Comparison of Milk Oligosaccharides Pattern in Colostrum of

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Comparison of milk oligosaccharides pattern in colostrum of different horse breeds Elisabetta Difilippo, H.A.M. Willems, Johannes C. Vendrig, Johanna Fink-Gremmels, Harry Gruppen, and Henk A. Schols J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b01127 • Publication Date (Web): 30 Apr 2015 Downloaded from http://pubs.acs.org on May 4, 2015

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Journal of Agricultural and Food Chemistry

Comparison of milk oligosaccharides pattern in colostrum of different horse breeds Difilippo, E.a, Willems, H.A.M a, Vendrig, J.C.b, Fink-Gremmels, J.b, Gruppen, Ha, Schols, H.A.a*

a

Wageningen University, Laboratory of Food Chemistry, P.O. 17, 6700 AA Wageningen, The

Netherlands.

b

Utrecht University, Faculty of Veterinary Medicine, Institute for Risk Assessment Sciences,

subdivision of Veterinary

Pharmacology,

Pharmacotherapy and Toxicology, The

Netherlands.

* Corresponding author. E-mail: [email protected]: Phone: +31 317 482239; Fax: +31 317 484893

1

ACS Paragon Plus Environment


1

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ABSTRACT

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Colostrum oligosaccharides are known to exhibit prebiotic and immunomodulatory

3

properties. Oligosaccharide composition is species-specific, and equine colostrum has been

4

reported to contain unique oligosaccharides. Therefore, equine oligosaccharides (EMOS)

5

from colostrum from different horse breeds were analyzed by CE-LIF, CE-MSn, HILIC-MSn,

6

and exoglycosidase degradation. Sixteen EMOS were characterized and quantified, of which

7

half were neutral and half were acidic. EMOS showed about 63% structural overlap with

8

human milk oligosaccharides, known for their bioactivity. Seven EMOS were not reported

9

before in equine oligosaccharides literature: neutral Gal(β1−4)HexΝΑc, Gal(β1-4)Hex-Hex,

10

β4’-galactosyllactose, and lactose-N-hexaose, as well as acidic 6’-Sialyl-Hex-Ac-HexNAc,

11

sialyllacto-N-tetraose-a, and disialylacto-N-tetraose (isomer no further specified). In all

12

colostrum samples, average oligosaccharide concentration ranged from 2.12 to 4.63 g/L; with

13

β 6’and 3’- galactosyllactose, 3’-sialyllactose, and disialyllactose as the most abundant of all

14

oligosaccharides (27-59, 16-37, 1-8, and 1-6%, respectively). Differences in presence and in

15

abundance of specific EMOS were evident; not only between the four breeds, but also within

16

the breed.

17

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Keywords: milk, CE-MS, HILIC-MS, exoglycosidases, mass analysis, chromatography

19

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INTRODUCTION

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Mammalian milk and colostrum contain many structures, which are essential for the

22

nutrition and health of the suckling neonate. Throughout the past decades, specific

23

oligosaccharides, which have been identified in milk and colostrum, have been shown to

24

beneficially affect health.1 For example, human milk oligosaccharides (HMOS) carrying the

25

so-called bifidogenic factor (Gal(β1-3)GlcNAc), have been illustrated to enhance the growth

26

of bifidobacteria in the intestine of neonates.2 When bifidobacteria selectively ferment

27

HMOS, they produce short chain fatty acids (SCFA), leading to a pH decrease in the

28

intestine. The lowered pH in the intestine creates a less favorable environment for the growth

29

of the pathogens and stimulates the production of mucin which in turn, enhance the host’s

30

resistance to infections.3 The production of SCFA also leads to a stimulation of the immune

31

system through a direct interaction with the immune cells of the intestine.3,

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increasing evidence has come forward to demonstrate direct immunomodulatory properties of

33

oligosaccharides derived from milk and colostrum. Some properties could derive from

34

interaction with specific receptors on intestinal epithelial cells and immune cells, such as

35

carbohydrate receptors and Toll-like receptors.4-6 In addition, milk oligosaccharides serve as

36

soluble ligands for pathogens, preventing viral, and bacterial attachment to the intestinal

37

mucosa.5

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The core structure of mammalian milk oligosaccharides (MMOs) is, in most cases, a lactose

39

unit (Gal(β1-4)Glc) at the reducing end. However, in cow’s, horse’s and goat’s milk, N-

40

acetyllactosamine (Gal(β1-4)GlcNAc) is also found at the reducing end instead of the lactose

41

unit.7, 8 Starting from these disaccharides, glycosyltransferases can elongate the core structure

42

with neutral and charged sugars. The neutral sugars that can be attached to the core structure

43

are D-galactose (Gal), L-fucose (Fuc), N-acetylglucosamine (GlcNAc) and, with the

44

exception of HMOS, also N-acetylgalactosamine (GalNAc).7

4

Moreover,

3



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The charged sugars that can decorate the core structure are sialic acids, both N-acetyl- and N-

46

glycolyl-neuraminic acids, and glucose or N-acetylglucosamine substituted with one

47

phosphoric group (e.g., in milk oligosaccharides of horses and cattle).7-9 Depending on the

48

linkage present in their chains, the MMOs are divided into two groups: Type I group contains

49

Gal(β1-3)GlcNAc, and Type II group contains Gal(β1-4)GlcNAc.10

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The reported mature-milk oligosaccharide concentration is 12-13 g/L for human milk and

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approximately 0.05 g/L for cow’s milk.8, 11 The oligosaccharide concentration in colostrum is

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higher than the concentration in mature milk: 24 g/L in human colostrum and more than 1 g/L

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in cattle’s colostrum.7, 12 So far, 43 equine milk oligosaccharides (EMOS) have been reported

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of which 31 are neutral and 12 are acidic.7, 8, 13 Compared to the HMOS, the EMOS have a

55

higher degree of sialylation (20-30% and 60%, respectively)11 and a lower degree of

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fucosylation (70% and 5%, respectively).11 The previously documented EMOS showed a

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17% overlap with oligosaccharides present in human milk. Although not reported so far, the

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presence and relative abundance of individual EMOS may vary between individual animal

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breeds, comparable to the variations in HMOS in different human populations.14-16

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This study focuses on the analysis of EMOS in colostrum samples of four different horse

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breeds: Dutch Warmblood horse, Shetland pony, Crossbred Arabian/New Forest pony and

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Friesian horse. After isolation from the colostrum samples, the EMOS were identified and

63

quantified, highlighting the inter-breed (EMOS for the different horse breeds) and the intra-

64

breed (EMOS for the same breed) variations.

