to 80% B for 15 min, held at 80% B for 1 min, 80% B to 5% B in 1 min, and re-equilibrated for 13 min at 5% B (A = 0.1% formic acid, B = 100% acetonitrile) and directly sprayed into the mass spectrometer. The mass spectrometer was operated using a standard top 10 method, where one survey scan was followed by 10 MS/MS scans of the most intense ions eluting from the column. Dynamic exclusion was enabled. Immunoblotting Analysis
MFGM proteins from each group were pooled, and 20 μg electrophoresed through 8% polyacrylamide gel, transferred onto nitrocellulose membrane at 350 mA for 60 min, and blocked overnight in PBS/0.1% Tween-20 (PBST) with 5% nonfat milk at 4 °C. Antibodies againt xanthine oxidase, insulin-like growth factor binding protein 2 (IGFBP2), and alpha-enolase were purchased from Santa Cruz Biotechnologies. Bands were detected using Super Signal Femto chemiluminescent reagent (Pierce) and quantified using the Chemi-doc gel quantification system (Bio-Rad). All data were normalized to β-actin.
Data Analysis
Database Searching. Tandem mass spectra were extracted by BioWorks version 3.3. Charge state deconvolution and deisotoping were not performed. All MS/MS samples were analyzed using X! Tandem (www.thegpm.org; version TORNADO (2010.01.01.4)). X! Tandem was set up to search the Uniprot human complete proteome set database (2010_07, 21 525 entries) and 110 nonhuman common laboratory contaminants from the common repository of adventitous proteins database (www.thegpm.org) plus an equal number of reverse sequences assuming the digestion enzyme trypsin. X! Tandem was searched with a fragment ion mass tolerance of 0.40 Da and a parent ion tolerance of 1.8 Da. Iodoacetamide derivative of cysteine was specified in X! Tandem as a fixed modification. Deamidation of asparagine and glutamine, oxidation of methionine and tryptophan, sulphone of methionine, tryptophan oxidation to formylkynurenin of tryptophan, and acetylation of the N-terminus were specified in X! Tandem as variable modifications. Criteria for Protein Identification. Scaffold (version Scaffold_3_00_03, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability as specified by the Peptide Prophet algorithm.21 Protein identifications were accepted if they could be established at greater than 95.0% probability and contained at least one identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm.22 Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Using these Scaffold criteria, a False discovery rate (FRD) was calculated as 0.1% on the peptide level and 10.0% on the protein level using Decoy/Target as discussed in ref 23. 3531
dx.doi.org/10.1021/pr200149t |J. Proteome Res. 2011, 10, 3530–3541
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Table 1. Identified Human Milk Fat Globule Membrane Proteinsa serial no.
identified proteins
UniProt
molecular
no. of unique
sequence
accession no.
weight (kDa)
peptides
coverage (%)
1
14-3-3 protein epsilon
P62258
29
2
16
2
14-3-3 protein zeta/delta
P63104
28
2
13
3
1-acylglycerol-3-phosphate O-acyltransferase ABHD5
Q8WTS1
39
2
11
4
40S ribosomal protein S18
P62269
18
3
14
5
45 kDa calcium-binding protein
Q9BRK5
42
2
8.6
6
4F2 cell-surface antigen heavy chain
P08195
68
2
4.1
7 8
