N-Glycoproteomic Analysis of Chicken Egg Yolk - Journal of

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N-glycoproteomic Analysis of Chicken Egg Yolk Fang Geng, Yunxiao Xie, Jinqiu Wang, Kaustav Majumder, Ning Qiu, and Meihu Ma J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04492 • Publication Date (Web): 11 Oct 2018 Downloaded from http://pubs.acs.org on October 15, 2018

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Journal of Agricultural and Food Chemistry

N-glycoproteomic Analysis of Chicken Egg Yolk

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Fang Geng1,2, Yunxiao Xie1,2, Jinqiu Wang1,2*, Kaustav Majumder3*, Ning Qiu4, Meihu

4

Ma4

5 6

1

7

University, No. 2025 Chengluo Avenue, Chengdu, 610106, P. R. China

Key Laboratory of Coarse Cereal Processing (Ministry of Agriculture), Chengdu

8 9 10

2

College of Pharmacy and Biological Engineering, Chengdu University, No. 2025

Chengluo Avenue, Chengdu, 610106, P. R. China

11 12

3

13

Street, Lincoln, NE 68588, USA

Food Science and Technology Department, University of Nebraska-Lincoln, 1400 R

14 15

4

16

Huazhong Agricultural University, No. 1 Shizishan Street, Wuhan, 430070, P. R. China

National R&D Center for Egg Processing, College of Food Science and Technology,

17 18

*Corresponding authors:

19

Dr. Jinqiu Wang, E-mail: [email protected]

20

Dr. Kaustav Majumder, E-mail: [email protected]

21

1

ACS Paragon Plus Environment


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ABSTRACT

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Posttranslational N-glycosylation of food proteins plays a critical role in their structure

24

and function. However, the N-glycoproteome of chicken egg yolk (CEY) has not been

25

studied yet. Glycopeptides hydrolyzed from CEY proteins were enriched, and

26

deglycosylation using PNGase F, then were identified using a shotgun glycoproteomics

27

strategy. A total of 217 N-glycosylation sites and 86 glycoproteins were identified in CEY,

28

and these glycoproteins are mainly involved in “binding”, “biological regulation”,

29

“catalytic activity”, and “metabolic process”. Among the identified CEY glycoproteins, 22

30

were recognized as proteases and protease inhibitors, suggesting that a proteinase/inhibitor

31

regulation system exists in CEY; 15 were members of the complement and immune

32

systems, which provide protection against potential threats during hatching. The study

33

provides important structural information about CEY glycoproteins and aids in the

34

understanding of the underlying mechanism of embryo development as well as changes in

35

CEY functional characteristics during storage and processing.

36 37

KEYWORDS

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Chicken egg yolk; Glycoproteome; N-glycosylation site; Mass spectrometry; Protease;

39

Immunology.

40

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INTRODUCTION

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Posttranslational modifications (PTMs) of proteins have important effects on the

43

correct folding, molecular structure, and physical and chemical properties of proteins.

44

PTMs have been found in food proteins, mainly in glycosylated and phosphorylated forms.

45

It has been proved that phosphorylation of muscle proteins is involved in glycolysis,

46

muscle contraction and the degradation of muscle proteins, thus influencing the quality of

47

the meat.1-3 Another study showed that phosphorylation reduced the allergenicity of cow

48

caseins in children.4 In addition to phosphorylation, the N-glycosylation of food proteins

49

has also been studied. Lactoferrins are one of the most well-studied food proteins, and their

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N-glycosylation profile shows high heterogeneity, which is considered the potential reason

51

for the characteristic differences between lactoferrins.5-6 Recently, omics analyses of N-

52

glycoproteins in human colostrum and mature milk were performed, and a total of 133 N-

53

glycosylation sites on 73 milk whey proteins and 912 N-glycosylation sites on 506 milk fat

54

globule membrane proteins were identified; these results provided basic and important

55

information on the understanding of the role of milk protein glycosylation during infant

56

development.7-8

57

N-glycosylation also plays an important role in the structure and properties of chicken

58

egg proteins and is involved in the egg white thinning, egg allergenicity, antibacterial

59

properties and embryo protection.9-12 Therefore, the N-glycosylation profiles of egg