65

4


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MATERIALS AND METHODS

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Materials. Colostrum samples of horses belonging to four different breeds were

68

obtained from private Dutch owners with consent. Five colostrum samples were available for

69

the Crossbred Arabian/New Forest pony (Nederlands Rijpaarden and Pony Stamboek), four

70

colostrum samples were available for the Dutch Warmblood horse (Royal Warmblood

71

Studbook of the Netherlands), two colostrum samples were available for the Friesian horse,

72

and one colostrum sample was available for the Shetland pony. Each colostrum was collected

73

within 12 hours postpartum and frozen (-20o C) until use.

74

The standards 3’-, and 6’-sialyllactose, 3’-, and 6’-sialyl-N-acetyllactosamine, lacto-N-

75

tetraose, sialyllacto-N-tetraose-a, and lacto-N-hexaose were purchased from Dextra

76

Laboratories (Reading, UK). The standards β 3’-, 4’- and 6’- galactosyllactose, lacto-N-

77

tetraose, and lacto-N-neotetraose were bought from Carbosynth (Compton, UK). The

78

labelling of the oligosaccharides was accomplished using the Carbohydrate Labelling &

79

Analysis Kit (Beckman Coulter, Fullerton, CA, USA). All other chemicals used were of

80

analytical grade. Millipore water (Millipore, Darmstadt, Germany) was referred as water in

81

the text.

82

Extraction of equine colostral sugars. The Dutch Warmblood colostrum samples

83

were available in larger quantities than those of other breeds. Therefore, a mixture of the

84

Dutch Warmblood colostrum samples was used to optimize the extraction method and to

85

obtain enough material to be loaded in the preparative Size Exclusion Chromatography.

86

Specifically, 1.25 mL of each colostrum sample was pooled together and mixed with a vortex

87

for 2 minutes. After method optimization, EMOS were extracted from 1mL of the 12

88

individual colostrum samples. The optimized sugar extraction from the colostrum samples

89

was performed by modification of the method of Nakamura et al.13 Briefly, colostrum (5 mL)

90

was treated with 4 volumes of chloroform. After two hours head over tail mixing, the sample 5



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was centrifuged (5000 x g, 30 min, 5 oC) and the supernatant was treated with 2 volumes of

92

methanol. After the same mixing and centrifugation procedure, the methanol-water solution

93

was concentrated by vacuum evaporation. The remaining water solution was freeze-dried.

94

Purification and fractionation of EMOS. Size Exclusion Chromatography (SEC)

95

was used to remove lactose from the extracted carbohydrates of the pooled Dutch

96

Warmblood colostrum, in order to obtain pools containing EMOS with difference degrees of

97

polymerization (DP). In total four pools were obtained and they were analyzed for the

98

characterization of the EMOS. The fractionation was carried out at 35 oC on three Superdex

99

30 Hiload 26/60 preparative grade columns (GE Healthcare, Pittsburgh, PA, USA) connected

100

in series on a AKTA Purifier (GE Healthcare). After manual injection, the EMOS (200 mg)

101

were eluted with water containing 0.5% (v/v) EtOH and collected in 9 mL fractions. The

102

fractions were pooled as follows: pool 1: 290- 492 mL, pool 2: 493- 688 mL, pool 3: 689 –

103

730 mL, and pool 4: 731- 758 mL. Using water solution as eluent, charged oligosaccharides

104

were excluded from the column material and were eluted first. Neutral oligosaccharides were

105

instead fractionated based on their size. Lactose and monomers were the last to be eluted and

106

were excluded from further analysis. The neutral oligosaccharides were pooled according to

107

the retention times shown by a mixture of neutral galacto-oligosaccharides (DP from 1 to 6)

108

(Vivinal® GOS syrup) (FrieslandCampina Domo, Borculo, The Netherlands), which was

109

considered a reference for its retention time. After freeze drying, the resulting powders were

110

rehydrated with 1mL of water prior to analysis.

111

Solid Phase Extraction (SPE) was used to reduce the lactose content from the 12 individual

112

colostrum samples. The extracted carbohydrates (1 mg) were solubilized in 1mL of water and

113

loaded onto activated graphitized carbon cartridge (150 mg bed weight, 4 mL tube size;

114

Grace, Deerfield, IL, USA). Elution with water (1.5 mL) was used to remove salts and 2%

115

(v/v) acetonitrile (ACN) (1.5 mL) was used in order to elute monomers and lactose. The 6


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EMOS, still bound to the graphitized carbon cartridge, were recovered with 1.5 mL of

117

40/60% (v/v) ACN/water solution containing 0.05% (v/v) trifluoroacetic acid. The obtained

118

EMOS were dried overnight under a stream of nitrogen and subsequently solubilized in

119

0.5mL of water.

120

Capillary electrophoresis with laser induced fluorescent and mass spectrometry

121

detection. The EMOS fractionated either by SEC or SPE were labelled with a fluorescent

122

label (9-aminopyrene-1,4,6-trisulfonate, APTS) for their analysis by capillary electrophoresis

123

(CE) with laser induced fluorescence (LIF) detection as reported previously with minor

124

modifications.17 Briefly, 100 µL of sample containing 5 nmol of xylose, as internal standard,

125

was dried and labelled with APTS. Ten nmol of each oligosaccharide standard, containing 5

126

nmol of xylose, was labelled with the APTS and run together with the EMOS samples. The

127

labelled samples were diluted 40 times before the analysis. The experiment was performed

128

using a ProteomeLab PA 800 system (Beckman Coulter), equipped with a LIF detector

129

(Beckman Coulter). During the run, the sample was loaded hydrodynamically (7 s at 0.5 psi)

130

into a polyvinyl alcohol (NCHO) coated capillary (50 µm x 50.2 cm (Beckman Coulter).

131

Resultant peaks were integrated manually using Chromeleon software 6.8 (Dionex,

132

Sunnyvale, CA, USA). In the CE technique, the oligosaccharides are linked in a molar ratio

133

of 1:1 with the fluorescent, negatively-charged label (APTS). The resulting migration times

134

of the linked oligosaccharides depend on their size and on their net charge.