4-trimethylaminobutyraldehyde dehydrogenase 78 kDa glucose-regulated protein
P49189 P11021
54 72
2 3
6.3 7.3
9
actin, alpha skeletal muscle
P68133
42
2
34
10
actin, cytoplasmic 2
P63261
42
19
59
11
adenosine monophosphate-protein transferase FICD
Q9BVA6
52
2
12
alcohol dehydrogenase [NADP+]
P14550
37
3
16
13
aldo-keto reductase family 1 member C1
Q04828
37
2
13
14
alpha-1-antichymotrypsin
P01011
48
16
30
15 16
alpha-1-antitrypsin alpha-amylase 1
P01009 P04745
47 58
15 4
42 13
17
alpha-enolase
P06733
47
9
38
18
alpha-lactalbumin
P00709
16
23
73
19
alpha-s1-casein
P47710
22
34
65
20
angiopoietin-related protein 4
Q9BY76
45
3
12
21
angiotensin-converting enzyme 2
Q9BYF1
92
2
22
annexin A1
P04083
39
3
13
23 24
annexin A2 annexin A5
P07355 P08758
39 36
5 3
21 14
25
antithrombin-III
P01008
53
3
26
apolipoprotein A-I
P02647
31
22
72
27
apolipoprotein A-II
P02652
11
7
50
28
apolipoprotein A-IV
P06727
45
6
18
29
apolipoprotein B-100
P04114
516
6
30
apolipoprotein D
P05090
21
8
37
31 32
apolipoprotein E ATP-binding cassette subfamily G member 2
P02649 Q9UNQ0
36 72
9 4
39 11
33
beta-1,4-galactosyltransferase 1
P15291
44
7
23
34
beta-2-microglobulin
P61769
14
3
36
35
beta-actin-like protein 2
Q562R1
42
2
18
36
beta-casein
P05814
25
83
94
37
bile salt-stimulated lipase
P19835
79
38
41
38
bone marrow stromal antigen 2
Q10589
20
2
14
39 40
butyrophilin subfamily 1 member A1 C4b-binding protein alpha chain
Q13410 P04003
59 67
40 5
56 12
41
carbonic anhydrase 6
P23280
35
4
25
42
carcinoembryonic antigen-related cell adhesion molecule 1
P13688
58
2
43
CD59 glycoprotein
P13987
14
5
33
44
CD81 antigen
P60033
26
2
15
45
CD9 antigen
P21926
25
3
20
46
cell death activator CIDE-A
O60543
25
6
32
47 48
cell division control protein 42 homologue chitinase-3-like protein 1
P60953 P36222
21 43
3 6
23 23
49
chordin-like protein 2
Q6WN34
47
19
43
50
clusterin
P10909
52
25
45
51
cofilin-1
P23528
19
4
43
52
collagen alpha-1(XXIV) chain
Q17RW2
175
2
2.2
53
collagen alpha-4(IV) chain
P53420
164
2
3.3
3532
4.8
3.9
6.5
2.6
4.8
dx.doi.org/10.1021/pr200149t |J. Proteome Res. 2011, 10, 3530–3541
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ARTICLE
Table 1. Continued serial no.
identified proteins
UniProt
molecular
no. of unique
sequence
accession no.
weight (kDa)
peptides
coverage (%)
187 193
43 36
54 55
complement C3 complement C4-B
P01024 P0C0L5
56
complement component C9
P02748
63
2
5.4
57
complement factor B
P00751
86
3
5.1
58
cystatin-C
P01034
16
3
59
cytoplasmic aconitate hydratase
P21399
98
3
7
60 61
cytosolic nonspecific dipeptidase dehydrogenase/reductase SDR family member 1
Q96KP4 Q96LJ7
53 34
7 2
28 15
62
dihydropyrimidinase-related protein 3
Q14195
62
2
63
DNA-binding protein SATB2
Q9UPW6
83
2
4
64
elongation factor 1-alpha 1
P68104
50
6
19
65
elongation factor 2
P13639
95
3
6.3
66
endoplasmin
P14625
92
2
4.1
67
erythrocyte band 7 integral membrane protein
P27105
32
7
68 69
eukaryotic initiation factor 4A-III ezrin
P38919 P15311
47 69
2 5
70
fatty acid synthase
P49327
273
69
40
71
fatty acid-binding protein, heart
P05413
15
9
63
72
fibrinogen alpha chain
P02671
95
5
73
fibrinogen beta chain
P02675
56
3
12
74
fibrinogen gamma chain
P02679
52
5
15
75
folate receptor alpha
P15328
30
8
55
76 77
formin-2 fructose-bisphosphate aldolase A