60

proteins need to be well characterized. In our previous work, the N-glycosylation proteome

61

of chicken egg white was studied, and a total of 71 N-glycosylation sites in 26 egg white

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glycoproteins were identified.13 However, the N-glycosylation proteome of chicken egg

63

yolk (CEY) has not been investigated. Egg white proteins are synthesized by oviduct during 3



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egg formation but most of the CEY proteins are synthesized as a precursor protein by the

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liver of the hen, and then are transferred into the oocyte through bloodstream.14 After

66

enzymatic cleavage, the major fragments of precursor proteins (apolipoprotein B, APOB;

67

vitellogenins, VTGs) and lipids are combined into lipoprotein complexes (high-density

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lipoprotein, HDL; low-density lipoprotein, LDL), which ultimately form yolk plasma and

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yolk granule.14-16 The main CEY proteins, including immunoglobulin Y (IgY), HDL, LDL,

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and phosvitin have all been reported as glycoproteins, and some of their glycosylation sites

71

have been clarified.17-18 Recently, a study of the CEY plasma peptidome demonstrated that

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a total of 13 CEY plasma proteins contain PTMs, including 9 N-glycosylation sites on

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apolipoprotein B, complement C4, histidine-rich glycoprotein, prothrombin, cathepsin EA-

74

like protein, and vitellogenin-2.19 The effect of glycosylation on the function of some CEY

75

proteins has been studied. A previous study revealed that the removal of N-glycan changed

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the conformation, storage stability, and resistance to guanidine hydrochloride and pepsin

77

digestion of the IgY molecule, suggesting that N-glycosylation plays an important role in

78

the molecular structure and function of IgY.20

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However, most of the other CEY glycoproteins have not been investigated. Therefore,

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the N-glycosylation proteome of chicken egg yolk was studied in the present work. The

81

CEY proteins were digested with trypsin, and the glycopeptides were enriched using a

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hydrophilic microcolumn, followed by deglycosylation using PNGase F in H218O water.

83

The

84

chromatography/nanoelectrospray ionization/mass spectrometry (UPLC-NSI-MS/MS),

85

and the N-glycosylation sites were identified based on the MS/MS data via the MaxQuant

deglycopeptides

were

identified

using

4


ultra-performance

liquid

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software. In addition, the function of the identified CEY glycoproteins were annotated

87

through Gene Ontology analysis.

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MATERIALS AND METHODS

91

Chicken Egg Yolk (CEY)

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Fresh chicken eggs laid within 24 h from White Leghorns (about 50-week-old, cage,

93

standard diets) were collected in the morning from the poultry farm of Sichuan Sundaily

94

Village Ecological Food Co., Ltd. (Mianyang, Sichuan) and were used in the study. The

95

fresh egg was broken, and the CEY was separated and rolled on a filter paper to eliminate

96

the egg white. After the chicken egg white was absorbed and the vitelline membrane was

97

adhered by the filter paper, a pipet tip was used to penetrate the vitelline membrane, and

98

the CEY was pulled.21 The yolks of 15 eggs from 3 repeated sampling were homogenized,

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the mixed CEY sample was frozen and stored with liquid nitrogen until further analysis.

100

Protein Extraction

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A standard protein extraction process was employed. In detail, the frozen CEY

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samples (1 mL) was ground with liquid nitrogen into powder and then transferred to a 10-

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mL centrifuge tube, and four volumes of lysis buffer (8 mol/L urea, 1% Triton-100, 10

104

mmol/L dithiothreitol, 10 μmol/L trichostatin A and 50 mmol/L nicotinamide) were added

105

to the tube, followed by sonicating three times (150 W, 30 s each time) on ice using a high-

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intensity ultrasonic processor (JY92-II, Ningbo Scientz Biotechnology Co., Ltd., Ningbo,

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China). The remaining insolubles were removed by centrifugation at 12,000 ×g at 4°C for

108

10 min. Then, the supernatant was precipitated overnight with the addition of 5 volumes of 5



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0.1 mol/L ammonium acetate/methanol. After centrifugation at 12,000 ×g and 4°C for 10

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min, the supernatant was discarded. The remaining precipitate was washed with cold