135

The SEC-fractionated, labelled EMOS were also analyzed for their masses by the PA 800

136

plus system (Beckman Coulter) coupled to a Velos Pro mass spectrometer (LTQ Velos Pro

137

ion trap MS, Thermo Scientific, Waltham, MA, USA), after 20 times dilution. The CE-MSn

138

experiment conditions were performed as reported by Albrecht et al. 18

7



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HILIC-ESI-MSn. The EMOS pools, derived from SEC, were also analyzed through

140

hydrophilic interaction liquid chromatography (HILIC) with mass spectrometry detection

141

(MSn) as described by Remoroza et al with some minor modifications.19

142

Briefly, an Accela UHPLC system (Thermo Scientific) coupled to a mass spectometer (LTQ

143

Velos Pro ion trap MS, Thermo Scientific) was used. The chromatographic separation was

144

performed on an Acquity HILIC BEH Amide column (1.7 µm, 2.1 mm × 150 mm) combined

145

with a Van Guard precolumn (1.7 µm, 2.1 mm × 5 mm; Waters Corporation, Milford, MA,

146

USA). The acquisition time was 72 min and the eluents had a flow rate of 600 µL/min. The

147

injection volume was 5µL. The composition of the three mobile phases were (A) water with

148

1% (v/v) ACN, (B) 100% (v/v) ACN and (C) 200 mM ammonium formate (pH 3.0). The

149

elution program was performed as follows: 2 min isocratic 80% B; 58 min linear gradient

150

from 80% to 30% B; followed by 12 min of column washing with a linear gradient from 30%

151

to 20% B and column re-equilibration form 20 to 80% B. The eluent C was kept at 5% during

152

the elution.

153

Exoglycosidase degradation. To determine the linkage type of the sialic acid

154

decorating the EMOS, two types of sialidases were used: Sialidase S and Sialidase T

155

(ProZyme, Hayward, CA, USA). Sialidase S is specific for the α2-3 linked neuraminic acid,

156

while Sialidase T splits α 2-3 and α 2-6 linked neuraminic acid. For the structural analysis of

157

the

158

4,6)galactosidase and Glykoβ-N-acetylhexosaminidase (ProZyme) were used. All reactions

159

were performed as suggested by the manufacturer. After the enzyme degradation at 37 oC

160

overnight, the solutions were centrifuged (5000 x g, 5 min, 25 oC) and the supernatants were

161

analyzed by HILIC-MSn.

neutral

EMOS

Glyco(β1-3)galactosidase,

Glyco(β1-4)galactosidase,

Glyco(β1-

162

Quantification of the EMOS. In CE-LIF the quantification of the oligosaccharides

163

was enabled by the use of an internal standard and by a 1:1 stoichiometry between the 8


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oligosaccharide and the APTS molecule. A linear correlation between concentration and CE-

165

LIF peak area has been shown.17 CE-LIF peak areas were converted to the corresponding

166

EMOS nanomoles and their concentrations in the colostrum samples (g/L). The samples were

167

run in duplicate and the quantification was validated with the help of labelled neutral and

168

acidic oligosaccharides standards. The quantification of 3’-sialyl-N-acetyllactosamine (3’-

169

SLN) and Gal(β1-4)Hex-Hex, annotated by HILIC-MSn and not assigned in the CE-LIF

170

electropherograms, was performed with HILIC-MSn. A calibration curve was made based on

171

the peak areas in mass spectrometry of the 3’-SLN, and β 4’-galactosyllactose (β 4’-GL)

172

standards, respectively. The curves fitting the 3’-SLN and β 4’-GL standards had a linear

173

correlation with R2 of 0.997 and 0.998, respectively.

9



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RESULTS AND DISCUSSION

175

Structural characterization of the equine milk oligosaccharides. In order to

176

elucidate the structure of the EMOS, a colostrum mixture was prepared using Dutch

177

Warmblood colostrum. After sugar extraction, part of the oligosaccharides was fractionated

178

using Size Exclusion Chromatography (SEC). A lactose-free EMO mixture was obtained

179

pooling part of the SEC fractions. Lactose-free EMO mixture and four SEC pools obtained

180

were labelled with the fluorescent dye APTS and subsequently analyzed by CE-LIF and CE-

181

MSn.

182

In Figure 1, CE electropherograms of labelled lactose-free EMO mixture and SEC pools are

183

shown. The migration times of labelled EMOS were compared with available standards and

184

with HMOS elution patterns, that have been described in literature.20 In total, twenty-four

185

peaks were counted of which eleven peaks were annotated (numbers 2-14, Figure 1).

186

Together with remaining lactose after SEC (symbol #, Figure 1), the annotation of five

187

oligosaccharides was confirmed by CE-MSn analysis. Acidic oligosaccharides were the first

188

eluted from the SEC columns and they were mainly pooled in pool 1. Pool 2 contained

189

sialyllactose together with neutral pentamer and hexamer oligosaccharides. Pool 3 and pool 4

190

contained neutral tetramer and trimer, respectively.

191

In the CE-MS2, the APTS-labelled oligosaccharides showed a specific fragmentation

192

behavior due to the fluorescent molecule attached at their reducing end and a multiple charge

193

state in their fragments spectrum. One example of CE-MSn identification is given for lacto-N-

194

novo-pentaose I, previously identified in equine milk, and shown in Figure 2.8, 21 APTS-lacto-

195

N-novo-pentaose I loses one terminal galactose yielding a APTS-tetramer fragment, with m/z

196

573 and m/z 382, having charge states of -2 and -3, respectively. The APTS-tetramer

197

fragment loses a terminal galactose producing the fragment GlcNAc-Gal-Glc-APTS with m/z

198

492 and 328, having charge states -2 and -3, respectively. The GlcNAc-Gal-Glc-APTS 10


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199

subsequently forms APTS-lactose, with m/z 391, having charge state -2, via the loss of the

200

terminal N-acetyl-glucosamine. From the fragmentation of the APTS-lactose, a molecule of

201

APTS-glucose was formed, with m/z 310, having charge state -2.

202

In order to enhance the characterization of the EMOS, the mass analysis was also performed

203

with HILIC-MSn with non-labelled oligosaccharides obtained after SEC fractionations. With

204

this technique five more EMOS were annotated. All found molecules had a charge state of -1.

205

One example for HILIC-MSn identification of 3'-sialyl-N-acetyllactosamine (3’-SLN) is

206

shown in Figure 3. Two peaks are highlighted for the sialyl-N-acetyllactosamine (Figure 3a).

207

The highest peak refers to 3’-SLN, while the lowest peak refers to 6’-SLN. Each of the

208

molecules were found as a double peak. It is hypothesized that the double peaks correspond

209

to the α/β conformation of the reducing end.22 The MS2 fragmentation of the 3’-SLN was

210

compared with existing literature and with MS2 fragmentation of available standard.23 The 3’-

211

SLN loses water (Figure 3b), forming thereby the fragment with m/z 655. A characteristic

212

intra-ring fragmentation of the molecule leads to the formation of the fragment with m/z 572.