Q9NZ56 P04075
180 39
2 2
78
galectin-3-binding protein
Q08380
65
7
79
gamma-glutamyltranspeptidase 1
P19440
61
5
12
80
gelsolin
P06396
86
4
10
81
glutamate receptor, ionotropic kainate 1
P39086
104
2
82
glutathione peroxidase 3
P22352
26
2
13
83
glyceraldehyde-3-phosphate dehydrogenase
P04406
36
5
26
84 85
Golgi-associated plant pathogenesis-related protein 1 G-protein coupled receptor family C group 5 member B
Q9H4G4 Q9NZH0
17 45
3 2
31 6.5
86
GTP-binding protein SAR1a
Q9NR31
22
4
27
87
guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
P62873
37
3
16
88
guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-2
P62879
37
3
12
89
guanine nucleotide-binding protein G(q) subunit alpha
P50148
42
3
10
90
haptoglobin
P00738
45
2
91
heat shock cognate 71 kDa protein
P11142
71
4
92 93
heat shock protein beta-1 heat shock protein HSP 90-alpha
P04792 P07900
23 85
5 8
36 13
94
hemoglobin subunit beta
P68871
16
2
13
95
HLA class II histocompatibility antigen, DR alpha chain
P01903
29
5
28
96
HLA class II histocompatibility antigen, DR beta 5 chain
Q30154
30
2
16
97
HLA class II histocompatibility antigen, DRB1-1 beta chain
P04229
30
3
22
98 99 100
HLA class II histocompatibility antigen, DRB1-15 beta chain hyaluronan and proteoglycan link protein 3 Ig alpha-1 chain C region
P01911 Q96S86 P01876
30 41 38
2 8 18
101
Ig alpha-2 chain C region
P01877
37
4
60
102
Ig gamma-1 chain C region
P01857
36
2
12
103 104
Ig kappa chain C region Ig kappa chain V III region GOL
P01834 P04206
12 12
8 3
89 39
105
Ig kappa chain V III region vg (Fragment)
P04433
13
2
23
106 107
Ig lambda-1 chain C regions Ig lambda-3 chain C regions
P0CG04 P0CG06
11 11
2 6
67 75
3533
36 33
19
3.3
43 5.8 9.7
6.9
3.1 6.9 19
4.6
9.1 8.4
8.3 29 62
dx.doi.org/10.1021/pr200149t |J. Proteome Res. 2011, 10, 3530–3541
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Table 1. Continued serial no.
identified proteins
UniProt
molecular
no. of unique
sequence
accession no.
weight (kDa)
peptides
coverage (%)
108
Ig mu chain C region
P01871
49
7
23
109
immunoglobulin J chain
P01591
18
6
53
110
insulin-like growth factor-binding protein 2
P18065
35
4
20
111
isocitrate dehydrogenase [NADP] cytoplasmic
O75874
47
2
112
kappa-casein
P07498
20
23
65
113
lactadherin, milk fat globule EGF factor 8 protein
Q08431
43
41
85
114
lactoperoxidase
P22079
80
2
5.5
115 116
lactoferrin lanosterol synthase
P02788 P48449
78 83
174 4
88 6.6
117
leucine-rich alpha-2-glycoprotein
P02750
38
5
118
lipoprotein lipase
P06858
53
9
31
119
L-lactate
P07195
37
3
11
120
long-chain-fatty-acid--CoA ligase 1
P33121
78
2
4.7
121
long-chain-fatty-acid CoA ligase 3
O95573
80
3
9.4
dehydrogenase B chain
7.2
22
122
long-chain-fatty-acid CoA ligase 4
O60488
79
7
14
123 124
lysozyme C macrophage mannose receptor 1
P61626 P22897
17 166
13 12
69 14
125
MARCKS-related protein
P49006
20
2
25
126
matrilin-3
O15232
53
3
12
127
metalloproteinase inhibitor 1
P01033
23
2
15
128
methyltransferase-like protein 7A
Q9H8H3
28
2
11
129
moesin
P26038
68
2
130
monocyte differentiation antigen CD14
P08571
40
10
131 132
mucin-1 mucin-4
P15941 Q99102
122 232
7 10
8 8.9
133
myosin-9
P35579
227
4
3.5
134
Na(+)/H(+) exchange regulatory cofactor NHE-RF1
O14745
39
2
6.7
135
neutral alpha-glucosidase AB