111

acetone three times. The protein was redissolved in 8 mol/L urea, and the protein

112

concentration was determined with a BCA kit according to the manufacturer’s

113

instructions.22-23

114

Digestion, Enrichment, and Deglycosylation of CEY Glycopeptides

115

The CEY proteins were processed based on a previous protocol with modifications.13,

116

24

117

for 30 min at 56°C and alkylated with 11 mmol/L iodoacetamide for 30 min at room

118

temperature in darkness. After diluting by adding 100 mM NH4HCO3 to urea with

119

concentration less than 2 mol/L, the CEY proteins were digested using trypsin (Sigma-

120

Aldrich) at a 1:50 trypsin-to-protein mass ratio for the first digestion overnight and 1:100

121

trypsin-to-protein mass ratio for a second 4-h digestion.

Briefly, the CEY protein solution (2 mg/mL) was reduced with 5 mmol/L dithiothreitol

122

The hydrolyzed CEY solution (50 μL) was freeze-dried and then reconstituted in 50

123

μL enrichment buffer (80% acetonitrile and 1% trifluoroacetic acid). The solution was

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transferred to a hydrophilic interaction liquid chromatography microcolumn (HILIC,

125

SeQuant, Southborough, MA, USA) and centrifuged at 4000 ×g for 15 min, and then, the

126

hydrophilic microcolumn was washed 3 times with enrichment buffer.25 The glycopeptides

127

were eluted with 10% acetonitrile, the eluate was collected and lyophilized. Afterwards,

128

the glycopeptides were reconstituted in 50 μL of the 50 mmol/L ammonium bicarbonate

129

buffer, which was prepared with H218O, and 2 μg of PNGase F (Roche, 11365185001,

130

Mannheim, Germany) was added and incubated at 37°C overnight.13 Finally, the salt was

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removed according to the C18 ZipTips instructions (Millipore, Billerica, MA, USA), and

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the enriched CEY glycopeptides were lyophilized.

133

Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) Analysis

134

Liquid chromatography separation and mass spectrometric measurement were

135

performed via UPLC-NSI-MS/MS using a Thermo Scientific™ Orbitrap Fusion™

136

Tribrid™ equipped with an EASY-nLC 1000 UPLC system (Thermo Fisher Scientific,

137

Bremen, Germany). The enriched CEY glycopeptides were dissolved in 0.1% formic acid

138

(solvent A) and loaded onto a Reprosil-Pur C18 reverse-phase analytical column (1.9 μm

139

particles, inner diameter 75 µm, 15 cm length). The gradient comprised an increase from 5%

140

to 20% solvent B (0.1% formic acid in 98% acetonitrile) over 42 min, 20% to 35% in 12

141

min and climbing to 80% in 3 min, then holding at 80% for the last 3 min. The separation

142

was performed at a constant flow rate of 700 nL/min. After LC separation, the CEY

143

glycopeptides were subjected to a nanoelectrospray ionization (nESI) source followed by

144

mass spectrometry (MS/MS) in the Orbitrap FusionTM. The electrospray voltage of the

145

nESI source was 2.0 kV, the m/z scan range was 350 to 1550 for the full scan, and intact

146

peptides were detected in the Orbitrap at a resolution of 60,000. The mass spectrometer

147

was operated in data-dependent acquisition mode, and the top20 most abundant peptides

148

from the MS data were subjected to fragmentation by high-energy collision-induced

149

decomposition. For the MS/MS analysis, the automatic gain control was set at 5E4, the

150

signal threshold at 5000 ions/s, the maximum injection time at 200 ms, and the dynamic

151

exclusion time for the tandem mass scan at 15 s.

152

Tandem Mass Spectrometry Data Analysis

7



153

The data were analyzed using MaxQuant software (1.5.2.8) and compared against the

154

UniProt and NCBI databases (organism: Gallus gallus, date: 2018.03.09). Trypsin was

155

specified as the cleavage enzyme, allowing up to 4 missing cleavages. The mass tolerances

156

for the precursor ions were set as 20 ppm in the first search and 5 ppm in the main search,

157

and the mass tolerance for the fragment ions was set as 0.02 Da. Carbamidomethyl on Cys

158

was specified as fixed modification, and deamidation with 18O (N) and oxidation of Met

159

were specified as variable modifications. For identification, the false discovery rate was

160

specified as 1% at both the peptide and site levels.