213

The fragment with the highest intensity refers to the N-acetylneuraminic acid, with m/z 290.

214

For four milk oligosaccharides enzymatic degradation was necessary to determine their

215

linkages. The neutral dimer Gal(β1−4) HexΝΑc and the trimer Gal(β1-4)Hex-Hex could be

216

characterized only by using Glyco-galactosidases specific for β1-3, 1-4 and 1-4, 6 linkages,

217

in addition using the Glykoβ-N-acetylhexosaminidase. The acidic 6’-sialyl-Hex[Ac]-

218

HexNAc and 3’-sialyllacto-N-tetraose a (LSTa), could be characterized only by using

219

Sialidase S (specific for the α2-3 linked N-acetylneuraminic acid), Sialidase T (specific for

220

α2-3 and 6 linked N-acetylneuraminic acid) and Glykoβ-N-acetylhexosaminidase. After their

221

enzyme degradation, the samples were analyzed by HILIC-MSn. As example, Figure 4

222

illustrates the enzymatic degradation of Gal(β1−4)HexΝΑc and LSTa prior to analysis by 11



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HILIC-MSn. The Gal(β1−4)HexΝΑc was found as double peak (Figure 4a), as consequence

224

of α/β conformation of the reducing end. Using the enzymes tested, Gal(β1−4)HexΝΑc was

225

digested by Glyco(β1-4)galactosidase (Figure 4b), while neither Glyco(β1-3)galactosidase

226

nor Glyko-N-acetylhexosaminidase showed any activity, indicating the presence of a β1-4

227

linked, terminal galactose. The LST molecule, as found in the mare colostrum samples, was

228

digested by Sialidase S (Figure 4f), proving the presence of a terminal N-acetylneuraminic

229

acid α 2-3 linked. Moreover, the identification of LST-a was confirmed by its migration time

230

in CE, similar to commercially available LST-a standard (Table S1, supporting information).

231

Through the combination of CE and HILIC mass analysis and enzyme degradation of the

232

SEC pools, it was possible to identify 16 EMOS. All identified EMOS are listed in Table 1,

233

and an overview of methods used in order to characterize them (CE migration time, mass

234

over charge values, and retention times in CE-MSn, and HILIC-MSn, and enzymes) are

235

described in supporting information (Table S1). Comparing the outcomes of this study with

236

previous data, seven EMOS were not reported previously, and were presented in Table 1 in

237

bold:

238

galactosyllactose, and lactose-N-hexaose, as well as the acidic oligosaccharides 6’-sialyl-

239

Hex-Ac-HexNAc, LST-a, and disialylacto-N-tetraose (isomer no further specified).7,

240

identification was possible for six of the novel EMOS, while partial identification was

241

possible for three of the novel EMOS. For 6’-sialyl-Hex-Ac-HexNAc, the presence of an

242

additional acetyl group (Ac) was confirmed by mass fragmentation. An additional acetyl

243

group linked to acidic milk oligosaccharides has previously been reported in equine

244

colostrum.8 The additional acetyl group was reported to be linked to the sialic acid group,

245

while in our study it was found to be linked to the hexose moiety.

the

neutral

oligosaccharides

Gal(β1−4)HexΝΑc,

Gal(β1-4)Hex-Hex,

β4’-

8

Full

246

12


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247

Comparison of the equine milk oligosaccharides per breed. After annotation of the

248

EMOS, peaks in the CE-LIF electropherogram of the lactose-free EMO mixture were

249

assigned. The peaks assigned were used as a reference for the identification of EMOS in the

250

CE profiles of the 12 colostrum samples of the four horse breeds. In Figure 5, the EMO

251

profiles of the four breeds are shown, including the peak assignments. The CE-LIF

252

electropherograms of the labelled EMOS showed high reproducibility and high peak

253

resolution.

254

Inter-breed EMOS comparison. The comparison of the EMO profiles for the four

255

breeds (A, B, C and D in Figure 5) reveals that the presence and relative abundance of the

256

different oligosaccharides varied depending on the breed. Quantification was carried out after

257

manual integration of CE-LIF peaks area. For two compounds, Gal(β1-4)Hex-Hex and 3'-

258

sialyl-N-acetyllactosamine, the assignment in the CE-LIF EMO profiles was not possible. For

259

these two molecules the quantification was obtained with HILIC-MSn.

260

The total concentrations of EMOS varied between the different colostrum samples: 4.63 g/L

261

for the Shetland pony, 1.81-6.71 g/L for the Crossbred Arabian/New Forest ponies, 1.42-2.75

262

for the Dutch Warmblood horses, and 1.76-2.48 g/L for the Friesian horses (Table 1).

263

Oligosaccharide concentrations found in this study are higher than concentration found in

264

cow colostrum (about 1 g/L).8 For other animals, the oligosaccharide concentration has only

265

been reported for mature milks with levels (0.02 to 0.25 g/L), as expected, lower than the

266

values obtained in this study for equine colostrum.24 Although a large variation in the total

267

concentration of EMOS was observed, it was noticed that the ponies’ colostrum samples

268

contained the highest concentration of EMOS, followed by the Dutch Warmblood horses’

269

and the Friesian horses’ colostrum samples.

13



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Twelve out of the 16 EMOS were present in all colostrum samples. Gal(β1-4)Hex-Hex and

271

3'-sialyl-N-acetyllactosamine were absent in the Friesian horses’ colostrum samples, β4'-

272

galactosyllactose was absent in the Crossbred ponies’ colostrum samples, and 6'-sialyl-N-

273

acetyllactosamine was absent in the Dutch Warmblood horses’ colostrum samples (Table 1).

274

In all breeds, the most abundant oligosaccharides were β3'-galactosyllactose, β6'-

275

galactosyllactose, 3'-sialyllactose, and disialyllactose (numbers 2, 4, 9, and 12 in Table 1).

276

The highest concentrations of β6'-galactosyllactose, β3'-sialyllactose, and disialyllactose

277

were found in the colostrum sample of the Shetland pony (concentrations of 1.91 g/L, 1.73

278

g/L, and 0.23 g/L, respectively). These values for EMOS present in Shetland pony colostrum

279

are only an indication, since only one colostrum sample was available for the analysis. The

280

highest concentration of the β3'-galactosyllactose was found in the colostrum samples of the

281

Crossedbred ponies, with an average concentration of 0.14 g/L.