Q14697
107
2
136
neutrophil defensin 1
P59665
10
2
19
137
nicotinamide phosphoribosyltransferase
P43490
56
3
12
138
nonspecific lipid-transfer protein
P22307
59
2
4.4
139 140
nucleobindin-2 olfactomedin-4
P80303 Q6UX06
50 57
5 2
15 8.4
141
osteopontin
P10451
35
7
26
142
parathyroid hormone-related protein
P12272
20
2
13
143
peptidyl-prolyl cis trans isomerase A
P62937
18
5
51
144
perilipin-2
Q99541
48
37
81
145
perilipin-3
O60664
47
15
63
146
peroxiredoxin-6
P30041
25
2
19
147 148
phosphoglucomutase-1 phosphoglycerate kinase 1
P36871 P00558
61 45
2 5
6.8 15
149
platelet glycoprotein 4
P16671
53
7
19
150
polymeric immunoglobulin receptor
P01833
83
23
37
151
proactivator polypeptide
P07602
18
152
probable cation-transporting ATPase 13A4
Q4VNC1
153
profilin-1
154 155 156 157
6.9 38
5.1
58
6
134
2
P07737
15
2
21
prolactin-inducible protein
P12273
17
3
29
protein disulfide-isomerase protein disulfide-isomerase A3 protein disulfide-isomerase A6
P07237 P30101 Q15084
57 57 48
6 4 2
21 8.5 6.6
158 159
Rab GDP dissociation inhibitor beta Ras-related C3 botulinum toxin substrate 1
P50395 P63000
51 21
3 2
9.4 19
160 161
Ras-related protein Rab-10 Ras-related protein Rab-18
P61026 Q9NP72
23 23
2 6
12 41
3534
2.6
dx.doi.org/10.1021/pr200149t |J. Proteome Res. 2011, 10, 3530–3541
Journal of Proteome Research
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Table 1. Continued serial no.
identified proteins
UniProt
molecular
no. of unique
sequence
accession no.
weight (kDa)
peptides
coverage (%)
162
Ras-related protein Rab-1A
P62820
23
3
21
163
Ras-related protein Rab-2A
P61019
24
2
14
164
Ras-related protein Rab-5C
P51148
23
2
12
165
Ras-related protein Ral-B
P11234
23
3
18
166
Ras-related protein Rap-1b
P61224
21
2
14
167 168
sclerostin domain-containing protein 1 selenium-binding protein 1
Q6 4U4 Q13228
23 52
3 2
23 5.9
169
serum albumin
P02768
69
25
170
sterol-4-alpha-carboxylate 3-dehydrogenase, decarboxylating
Q15738
42
2
171
sulfhydryl oxidase 1
O00391
83
10
172
synaptic vesicle membrane protein VAT-1 homologue
Q99536
42
2
173
synaptosomal-associated protein 23
O00161
23
2
13
174
syntaxin-3
Q13277
33
3
20
175 176
syntaxin-binding protein 2 syntenin-1
Q15833 O00560
66 32
2 4
7.8 28
177
tenascin
P24821
241
119
178
thrombospondin-1
P07996
129
7
7
179
transforming protein RhoA
P61586
22
2
17
180
transthyretin
P02766
16
3
44
181
triosephosphate isomerase
P60174
27
2
12
182
tubulin alpha-1B chain
P68363
50
2
8
183 184
tumor necrosis factor receptor superfamily member 11B ubiquitin
O00300 P62988
46 9
3 4
12 62
52 8.6 19 6.9
58
185
UTP glucose-1-phosphate uridylyltransferase
Q16851
57
8
23
186
vitamin-D-binding protein
P02774
53
3
13
187
vitronectin
P04004
54
6
18
188
von Willebrand factor A domain-containing protein 1
Q6PCB0
47
2
189
xanthine dehydrogenase/oxidase
P47989
146
78
190
zinc finger protein 687
Q8N1G0
130
2
191
zinc-alpha-2-glycoprotein
P25311
34
10
9.2 59 3.3 40
a
One hundred and ninety-one proteins were identified and listed alphabetically. Accession numbers were assigned according to UniProt Database Release 15.14. The complete identification statistics can be found in the Scaffold located in the proteome data set uploaded to the Tranche proteome commons repository (Materials and Methods).