161

Bioinformatics Analysis

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The potential N-glycosylation sites of the identified CEY glycoproteins were

163

predicted by the NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/) based

164

on the sequences obtained from the UniProt database (http://www.uniprot.org/). Gene

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Ontology (GO) analysis of the identified CEY glycoproteins was derived from the UniProt-

166

Gene Ontology Annotation database (http://www.ebi.ac.uk/GOA). The locations of the N-

167

glycosylation sites of Vitellogenins (VTGs) were visualized and presented by Illustrator

168

for Biological Sequences (IBS, version 1.0).26

169 170

RESULTS AND DISCUSSION

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Characterization of the identified N-glycosylation sites in CEY

172

Glycopeptides derived from the CEY proteins were enriched by the HILIC column

173

and deglycosylated by PNGase F in H218O, resulting in the conversion of the originally

174

glycosylated asparagine to aspartic acid (occupied by -18OH) with a 2.99 Da increase in

175

molecular weight, which can be exploited to identify the N-glycosylation sites.27-28 Of the 8


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28883 spectra produced by the mass spectrometer, a total of 4341 spectra matched with the

177

alignment protein, and 1109 of these spectra were identified as glycopeptides. All the

178

glycopeptides were identified with high precision, and the mass tolerance of the peptide

179

ions was less than 5 ppm (Figure 1A). After clearing the repeated sequence, a total of 208

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unique CEY glycopeptides were obtained, which contain 217 N-glycosylation sites and

181

belong to 86 glycoproteins (Table S1).

182

In the present study, the number of MS/MS spectra that matched a certain unique

183

glycopeptide was called the “MS/MS count”, which was used to approximate the

184

abundance of the glycopeptides. Among the identified CEY glycopeptides, about half of

185

the identified CEY glycopeptides had an MS/MS count of 1, indicating that these N-

186

glycosylation sites are low in abundance. However, 17% of the identified CEY

187

glycopeptides had a large MS/MS count (5 or more), indicating that these N-glycopeptides

188

are relatively high in abundance (Figure 1B).

189

The canonical N-glycosylation sequence is N-X-[S/T] (where X is not proline). Here,

190

Web-Logo was employed to visualize the sequence motifs around the N-glycosylation sites

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(5 amino acids on each side).29 As shown in Figure 1C, threonine (T) and serine (S) were

192

significantly overrepresented at the +2 position, suggesting that most of the identified egg

193

white N-glycosylation sites were localized at the canonical sequence. More specifically,

194

based on the unique peptide, the N-glycosylation sites that matched with N-X-T (75 unique

195

peptides, accounting for 35%) occurred more frequently than those that matched with N-

196

X-S (62 unique peptides, 29%). However, based on the MS/MS count, the number of

197

MS/MS spectra with N-X-T (391 MS/MS count, accounting for 36%) was lower than that

9



198

with N-X-S (567 MS/MS count, 52%) (Figure 1C). This indicated that CEY glycoproteins

199

that contain the N-X-S sequence have a higher abundance.

200

Gene Ontology analysis of the identified CEY glycoproteins

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The identified CEY glycoproteins were classified according to their GO annotation.

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As shown in Figure 2, in terms of “molecular function,” 35 CEY glycoproteins are involved

203

in “binding” (GO:0005488) at GO level 2. For the development of the embryo, CEY is rich

204

in a variety of nutrients, such as lipids, proteins, minerals, and bioactive molecules.30

205

Therefore, many CEY glycoproteins have binding abilities, including “ion binding” (19

206

glycoproteins), “protein binding” (15), and “lipid binding” (5) at GO level 3. A total of 22

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CEY glycoproteins are involved in “catalytic activity” (GO:0003824), which mainly refers

208

to “hydrolase activity” (GO:0016787, 17) and “catalytic activity, acting on a protein”

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(GO:0140096, 16) at GO level 3; in particular, 14 CEY glycoproteins are classified as

210

having “peptidase activity” (GO:0070011). These results indicated that many CEY

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glycoproteins are involved in protein hydrolysis/digestion, which can provide peptides and

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amino acids for embryonic development. Another well-represented category is “molecular

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function regulator” (19); notably, 17 of these 19 CEY glycoproteins were annotated as

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having “endopeptidase inhibitor activity” (GO:0004866). The number of inhibitors was

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equivalent to that of hydrolases, suggesting that there is a proteinase/inhibitor regulation

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system in CEY.