282

In profiles A1, and B1-5 (Figure 5) high concentrations of non-characterized EMOS,

283

migrating at 5.6 and 6.2 min., were observed. From the CE migration time it could be

284

suggested that these are neutral trimers and pentamers, respectively. The unidentified peaks

285

with a retention time of 6.5, and 6.6 min (Figure 5), suggesting acidic or neutral hexamers,

286

were present in all colostrum samples.

287

The oligosaccharide with the lowest observed concentration in all colostrum samples was 3'-

288

sialyl-N-acetyllactosamine with an average concentration of 0.02 g/L.

289

Intra-breed EMOS comparison. Evaluating the EMOS profiles within the same

290

breed, differences were also noticed. Specifically, for the Dutch Warmblood profiles, β6'-

291

galactosyllactose and 3'-sialylactose (numbers 2 and 9 in Table 1) were the most abundant

292

oligosaccharides and their concentrations varied by a factor 3.2 and 4, respectively. β6'-

14


Page 15 of 32


293

galactosyllactose was present with a concentration of 0.65-2.08 g/L, while 3'-sialylactose

294

with was present with a concentration of 0.29-1.16 g/L.

295

Looking at the overall electropherograms, the Crossbred ponies’ colostrum samples

296

contained the same EMOS structures, although they highly varied in concentration. β4'-

297

Galactosyllactose, however, was not detected in three colostrum samples (B3-5, Table 1). In

298

the Crossbred ponies’ colostrum samples, similarly to the Dutch Warmblood horses’

299

colostrum samples, the most abundant oligosaccharides were the β6'-galactosyllactose and

300

the 3'-sialylactose. The β6'-galactosyllactose was present in a concentration of 0.55-3.15 g/L,

301

while the 3'-sialylactose with was present in a concentration of 0.38-1.75 g/L, exhibiting a 5.7

302

and a 4.6 factor, respectively. Intra-breed analysis showed relatively high variation in EMOS

303

concentration (Table 1). Most probably the variation in EMOS concentration could be

304

dependent on the intrinsic variation in composition and in quality of colostrum among

305

individuals, as shown before for cow milk and colostrum.25, 26

306

Presence of equine milk oligosaccharides structures in comparison with the

307

profile in milk of other mammals. Ten out of the 16 EMOS found in this study were also

308

found in milk samples from other domestic animals (Table 2). So far, β6'- and 3'-

309

galactosyllactose, 3'-sialylactose, and 3'-sialyl-N-acetyllactosamine have been found in

310

bovine, caprine, and ovine milk, while 6'-sialyllactose and disialyllactose have been found

311

both in bovine and caprine milk.7,

312

while β4'-galactosyllactose, lacto-N-novo-pentaose I, and 6'-sialyl-N-acetyllactosamine were

313

present in bovine milk.7, 9, 27 Ten out of 16 milk oligosaccharide structures present in equine

314

colostrum were previously reported to be present in human milk: the neutral trimers,

315

tetramer, and hexamer, and the acidic 3’-, 6’-sialyllactose, tetramer, pentamer, and

316

hexamer.10, 20

27

Lacto-N-hexaose was characteristic for caprine milk,

15



Page 16 of 32

317

The presence of milk oligosaccharides carrying the bifidogenic factor (Gal(β1-3)GlcNAc)

318

(number 6, 7, and 13 in Table 1) and the presence of eight EMOS decorated with sialic acids

319

suggest bioactivity of the EMOS, such as being bifidogenic and soluble ligands for intestinal

320

pathogens.2, 28-31 In this study, EMOS decorated with N-glycolylneuraminic acid (Neu5Gc)

321

were not detected, while they were reported to count for less than 1% in previous study.8 The

322

presence of Neu5Gc linked to milk oligosaccharides is not rare in oligosaccharides derived

323

from domestic animals. For example, such oligosaccharides account for about 6% of the

324

oligosaccharides in cows’ colostrum.8 On the contrary, milk oligosaccharides decorated with

325

Neu5Gc are absent in human colostrum, which is considered as beneficial since blood

326

antibodies against Neu5Gc are present in several diseases such as acute and chronic hepatitis,

327

hyperacute transplant rejection, and cancer. 10, 31-33

328

In summary, oligosaccharide profile of colostrum from horses showed significant differences

329

between breeds as well as per individual. EMOS identified showed quite some overlap with

330

oligosaccharides present in milk from human and other mammals, although also horse

331

specific structures were identified. The presence of EMOS carrying the bifidogenic factor

332

and/or the N-acetylneuraminic acid, together with low concentrations of EMOS with N-

333

glycolylneuraminic acid, could make equine colostrum a good source for further research into

334

in vitro bioactive effects of milk oligosaccharides.

16


Page 17 of 32


335

ABBREVIATIONS

336

DSL: Disialyllactose

337

DS-LNnT: Di-sialyllacto-N-neotetraose

338

Fuc: Fucose

339

Gal: D-Galactose

340

GalNAc: N-Acetylgalactosamine

341

β3'-GL: β3'-Galactosyllactose

342


343


344

Glc: D-Glucose

345

GlcNAc: N-Acetylglucosamine

346

Hex: Hexaose

347

HexNAc: N-Acetyl-hexosamine

348

LNH: Lacto-N-hexaose

349

LNnT: Lacto-N-neo-tetraose

350

LNnP I: lacto-N-novo-pentaose I

351

LSTa: Sialyllacto-N-tetraose a

352

3'-SL: 3'-Sialylactose

353

6'-SL: 6'-Sialyllactose

354

3'-SLN: 3'-Sialyl-N-acetyllactosamine

355

6'-SLN: 6'-Sialyl-N-acetyllactosamine 17



356

357 358

Page 18 of 32

ACKNOWLEDGEMENT We would like to thank Edwin Bakx (Laboratory of Food Chemistry, Wageningen University) for the fruitful discussions on the interpretation of the mass spectra.

359

360

SUPPORTING INFORMATION AVAILABLE: Analytical techniques used for the

361

identification of EMOS in the Dutch Warmblood horses’ colostrum, and EMOS’ liquid

362

chromatography and mass spectrometry characteristics. This material is available free of

363

charge via the Internet at http://pubs.acs.org.