Spectral Counting and Shared Peptide Refinement. Label free quantitation was performed using a standard spectral counting method. Scaffold 3.3 was used to sum spectral counts and group peptides into proteins. An in house script was written to refine spectral counts from peptides shared across multiple proteins according to the method in ref 24. The refined spectral counting data was filtered so a protein required a minimum of four spectral counts in any one category, and a heat map was generated using the hierarchical clustering tool in Spotfire 3.2 using an unweighted pair-group method with arithmetic mean (UPGMA) method. The most differentiated proteins were identified by calculating a P value using a one-way ANOVA analysis in Spotfire 3.2. Proteomics Data Set. The data associated with this manuscript may be downloaded from ProteomeCommons.org Tranche using the following hash: wultCo8 1POOWyJXSTj1LVYuONCJKoRFPF93MAK1q0MUlB25H5JnmIJRy1iqYZRyVW+6 kWJe9 kWrPyMfVzgsK8WZwLUAAAAAAAAaLw== The hash may be used to prove exactly what files were published as part of this paper’s data set, and the hash may also be used to check that the data has not changed since publication.
’ RESULTS AND DISCUSSION The MFGM fraction is a rich source of milk proteins. The present study characterized the proteins from human MFGM, and 191 proteins were identified (see Table 1) with their relative expression during a 12 month lactation period. The human MFGM protein compartment, though quantitatively a minor fraction of total milk protein, contains a wide variety of proteins, and their functions suggest that they are involved in multiple biological functions. In this study, proteins supporting growth and maintenance represent 8.4% of the total identified proteins, and those involved in immune function represent 19.9%, suggesting novel functions of the MFGM proteins. Among those proteins are the polymeric immunoglobulin receptor and human leukocyte antigens (HLA). Polymeric immunoglobulin receptor is expressed specifically on glandular epithelia, and in the mammary gland it is a key member of the mucosal immune system, facilitating the epithelial transcytosis of sIgA.25 One class of HLA was detected, HLAII, but not HLAI, HLAIII. These proteins control the antigen (endogenous and exogenous) presentation to T cells and may possibly be involved 3535
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Journal of Proteome Research in the differences in immune function observed between breastfed and formula-fed infants.26 As can be seen in Figure 1, proteins
Figure 1. Pie graph representation of functional characteristics of human milk fat globule membrane proteins. Abbreviations used: C;S (cell communication;signal transduction); N (nucleobase, nucleoside, nucleotide and nucleic acid regulation); I (immune response); G;M (cell growth and/or maintenance); T (transport); P (protein metabolism); M;E (metabolism;energy pathways); M (multiple).
Figure 2. Pie graph representation of subcellular localization of human milk fat globule membrane proteins.