217

In terms of “biological process” at GO level 2, the CEY glycoproteins related to

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“biological regulation” (GO:0065007, 39), “metabolic process” (GO:0008152, 21),

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“cellular process” (GO:0009987, 19), “response to stimulus” (GO:0050896, 18), and

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“immune system process” (GO:0002376, 10) accounted for the largest portion. As an

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independent organ, the chicken egg needs to perform a variety of biological processes alone,

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and thus, many CEY glycoproteins are involved in regulatory and metabolic processes.

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During hatching, the chicken egg is under different types of stress and environmental

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factors;31 therefore, proteins that respond to stimuli account for a large proportion of the

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CEY glycoproteins.

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Regarding the cellular components (at GO level 2), the most well-represented

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category was “extracellular region part” (GO:0044421, 40), “cell part” (GO:0044464, 25),

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“protein-containing complex” (GO:0032991, 13). The egg yolk plasma and granules,

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which account for most of the egg yolk, do not belong to the cell part, and thus most of the

230

CEY glycoproteins are classified as “extracellular region part.” Low-density lipoprotein,

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high-density lipoprotein, and yolk granules are all complexes, and thus “protein-containing

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complex” accounts for a high proportion of the CEY glycoproteins.

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The primary glycoproteins in CEY

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Of the 86 identified CEY N-glycoproteins, 39 (approximately 49%) carried single N-

235

glycosylation sites, and the other 47 proteins had multiple N-glycosylation sites.

236

Apolipoprotein B (APOB) was the most heavily N-glycosylated protein in CEY with 35

237

N-glycosylation sites, followed by Vitellogenin-1 (VTG1), Vitellogenin-2 (VTG2) and

238

serum albumin with 15, 13 and 7 N-glycosylation sites, respectively (Figure 3A). However, 11



239

according to the abundance (MS/MS count) of the CEY glycopeptides, APOB is the most

240

abundant glycoprotein in CEY (443 MS/MS count), followed by VTG-2 (163 MS/MS

241

count), VTG-1 (89 MS/MS count) and IgY (67 MS/MS count, including light chain P20763

242

and heavy chain AHX37590.1) (Figure 3B).

243

Apolipoprotein B

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As the most heavily glycosylated protein in CEY, APOB is a 523 kDa (4,631 AA)

245

protein that is enzymatically cleaved and results in 7 kinds of fragments during its transfer

246

from chicken serum into the yolk.14 These APOB fragments, together with apovitellenin-1

247

(which was also identified as a glycoprotein with an N-glycosylation site at N75 in the

248

present study) and lipids, assemble and form CEY LDL.15-16 APOB contains 19 potential

249

N-glycosylation sites predicted by NetNGlyc 1.0, two of which have been identified

250

recently (N1369 and N3697).19 Here, a total of 35 N-glycosylation sites of APOB were

251

identified, and 12 of them were also predicted to be modified by NetNGlyc 1.0, while the

252

other 23 sites were unpredicted, and all of them localized at the noncanonical sequence.

253

Meanwhile, 7 of the predicted N-glycosylation sites of APOB were not identified in the

254

current experiment (Figure 4). These results indicated that the low-frequency noncanonical

255

N-glycosylation modifications cannot be predicted, while the predicted N-glycosylation

256

sites based on the canonical N-X-[S/T] sequence need to be confirmed through

257

experimental analysis. However, the 12 APOB N-glycopeptides, which were both

258

predicted and identified, had a large MS/MS count (Table S1), suggesting that high-

259

frequency N-glycosylation tends to occupy the canonical N-X-[S/T] sequence.