18


Page 19 of 32


364

References

365

1. German, J. B.; Freeman, S. L.; Lebrilla, C. B.; Mills, D. A., Human milk oligosaccharides: evolution, structures and bioselectivity as substrates for intestinal bacteria. Nestle Nutr Workshop Ser Pediatr Program 2008, 62, 205-18; discussion 218-22. 2. Asakuma, S.; Hatakeyama, E.; Urashima, T.; Yoshida, E.; Katayama, T.; Yamamoto, K.; Kumagai, H.; Ashida, H.; Hirose, J.; Kitaoka, M., Physiology of consumption of human milk oligosaccharides by infant gut-associated bifidobacteria. J Biol Chem 2011, 286, 3458392. 3. Preidis, G. A.; Versalovic, J., Targeting the human microbiome with antibiotics, probiotics, and prebiotics: gastroenterology enters the metagenomics era. Gastroenterology 2009, 136, 2015-31. 4. Seifert, S.; Watzl, B., Inulin and oligofructose: review of experimental data on immune modulation. J Nutr 2007, 137, 2563S-2567S. 5. Bode, L., Human milk oligosaccharides: every baby needs a sugar mama. Glycobiology 2012, 22, 1147-62. 6. He, Y.; Liu, S.; Kling, D. E.; Leone, S.; Lawlor, N. T.; Huang, Y.; Feinberg, S. B.; Hill, D. R.; Newburg, D. S., The human milk oligosaccharide 2'-fucosyllactose modulates CD14 expression in human enterocytes, thereby attenuating LPS-induced inflammation. Gut 2014. 7. Urashima, T.; Saito, T.; Nakamura, T.; Messer, M., Oligosaccharides of milk and colostrum in non-human mammals. Glycoconj J 2001, 18, 357-71. 8. Albrecht, S.; Lane, J. A.; Marino, K.; Al Busadah, K. A.; Carrington, S. D.; Hickey, R. M.; Rudd, P. M., A comparative study of free oligosaccharides in the milk of domestic animals. Br J Nutr 2014, 111, 1313-28. 9. Parkkinen, J.; Finne, J., Isolation of sialyl oligosaccharides and sialyl oligosaccharide phosphates from bovine colostrum and human urine. Methods Enzymol 1987, 138, 289-300. 10. Urashima, T.; Asakuma, S.; Messer, M., Milk oligosaccharides. In Comprehensive glycoscience. From chemistry to system biology, Kamerling, J. P.; Boons, G. J.; Lee, Y. C.; Suzuki, A.; Taniguchi, N.; Voragen, A. G. J., Eds. Elsevier: Oxford, UK, 2007; Vol. 4, pp 695-722. 11. Tao, N.; DePeters, E. J.; German, J. B.; Grimm, R.; Lebrilla, C. B., Variations in bovine milk oligosaccharides during early and middle lactation stages analyzed by highperformance liquid chromatography-chip/mass spectrometry. J. Dairy Sci. 2009, 92, 29913001. 12. Albrecht, S. Gastrointestinal-active oligosaccharides from human milk and functional foods. PhD. thesis Wageningen University, Wageningen, The Netherlands, 2011. 13. Nakamura, T.; Amikawa, S.; Harada, T.; Saito, T.; Arai, I.; Urashima, T., Occurrence of an unusual phosphorylated N-acetyllactosamine in horse colostrum. Biochim Biophys Acta 2001, 1525, 13-8. 14. Chaturvedi, P.; Warren, C. D.; Altaye, M.; Morrow, A. L.; Ruiz-Palacios, G.; Pickering, L. K.; Newburg, D. S., Fucosylated human milk oligosaccharides vary between individuals and over the course of lactation. Glycobiology 2001, 11, 365-72. 15. Kunz, C.; Rudloff, S.; Baier, W.; Klein, N.; Strobel, S., Oligosaccharides in human milk: structural, functional, and metabolic aspects. Annu Rev Nutr 2000, 20, 699-722. 16. Erney, R. M.; Malone, W. T.; Skelding, M. B.; Marcon, A. A.; Kleman-Leyer, K. M.; O'Ryan, M. L.; Ruiz-Palacios, G.; Hilty, M. D.; Pickering, L. K.; Prieto, P. A., Variability of human milk neutral oligosaccharides in a diverse population. J Pediatr Gastroenterol Nutr 2000, 30, 181-92.

366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411

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412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459

Page 20 of 32

17. Albrecht, S.; van Muiswinkel, G. C. J.; Schols, H. A.; Voragen, A. G. J.; Gruppen, H., Introducing capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) for the characterization of konjac glucomannan oligosaccharides and their in vitro fermentation behavior. J Agric Food Chem 2009, 57, 3867-3876. 18. Albrecht, S.; Schols, H. A.; van Zoeren, D.; van Lingen, R. A.; Groot Jebbink, L. J. M.; van den Heuvel, E. G. H. M.; Voragen, A. G. J.; Gruppen, H., Oligosaccharides in feces of breast- and formula-fed babies. Carbohyd. Res. 2011, 346, 2173-2181. 19. Remoroza, C.; Buchholt, H. C.; Gruppen, H.; Schols, H. A., Descriptive parameters for revealing substitution patterns of sugar beet pectins using pectolytic enzymes. Carbohydr. Polym. 2014, 101, 1205-1215. 20. Albrecht, S.; Schols, H. A.; van den Heuvel, E. G. H. M.; Voragen, A. G. J.; Gruppen, H., Occurrence of oligosaccharides in feces of breast-fed babies in their first six months of life and the corresponding breast milk. Carbohyd. Res. 2011, 346, 2540-2550. 21. Urashima, T.; Saito, T.; Kimura, T., Chemical structures of three neutral oligosaccharides obtained from horse (thoroughbred) colostrum. Comparative Biochemistry and Physiology Part B: Comparative Biochemistry 1991, 100, 177-183. 22. Hernandez-Hernandez, O.; Calvillo, I.; Lebron-Aguilar, R.; Moreno, F. J.; Sanz, M. L., Hydrophilic interaction liquid chromatography coupled to mass spectrometry for the characterization of prebiotic galactooligosaccharides. J Chromatogr A 2012, 1220, 57-67. 23. Wheeler, S. F.; Harvey, D. J., Negative ion mass spectrometry of sialylated carbohydrates: discrimination of N-acetylneuraminic acid linkages by MALDI-TOF and ESITOF mass spectrometry. Anal. Chem. 2000, 72, 5027-5039. 24. Martinez-Ferez, A.; Rudloff, S.; Guadix, A.; Henkel, C. A.; Pohlentz, G.; Boza, J. J.; Guadix, E. M.; Kunz, C., Goats' milk as a natural source of lactose-derived oligosaccharides: Isolation by membrane technology. Int Dairy J 2005, 16, 173-181. 25. Parrish, D. B.; Wise, G. H.; Hughes, J. S.; Atkeson, F. W., Properties of the Colostrum of the Dairy Cow. V. Yield, Specific Gravity and Concentrations of Total Solids and its Various Components of Colostrum and Early Milk1. J. Dairy Sci. 1950, 33, 457-465. 26. Kehoe, S. I.; Jayarao, B. M.; Heinrichs, A. J., A Survey of Bovine Colostrum Composition and Colostrum Management Practices on Pennsylvania Dairy Farms1. J. Dairy Sci. 2007, 90, 4108-4116. 27. Kiskini, A.; Difilippo, E., Oligosaccharides in goat milk: structure, health effects and isolation. Cell Mol Biol (Noisy-le-grand) 2013, 59, 25-30. 28. Kitaoka, M.; Tian, J.; Nishimoto, M., Novel putative galactose operon involving lacto-N-biose phosphorylase in Bifidobacterium longum. Appl Environ Microbiol 2005, 71, 3158-62. 29. Wada, J.; Ando, T.; Kiyohara, M.; Ashida, H.; Kitaoka, M.; Yamaguchi, M.; Kumagai, H.; Katayama, T.; Yamamoto, K., Bifidobacterium bifidum lacto-N-biosidase, a critical enzyme for the degradation of human milk oligosaccharides with a type 1 structure. Appl Environ Microbiol 2008, 74, 3996-4004. 30. Sela, D. A.; Li, Y.; Lerno, L.; Wu, S.; Marcobal, A. M.; German, J. B.; Chen, X.; Lebrilla, C. B.; Mills, D. A., An infant-associated bacterial commensal utilizes breast milk sialyloligosaccharides. J Biol Chem 2011, 286, 11909-18. 31. Bode, L., Recent advances on structure, metabolism, and function of human milk oligosaccharides. J Nutr 2006, 136, 2127-30. 32. Morito, T.; Nishimaki, T.; Masaki, M.; Yoshida, H.; Kasukawa, R.; Nakarai, H.; Kano, K., Studies on Hanganutziu-Deicher Antigens-Antibodies. Int Arch Allergy Immunol 1986, 81, 204-209.