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involved in metabolism and energy production are the major category of the profile and account for 21.5% of all identifed proteins, indicating a role for MFGM proteins specifically in mammary cell energy metabolism. Proteins in this category not identified previously include alcohol dehydrogenase [NADP+], 4-trimethylaminobutyraldehyde dehydrogenase, aldo-keto reductase family 1 member C1, cytoplasmic aconitate hydratase, dehydrogenase/reductase SDR family member 1, isocitrate dehydrogenase [NADP] cytoplasmic, lanosterol synthase, longchain-fatty-acid CoA ligase 1/3/4, myosin-9, neutral alphaglucosidase AB, proxiredoxin-6, phosphoglucomutase-1, phosphoglycerate kinase 1, sterol-4-alpha-carboxylate 3-dehydrogenase, decarboxylating, glutathione peroxidase 3, angiotensin-converting enzyme 2, 1-acylglycerol-3-phosphate O-acyltransferase ABHD5, gamma-glutamyltranspeptidase 1, perilipin-2/3, probable cationtransporting ATPase 13A4, and adenosine monophosphate-protein transferase FICD. Three caseins were identified in the human MFGM, including alpha-s1-casein, beta-casein, and kappa-casein. Milk caseins supply the neonate with amino acids, calcium, and phosphate and possibly contribute to the high bioavailability of milk calcium.27 When examining mammary gland epithelial cells, particularly the edges of the alveolar boundary, membrane bound vesicles and flattened sacs which bulge into the lumen of the alveolus were frequently found to contain casein granules.28 These casein granules were suggested to aid in separating the droplet from the alveolar lumen in this region. Therefore, one function of caseins in MFGM might be to facilitate the globule membrane being secreted and dissociated from the synthesis machinery. In fact, xanthine oxidase has also been suggested to have functions different from its traditional assignment. Xanthine oxidase functions in the oxidative metabolism of purines in
Figure 3. Heat map presentation of spectral counting data. Hierarchical clustering analysis was performed using Euclidian distance. Each column represents the summed total spectral count of each group, and each row represents indivudual proteins. Proteins with >4 spectral counts in one group (149 total) are included. Colors represent the scaled fold-change of spectrum counts between samples within a row. Red color represents higher expression and blue color represents lower expression. 3536
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Figure 4. Continued on next page 3537
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Figure 4. Box plot of differential expression for the following proteins: insulin-like growth factor-binding protein 2 (IGFBP2), annexin A2, tenascin, long-chain-fatty-acid CoA ligase 4, butyrophilin subfamily 1 member A1, HLA class II histocompatibility antigen, DR alpha chain, complement C3, xanthine dehydrogenase/oxidase, and alpha-enolase. Each graph represents the median spectral counts and standard error of individual replicate. There were five replicates for each protein.
the liver,29 whereas it forms a complex with butyrophilin and adipophilin at the inner MFGM and is responsible for lipid droplet formation/secretion.30,31 Lipid homeostasis is important for the milk fat globule, and it should be noted that 25 proteins (13.1% of all proteins) specifically involved in lipid metabolism were identified in this fraction. These proteins span a variety of processes in lipid metabolism, including lipid
de novo synthesis (1-acylglycerol-3-phosphate O-acyltransferase ABHD5, fatty acid synthase, lanosterol synthase, sterol-4-alphacarboxylate 3-dehydrogenase , and triosephosphate isomerase); lipid transport (apolipoprotein A-I, apolipoprotein A-II, apolipoprotein A-IV, apolipoprotein B-100, apolipoprotein D, apolipoprotein E, ATP-binding cassette subfamily G member 2, and fatty acid-binding protein). We also 3538
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Figure 5. Immunoblotting confirmation for relative expression of MFGM proteins. Xanthine oxidase, insulin-like growth factor binding protein 2, and alpha-enolase protein in human MFGM extracted from colostrum, and milk samples from 1, 2, 3, 6, and 12 months of lactation were analyzed. Values are means ( SEM run in triplicates, and β-actin served as internal control.