260

The large number of low-frequency noncanonical N-glycosylation modifications was

261

a noteworthy phenomenon. How does this low-frequency N-glycosylation modification 12


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affect the structure and function of proteins? Is it a random and meaningless modification

263

or important for biological processes? These questions should be of concern and studied in

264

the future.

265

Vitellogenins

266

VTG1, VTG2 and VTG3 are the three major vitellogenin proteins in CEY, and they

267

have similar domains (lipovitellin-1, lipovitellin-2, phosvitin and YGPs) but low sequence

268

homology (34% identity by the Alignment analysis of UniPort). VTGs are synthesized in

269

the liver and transferred to the CEY through the blood circulation, and then they are cleaved

270

to lipovitellin-1, phosvitin, lipovitellin-2 and YGP42/40/30. Lipovitellin-1, lipovitellin-2

271

and lipids compose the CEY HDL, and HDL together with phosvitins assemble the CEY

272

granules.14, 30

273

According to sequence analysis, VTG1, VTG2 and VTG3 contain 9, 9 and 5 potential

274

N-glycosylation sites, respectively. However, similar to the case of APOB, only some of

275

these predicted sites (4 sites of VTG1, 5 sites of VTG2, 1 sites of VTG3) were confirmed

276

to have undergone N-glycosylation in the present experiment. In addition, 11 unpredicted

277

sites on VTG1, 8 unpredicted sites on VTG2 and 5 unpredicted sites on VTG3 (all of which

278

were located at the noncanonical sequence) were identified as N-glycosylation sites.

279

Among the 15 total identified N-glycosylation sites of VTG1, 7 are located on lipovitellin-1,

280

8 are located on YGP42, and no sites are located on lipovitellin-2. In contrast, among the

281

13 total identified N-glycosylation sites of VTG2, 7 are located on lipovitellin-1, 1 is

282

located on lipovitellin-1, and 5 are located on YGP40 (Figure 5). The difference in the

283

distribution of these N-glycosylation sites may be an important reason for the difference in

284

the properties of VTG1 and VTG2. Another noteworthy result was that the total number of 13



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MS/MS counts that matched with VTG1, VTG2 and VTG3 was 89, 163 and 14,

286

respectively, suggesting that VTG2 was the most abundant CEY VTG, which was

287

consistent with previous research.32-33

288

Although phosvitin has been reported to undergo glycosylation on the asparagine at

289

1280,34 none of the N-glycosylation sites was identified in the phosvitin region of the VTGs

290

in the present study. This may be because the high phosphorylation weakens the affinity

291

between the phosvitin peptides and HILIC column.

292

Protease and its inhibitor

293

A total of 8 and 14 identified CEY glycoproteins were recognized as proteases and

294

protease

295

carboxypeptidase B2, cathepsin EA-like protein, gamma-glutamyl hydrolase, plasminogen

296

(F1NWX6, and R4GMH5), protein C, and prothrombin; the protease inhibitors included:

297

antithrombin-III, kininogen 1, serpin family A member 10, serpin family G member 1,

298

ovoinhibitor, and several uncharacterized proteins (A0A1D5PBU0, A0A1D5PCD2,

299

E1C206, E1C7T1, F1NEQ4, F1NK40, F1NL38, F1NPN5, and F1P587) (Table S1). Of

300

these proteases and its inhibitors, carboxypeptidase was the most heavily N-glycosylated

301

proteases, with 2 N-glycosylation sites and an MS/MS count of 9; while ovoinhibitor was

302

the most heavily N-glycosylated inhibitors, with 4 N-glycosylation sites and an MS/MS

303

count of 15. More specifically, the ovoinhibitor, a member of the Kazal family of protease

304

inhibitors with inhibitory activity against serine proteinase, contains three predicted N-

305

glycosylation sites at N27, N141 and N461. In our previous study, two N-glycosylation sites

306

at N62 and N141 had been experimentally verified;13 here, another two sites (N193 and N461)

307

were identified to have undergone N-glycosylation.

inhibitors,

respectively.

The

proteases

14


included:

carboxypeptidase,

Page 15 of 34


308

Covalently bonded N-glycans can alter the folding and the surface properties of the

309

protease or inhibitor, thus affecting the recognition, specificity, and binding affinity.