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Page 21 of 32

460 461 462 463 464


33. Tangvoranuntakul, P.; Gagneux, P.; Diaz, S.; Bardor, M.; Varki, N.; Varki, A.; Muchmore, E., Human uptake and incorporation of an immunogenic nonhuman dietary sialic acid. Proceedings of the National Academy of Sciences 2003, 100, 12045-12050. 34. Urashima, T.; Saito, T.; Nakamura, T.; Messer, M., Oligosaccharides of milk and colostrum in non-human mammals. Glycoconj J 2001, 18, 357-71.

465

21



Page 22 of 32

466

Note

467

This project is jointly financed by the European Union, European Regional Development

468

Fund and The Ministry of Economic Affairs, Agriculture and Innovation, Peaks in the Delta,

469

the Municipality of Groningen, the Provinces of Groningen, Fryslân and Drenthe, the Dutch

470

Carbohydrate Competence Center (CCC WP25; www.cccresearch.nl), Danone Nutricia

471

Research and FrieslandCampina.

472

22


Page 23 of 32


473

Figure Captions

474

Figure 1: CE-LIF electropherograms of lactose-free EMO mixture and the EMO SEC pools1-

475

4. *: Internal standard xylose, #: Lactose, 1-14: identified peaks as named in Table 1.

476 477

Figure 2. Selected base peak for the lacto-N-novo-pentaose-I (LNnP-I) in CE-MSn as present

478

in lactose-free EMO mixture (a). Corresponding MS2 fragmentation patterns and structural

479

composition of LNnP-I (b). m/z 436 precursor ion in CE-MSn. [-2], [-3]: charge state of the

480

molecules formed after fragmentation.

481 482

Figure 3. Selected base peak for the 3'-sialyl-N-acetyllactosamine (3’-SLN) in HILIC-MSn as

483

present in lactose-free EMO mixture (a). Corresponding MS2 fragmentation patterns and

484

structural composition of 3’-SLN as found in the lactose-free EMO mixture (b). m/z 673

485

precursor ion in HILIC-MSn. [-1]: charge state of molecules

486 487

Figure 4. Selected base peak for the Gal(β1-4)HexNAc in HILIC- MSn (a) as present in

488

lactose-free EMO mixture. Corresponding base peak after degradation by Glyco-

489

galactosidases specific for β1-4 linkage (b), by Glyco-galactosidases specific for β1-3 linkage

490

(c), by Glykoβ−N-acetylhexosaminidase (d). Selected base peak for the 3’-sialyllacto-N-

491

tetraose-a (LSTa) in HILIC- MSn as present in lactose-free EMO mixture (e). Corresponding

492

base peak after degradation by Sialidase S specific for the α2-3 linkage (f). m/z 382 precursor

493

ion for Gal(β1-4)HexNAc in HILIC-MSn; m/z 998 precursor ion for LSTa in HILIC-MSn.

494 495

Figure 5: CE-LIF electropherograms of APTS-derivatised oligosaccharides for the four

496

breeds. A: Dutch Warmblood horse; B: Crossbred Arabian/New Forest pony; C: Shetland

23



Page 24 of 32

497

Pony; D: Friesian horse. *: Internal standard xylose, #: Lactose, 1-14: identified peaks as

498

named in Table 1.

499

24


Page 25 of 32


Tables Table 1. Presence and Concentration of Equine Milk Oligosaccharides for the Four Breeds’ Colostrum Samplesa CONCENTRATION OF EQUINE MILK OLIGOSACCHARIDE (g/L) Degree of polymerization

EMOS structures

Abbreviation

MW

Peak CE-LIF (Figure 1)

Crossbred Arabian/ New Forest ponies

Dutch Warmblood

Shetland pony

Friesian

A1

A2

A3

A4

B1

B2

B3

B4

B5

C1

D1

D2

1 na 2 3 4 5 6 7

0.11 ++ 0.98 tr 0.16 0.02 0.03 0.03

0.14 ++ 2.08 tr 0.16 tr 0.08 0.05

0.05 ++ 0.65 tr 0.10 0.13 0.09 0.02

0.08 + 0.84 tr 0.09 0.02 tr tr

0.14 + 0.83 tr 0.23 0.39 0.05 0.04

0.11 + 0.55 tr 0.08 0.21 0.04 0.03

0.38 + 1.64

0.09 ++ 0.83

0.51 + 3.15

0.22 + 1.91

0.29

0.13

─

─

─

─

0.04 0.13 0.04 0.02

0.14 0.26 0.04 0.04

0.23 0.09 0.11 0.07

0.11 0.10 0.06 0.04

0.07 0.05 1.00 0.10 tr

─

─

0.03 1.16 0.15 tr

0.02 0.71 0.07 tr

0.01 0.02 0.29 0.03 tr

0.12 0.01 0.48 0.12 0.10

0.02 0.003 0.38 0.06 0.05

0.06 0.01 0.69 0.12 0.01

0.01 0.02 0.44 0.03 0.06

0.02 0.03 1.75 0.23 0.02

0.02 0.02 1.73 0.09 0.03

NEUTRAL DIMER

TRIMER

TETRAMER PENTAMER ESAMER

TRIMER

Gal(β β1-4)HexNAc Gal(β β1-4)Hex-Hex β6'-Galactosyllactose β4'-Galactosyllactose β4 β3'-Galactosyllactose Lacto-N-neotetraose Lacto-N-novo-pentaose I Lacto-N-hexaose ACIDIC 6'-Sialyl-N-acetyllactosamine 3'-Sialyl-N-acetyllactosamine 3'-Sialylactose 6'-Sialyllactose 6’-Sialyl-Hex-Ac-HexNAc