found proteins involved in milk fat globule lipid droplet formation and secretion (butyrophilin and xanthine dehydrogenase/ oxidase), lipid storage (perilipin-2 and perilipin-3), hydrolysis of milk triacylglycerols (bile salt- stimulated lipase), fatty acid uptake by the mammary gland from the circulation (lipoprotein lipase), activation of long-chain fatty acid for both cellular lipids synthesis and oxidation (long-chain-fatty-acid-CoA ligase 1/3/4), lipid degradation (peroxiredoxin-6), bile acid and bile salt transport, and cholesterol homeostasis (aldo-keto reductase family 1 member C1). The cellular distribution data for the identified proteins show that 24.1% are membrane associated proteins, 30.9% cytoplasmic proteins, 35.1% extracellular proteins, 4.2% nuclear proteins, and
5.8% are typically located at multiple subcellular compartments (Figure 2). Cavaletto et al. proposed a molecular architecture for the human MFGM (an average globule diameter of 4 μm) with a center triacylglycerol core, an outer membrane, and an aqueous domain in between.2 In this study, after extensively washing away whey proteins on the fat globule, detergent was used to permeabilize the outer lipid bilayer. The large proportion of nonmembrane proteins identified could be a result of efficiently catching the trapped proteins between the outer fat globule membrane and the triacylglycerol core of the milk fat. The possibility of whey protein contamination is expected to be minimal, as results obtained from this study are significantly different from our previous study on human milk whey proteins.19 3539
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Journal of Proteome Research Relative quantification of the identified MFGM proteins during the course of lactation was performed by label free spectral counting and differentiation expression analysis, and data for part of the minor proteins are shown in Figures 3 and 4. The following proteins are expressed at higher abundance during early lactation: alpha-1-antitrypsin, alpha-amylase, apolipoprotein D, apolipoprotein E, bone marrow stromal antigen 2, chordinlike protein 2, gamma-glutamyltranspeptidase 1, IGFBP2, long chain fatty acid-coA ligase 4, MARCKS-related protein, matrilin3, and ubiquitin. And the following proteins are expressed at higher abundance during late lactation: CD9 antigen, fatty acidbinding protein, folate receptor alpha, gelsolin, glutathione peroxidase 3, heat shock protein beta-1, lysozyme C, nonspecific lipid transfer protein, and proactivator polypeptide. Figure 4 shows the most differentially expressed proteins: annexin, tenascin, longchain-fatty-acid CoA ligase 4, IGF-BP2, butyrophilin, HLA class II histocompatibility antigen, DR alpha chain, xanthine oxidase, alphaenolase, and complement C3. Figure 5 shows the immunoblotting confirmation for the presence and relative abundance of xanthine oxidase, IGFBP2, and alpha-enolase. In summary, by using LC-MS/MS proteomics analysis, 191 proteins were identified in the human MFGM. The human MFGM proteins are distributed between intracellular, extracellular, and membrane associated proteins, and they are mainly involved in cell communication and signal transduction, immune function, metabolism, and energy production; it is also likely that many of these proteins are involved in, and reflective of, mammary gland metabolic activities and milk biosynthesis. The present study identifies proteins endogenous to the human MFGM and provides further insights into this unique human milk component which may also help unveiling functional applications for this fraction from bovine milk. Milk-based infant formulas have to date been based on skim milk and whey protein concentrate and are thus void of MFGM proteins; further knowledge about their composition and biological significance in human milk may provide support for adding this fraction from bovine milk, which is now commercially available.
’ CONCLUSIONS The present study enhances our knowledge about the complexity of MFGM proteins in milk from healthy lactating mothers. The protein profile and composition of the milk fat globule membrane differ vastly from milk whey, implicating differences in their biological functions such as milk biogenesis and supporting infant growth, health, and development. The data obtained show that MFGM components are involved in lipid metabolism and energy production, important in milk synthesis and secretion, but also in immune function, possibly implicating a role in the known difference in prevalence of infections between breast-fed infants and formula-fed infants, as infant formulas are devoid of MFGM proteins. In summary, this study is the first attempt to use a quantitative proteomic approach which enables a more comprehensive understanding of the human MFGM proteome.
’ AUTHOR INFORMATION Corresponding Author
*Mailing address: Department of Nutrition, University of California, Davis, One Shields Avenue, Davis, CA 95616, USA.
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Telephone: 530-752-8347. Fax: 530-752-3564 E-mail: bllonnerdal@ ucdavis.edu.