310

Therefore, N-glycosylation modifications are important for the stability, activation, and

311

catalytic activity of proteases and their inhibitors.35 However, the effects of glycosylation

312

on CEY proteases and inhibitors are not yet clear, and they remain one of the concerned

313

topics in the field of egg science and need to be studied in the future.

314

Members of the Complement and Immune Systems

315

CEY is an important part of the embryo's defensive system and needs to provide

316

protection against potential internal or invasive threats. Therefore, many CEY

317

glycoproteins are involved in the immune and complement systems, which allow for the

318

direct killing of microbes, the disposal of ineffective complexes, and the self-regulation of

319

immune processes. Eight CEY glycoproteins were identified as components of the

320

complement system: complement C2, C3, C4, C6, C8, complement factors H and I, as well

321

as an uncharacterized protein (F1P587, gene name is LOC418892). Complement C3 was

322

most heavily N-glycosylated, with two N-glycosylation sites at N957 and N1428, which were

323

predicted by NetNGlyc 1.0. The matching MS/MS counts of N957 and N1428 were 7 and 10,

324

suggesting that complement C3 is a highly abundant complement glycoprotein in CEY.

325

The alignment analysis (provided by UniProt) revealed that the sequence identity between

326

CEY complement C3 and human complement C3 (P01024) is 53.8%; however, these two

327

proteins do not share an N-glycosylation site (data not shown). The high sequence identity

328

and low modification similarity of homologous glycoproteins have also been found in our

329

previous study of chicken egg white ovomacroglobulin.24 This phenomenon is interesting

330

and noteworthy, showing a balance of the conservation and diversity of protein molecules. 15



331

There were 7 identified immune-related glycoproteins, including the light chain (Ig

332

lambda chain C region, P20763) and heavy chain (AHX37590.1) of IgY, Fc fragment of

333

IgG binding protein (A0A1D5P6F4), Ig mu chain C region (P01875), immunoglobulin

334

heavy chain variable region (CAO79238.1), and immunoglobulin light chain variable

335

region (AJQ23642.1). Among these glycoproteins, IgY was the most abundant immune-

336

related glycoprotein, containing 3 N-glycosylation sites (N156, N178 and N255 of

337

AHX37590.1) on the heavy chain constant region and 2 N-glycosylation sites (N61 and N90

338

of P20763) at the light chain constant region. The total MS/MS count of IgY glycopeptides

339

was 67, suggesting that IgY is a highly abundant immune glycoprotein in CEY. In a

340

previous work, two sites (N308 and N407) at the heavy chain of IgY were identified as N-

341

glycosylated, which corresponded the N156 and N255 of AHX37590.1 in the present study.18

342

In addition to these 2 sites, other 3 newly identified N-glycosylation sites of IgY provided

343

additional information of the glycosylation profile of IgY.

344

Other CEY glycoproteins

345

Ovotransferrin, which is the second most abundant protein in chicken egg white, has

346

also been found in egg yolk.19, 36-37 The N-glycosylation sites at N492 and N637 had been

347

reported in a previous study,13 and another two N-glycosylation sites at N233 and N514 were

348

found in the present study.

349

Albumin, also known as α-livetin, which is transferred from the blood serum to CEY,

350

has been identified as one of the major egg allergens. In albumin, MS/MS analysis

351

ascertained seven N-glycosylation sites, namely, N126, N158, N291, N393, N400, N498, and N500,

352

while only the N500 residue was predicted by NetNGlyc 1.0. Among the six unpredicted N-

16


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353

glycosylation sites, N291 and N498 have MS/MS counts of 4 and 2, respectively, implying

354

that these two sites are relatively high in abundance.

355

In summary, the N-glycoproteome of CEY was studied using a shotgun

356

glycoproteomics strategy, a total of 86 glycoproteins were identified in CEY and their

357

biological function were annotated and discussed. The N-glycosylated modification, as

358

well as other types of post-translational modifications, are extremely important for

359

understanding the structures, functions, and bioactivities of the food proteins. Because

360

these modifications could alter the molecular weight, surface charge and folding of proteins,

361

or as a marker for interaction and signaling. Therefore, in the research of food proteome,

362

modification of proteins is another basic dimension in addition to the protein species and

363

abundance, which need to be given more attention in the further.