TETRAMER

Disialyllactose

PENTAMER HEXAMER

Sialyllacto-N-tetraose-a Disialyllacto-N-tetraose*

TOTAL OLIGOSACCHARIDES CONCENTRATION AVERAGE OLIGOSACCHARIDES CONCENTRATION PER BREED

383 β6'-GL β4'-GL β3'-GL LNnT LNnP I LNH 6'-SLN 3'-SLN 3'-SL 6'-SL

504

708 870 1073

─

─

1.20 tr 0.05 0.07 tr tr

0.88 tr 0.05 0.03 tr 0.01

0.07

0.19

716

8 na 9 10 11

DSL

925

12

0.16

0.13

0.04

0.02

0.09

0.06

0.21

0.02

0.40

0.23

0.13

0.07

LSTa DS-LNT*

999 1290

13 14

0.02 0.02

0.03 tr

0.06 tr

0.02 tr

0.27 0.15

0.16 0.06

0.83 0.03

0.16 0.05

0.06 0.04

0.07 tr

0.05 tr

tr 0.03

2.75

4.01

1.94

1.42

3.02

1.81

4.21

2.19

6.71

4.63

2.48

1.76

675 633

2.52

3.58

4.63

─

─

0.53 0.09 tr

0.33 0.02 0.02

2.12

a

na= peak not assigned in CE-LIF and quantified by HILIC-MSn, ++= 0.001-0.002; += 0.001-0.0002 g/L; tr=detectable but not quantifiable; ─ = not detected, * = isomer not further specified. Structures in bold: oligosaccharides novel for the equine milk literature.8, 21, 34

25



Page 26 of 32

Table 2. EMOS Sound in the Dutch Warmblood Horses’ Colostrum and Their Presence in Other Mammalian Milk Samples b

MILK DEGREE OF POLYMERIZATION

EMOS structures (Dutch Warmblood milk)

COW

SHEEP

GOAT

HUMAN

7, 9

7, 27

7, 27

10, 20

Gal(β β 1-4)HexNAc

─

─

─

─

Gal(β β 1-4)Hex-Hex

─

─

─

─

8, 21, 34

NEUTRAL EQUINE MILK OLIGOSACCHARIDES DIMER

TRIMER

β6'-Galactosyllactose

X

X

X

X

β4'-Galactosyllactose β4

X

─

─

X

β3'-Galactosyllactose

X

X

X

X

TETRAMER

Lacto-N-neotetraose

─

─

─

X

PENTAMER

Lacto-N-novo-pentaose I

X

─

─

─

HEXAMER

Lacto-N-hexaose

─

─

X

X

6'-Sialyl-N-acetyllactosamine

X

─

─

─

3'-Sialyl-N-acetyllactosamine

X

X

X

─

3'-Sialylactose

X

X

X

X

6'-Sialyllactose

X

─

X

X

6’-Sialyl-Hex-Ac-HexNAc

─

─

─

─

TETRAMER

Disialyllactose

X

─

X

X

PENTAMER

Sialyllacto-N-tetraose-a

─

─

─

X

HEXAMER

Disialyllacto-N-tetraose*

─

─

─

X

ACIDIC EQUINE MILK OLIGOSACCHARIDES

TRIMER

b

X = present; ─ = not present, * oligosaccharides novel for

=

isomer not further specified. Structures in bold: 21, 34 the equine milk literature.8,

26


Page 27 of 32


Figure 1

# 2

9

4

12

*

LIF Intensity

8

10

14

7

SEC-pool 1

6 SEC-pool 2 3

5

SEC-pool 3 SEC-pool 4 Lactose-free EMO mixture

4.2

5.00

6.00

7.00

Time (min)

27



Page 28 of 32

Figure 2 500

-3

501 502

APTS

503

LNnP-I

a)

504

m/z 436

100

505

Relative abundance

506 507 508

50

509 510 511 512

0

31.0

33.0

513

35.0

Time (min)

514 -3

515

b)

APTS

-2 382 391

100

516

APTS

517 518 519

Relative Abundance

520 521

-3 APTS

522

50 -2

523

APTS

524 -2

APTS

-3

-2

310 APTS APTS

-3

260 0

526 APTS 472

314

492

400

galactose

527 573

328 300

glucose

525 -2

APTS

m/z

500

528

580

N-acetyl-glucosamine

28


Page 29 of 32


Figure 3

3’-SLN

a)

m/z 673 -1

Relative Abundance

100

6’-SLN

50

m/z 673

0 0

5 10 Time (min) -H2O

b)

290

100

Relative Abundance

15

1

572

-CO2 5

655

50 3

-CO2 308 0

-H2O

3

200

300

408

468

400

500

600

700

m/z galactose

N-acetyl-glucosamine

N-acetylneuraminic acid

29



Page 30 of 32

Figure 4

Gal(β β1-4)NAcHex

LST a

m/z 382

a)

50

50

e)

0

0

100

100 Relative Abundance

m/z 998

100

100

b)

50

f) 50

0

0 13.0

100

15.0 17.0 Time (min)

c)

50 0 100

d)

50 0 0

3.00

6.00

9.00

Time (min)

30


Page 31 of 32


Figure 5 *

# 2

min 5.6

9

min 6.2

1 4 8 12 10 11 3

13 6

7

LIF Intensity

5

min min 14 6.5 6.6 A 1 2 3 4

B 1 2 3 4 5

EOS26Bcrossed

C 1 D 1 2

4.2

5.00

6.00

7.00

8.40

Time (min)

31



Page 32 of 32

Table of content

32


Comparison of Milk Oligosaccharides Pattern in Colostrum of

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