’ ACKNOWLEDGMENT The authors would like to thank Dr. Deshanie Rai for constructive discussions and Mead Johnson Nutrition for supporting this study. We also want to thank Dr. Ian J. Griffin for milk sample collections; Mrs. Xiaogu Du for milk MFGM protein extractions; and Diana Tran and Bonnie Ching for LC-MS/MS sample preparations. ’ REFERENCES (1) L€ onnerdal, B.; Woodhouse, L.; Glazier, C. Compartmentalization and quantitation of protein in human milk. J. Nutr. 1987, 117 (8), 1385–1395. (2) Cavaletto, M.; Giuffrida, M. G.; Conti, A. The proteomic approach to analysis of human milk fat globule membrane. Clin. Chim. Acta 2004, 347 (1 2), 41–48. (3) Peterson, J. A.; Patton, S.; Hamosh, M. Glycoproteins of the human milk fat globule in the protection of the breast-fed infant against infections. Biol. Neonate 1998, 74 (2), 143–162. (4) Imam, A.; Drushella, M. M.; Taylor, C. R.; Tokes, Z. A. Preferential expression of a Mr 155,000 milk-fat-globule membrane glycoprotein on luminal epithelium of lobules in human breast. Cancer Res. 1986, 46 (12 Pt 1), 6374–6379. (5) Snow, D. R.; Jimenez-Flores, R.; Ward, R. E.; Cambell, J.; Young, M. J.; Nemere, I.; Hintze, K. J. Dietary milk fat globule membrane reduces the incidence of aberrant crypt foci in Fischer-344 rats. J. Agric. Food Chem. 2010, 58 (4), 2157–2163. (6) Holmgren, J.; Svennerholm, A.-M.; Lindblad, M.; Strecker, G., Inhibition of bacterial adhesion and toxin binding by glycoconjugate and oligosaccharide receptor analogues in human milk. In Human Lactation, 3rd ed.; Goldman, A. S., Ed.; Perseus Publishing: 1987. (7) Mantle, M.; Basaraba, L.; Peacock, S.; Gall, D. Binding of Yersinia enterocolitica to rabbit intestinal brush border membranes, mucus and mucin. Infect. Immun. 1989, 57, 3292–3299. (8) Yolken, R.; Peterson, J.; Vonderfecht, S.; Fouts, E.; Midthun, K.; Newburg, D. Human milk mucin inhibits rotavirus replication and prevents experimental bacteriostasis. J. Clin. Invest. 1992, 90, 1984–1991. (9) Newburg, D.; Peterson, J.; Ruiz-Palacios, G.; Matson, D.; Morrow, A.; Shults, J.; Guerrero, M.; Chaturvedi, P.; Newburg, S.; CD., S.; MR., T.; RL., C.; LK., P. Role of human milk lactadherin in protection against symptomatic rotavirus infection. Lancet 1998, 18 (351), 1190–1194. (10) Bojsen, A.; Buesa, J.; Montava, R.; Kvistgaard, A.; Kongsbak, M.; Petersen, T.; Heegaard, C.; Rasmussen, J. Inhibitory activities of bovine macromolecular whey proteins on rotavirus infections in vitro and in vivo. J. Dairy Sci. 2007, 90, 66–74. (11) Spitsberg, V. L. Invited review: Bovine milk fat globule membrane as a potential nutraceutical. J. Dairy Sci. 2005, 88 (7), 2289–2294. (12) Zavaleta, N.; Kvistgaard, A. S.; Graverholt, G.; Respicio, G.; Guija, H.; Valencia, N.; L€onnerdal,, B., Efficacy of a complementary food enriched with a milk fat globule membrane protein fraction on diarrhea, anemia and micronutrient status in infants. J. Pediatr. Gastroenterol. Nutr. Epub 2011 May 30. (13) Reinhardt, T. A.; Lippolis, J. D. Developmental changes in the milk fat globule membrane proteome during the transition from colostrum to milk. J. Dairy Sci. 2008, 91 (6), 2307–2318. (14) Fong, B. Y.; Norris, C. S. Quantification of milk fat globule membrane proteins using selected reaction monitoring mass spectrometry. J. Agric. Food Chem. 2009, 57 (14), 6021–6028. (15) Charlwood, J.; Hanrahan, S.; Tyldesley, R.; Langridge, J.; Dwek, M.; Camilleri, P. Use of proteomic methodology for the characterization of human milk fat globular membrane proteins. Anal. Biochem. 2002, 301 (2), 314–324. 3540
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