364 365

Abbreviations Used

366

APOB, Apolipoprotein B; CEY, chicken egg yolk; GO, Gene Ontology; HDL, high-

367

density lipoprotein; HILIC, Hydrophilic interaction liquid chromatography; IgY,

368

immunoglobulin Y; LDL, low-density lipoprotein; nESI, nanoelectrospray ionization;

369

PTMs, Posttranslational modifications; VTG, Vitellogenin.

370 371

Funding

372

National Key Research and Development Program of China (2018YFD0400302)

373

National Natural Science Foundation of China (No. 31601490; No. 31871732)

374

Supporting Information Description

17



375

The details of identified CEY N-glycopeptides and N-glycoproteins resulting from a

376

database search of LC-MS/MS data are shown in Supporting file (Table S1). This

377

information is available free of charge via the Internet at http://pubs.acs.org.

378 379

Notes

380

The authors declare no competing financial interests.

381 382

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383

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474

Figure Captions

475 476

Figure 1. Characteristics of the identified N-glycopeptides in CEY. A, mass error

477

distribution of identified N-glycopeptides; B, distribution of the MS/MS count number of

478

identified N-glycopeptides; C, N-glycosylation unique sequences as derived using Web-

479

Logo; D, distribution of recognized sequence motifs based on the unique sequence or

480

MS/MS count of the identified N-glycopeptides (X≠P).

481 482

Figure 2. GO analysis (level 2) of identified CEY glycoproteins.

483 484

Figure 3. Distribution of identified N-glycosylation sites (A) or matched MS/MS count

485

(B) in CEY proteins.

486 487

Figure 4 Overlapping Venn diagram of predicted and identified N-glycosylation sites of

488

APOB.

489 490

Figure 5. Comparison of the distributions of identified N-glycosylation sites on VTG1 and

491

VTG2.

492

22


Page 22 of 34

Page 23 of 34


493 494

Figure 1. Characteristics of the identified N-glycopeptides in CEY. A, mass error

495

distribution of identified N-glycopeptides; B, distribution of the MS/MS count number of

496

identified N-glycopeptides; C, N-glycosylation unique sequences as derived using Web-

497

Logo; D, distribution of recognized sequence motifs based on the unique sequence or

498

MS/MS count of the identified N-glycopeptides (X≠P).

499

23



500

Figure 2. GO analysis (level 2) of identified CEY glycoproteins.

501

24


Page 24 of 34

Page 25 of 34


502

Figure 3. Distribution of identified N-glycosylation sites (A) or matched MS/MS count

503

(B) in CEY proteins.

504

25



505

Figure 4 Overlapping Venn diagram of predicted and identified N-glycosylation sites of

506

APOB.

507

26


Page 26 of 34

Page 27 of 34


508

Figure 5. Comparison of the distribution of identified N-glycosylation sites on VTG1 and

509

VTG2.

510

27



511

Graphic for table of contents

512

28


Page 28 of 34

Page 29 of 34


Figure 1. Characteristics of the identified N-glycopeptides in CEY. A, mass error distribution of identified Nglycopeptides; B, distribution of the MS/MS count number of identified N-glycopeptides; C, N-glycosylation unique sequences as derived using Web-Logo; D, distribution of recognized sequence motifs based on the unique sequence or MS/MS count of the identified N-glycopeptides (X≠P). 190x139mm (300 x 300 DPI)



Figure 2. GO analysis (level 2) of identified CEY glycoproteins. 153x94mm (300 x 300 DPI)


Page 30 of 34

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Figure 3. Distribution of identified N-glycosylation sites (A) or matched MS/MS count (B) in CEY proteins. 123x137mm (300 x 300 DPI)



Figure 4 Overlapping Venn diagram of predicted and identified N-glycosylation sites of APOB. 74x54mm (300 x 300 DPI)


Page 32 of 34

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Figure 5. Comparison of the distributions of identified N-glycosylation sites on VTG1 and VTG2. 190x64mm (300 x 300 DPI)



Graphic for table of contents 168x107mm (300 x 300 DPI)


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N-Glycoproteomic Analysis of Chicken Egg Yolk - Journal of

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