Characterization of rat and mouse acidic milk oligosaccharides based on hydrophilic interaction chromatography coupled with electrospray tandem mass spectrometry

Oligosaccharides are one of the most important components in mammalian milk. Milk oligosaccharides can promote colonization of gut microbiota and protect newborns from infections. The diversity and structures of MOs differ among mammalian species.

Available online 2 February 2021

0144-8617/© 2021 The Authors Published by Elsevier Ltd This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

Characterization of rat and mouse acidic milk oligosaccharides based on

hydrophilic interaction chromatography coupled with electrospray tandem

mass spectrometry

Jiaqi Lia,b,1, Maorong Jiangc,1, JiaoRui Zhoud, Junjie Dinga, Zhimou Guoa,b, Ming Lid,

Fei Dingc, Wengang Chaie, Jingyu Yana,b,* , Xinmiao Lianga,b,*

aDalian Institute of Chemical Physics, Chinese Academy of Sciences, Key Laboratory of Separation Science for Analytical Chemistry, Dalian, 116023, China

bUniversity of Chinese Academy of Sciences, Beijing, 100049, China

cKey Laboratory of Neuroregeneration of Jiangsu and Ministry of Education, Co-Innovation Center of Neuroregeneration, Nantong University, Nantong, Jiangsu,

226001, China

dDepartment of Microecology, College of Basic Medical Science, Dalian Medical University, Dalian, 116044, China

eGlycosciences Laboratory, Faculty of Medicine, Imperial College London, Hammersmith Campus, London, W12 0NN, United Kingdom

A R T I C L E I N F O

Keywords:

Milk oligosaccharides

Rat and mouse

Structural characterization

Electrospray mass spectrometry

A B S T R A C T Oligosaccharides are one of the most important components in mammalian milk Milk oligosaccharides can promote colonization of gut microbiota and protect newborns from infections The diversity and structures of MOs differ among mammalian species MOs in human and farm animals have been well-documented However, the knowledge on MOs in rat and mouse have been very limited even though they are the most-widely used models for studies of human physiology and disease Herein, we use a high-sensitivity online solid-phase extraction and HILIC coupled with electrospray tandem mass spectrometry to analyze the acidic MOs in rat and mouse Among the fifteen MOs identified, twelve were reported for the first time in rat and mouse together with two novel sulphated oligosaccharides The complete list of acidic oligosaccharides present in rat and mouse milk is the baseline information of these animals and should contribute to biological/biomedical studies using rats and mice as models

1 Introduction

Breast milk is the primary source of nutrition for the mammals and

plays pivotal roles for their growth and development (Ballard &

Morrow, 2013; Victora et al., 2016) In humans, oligosaccharides are

one of the most abundant components in milk in addition to proteins and

fats (Bode, 2012; Kunz, Rudloff, Baier, Klein, & Strobel, 2000) They are

involved in numerous functions such as balancing infant’s gut

micro-biota as prebiotic (Bode, 2012; Marcobal et al., 2010), antiadhesive

antimicrobials (Bode, 2012; Craft, Thomas, & Townsend, 2019; Lin

et al., 2017), immune system modulators (Comstock et al., 2017;

Newburg, 2009; Zuurveld et al., 2020) and nutrients for brain

development (Charbonneau et al., 2016; Wang et al., 2019)

In recent years, there have been an increasing number of reports describing the contents, diversities and differences of oligosaccharides from different mammalian milk (Fukuda et al., 2010; Kumar & Deepak,

2019; Mineguchi et al., 2018; Tao, Ochonicky, German, Donovan, & Lebrilla, 2010; Verruck, Santana, de Olivera Müller, & Prudencio,

2018) The major difference has been found in milk between human and non-human mammals, e.g bovine, ovine, chimpanzee, and other farm and nonfarm mammals (Urashima, Saito, Nakamura, & Messer, 2001) Compared to the human milk, non-human mammalian milk contains much less oligosaccharides, in which sialylated milk oligosaccharides (SMOs) are the major components (Albrecht, Lane, Marino, Al Busadah,

Abbreviations: MOs, milk oligosaccharides; SMOs, sialylated milk oligosaccharides; SPE, solid-phase extraction; HILIC, hydrophilic interaction chromatography;

ESI-MS, electrospray mass spectrometry; CID, collision-induced dissociation; PBS, phosphate-buffered saline; ACN, acetonitrile; TIC, total ion chromatogram; Glc,

glucose; Gal, galactose; GlcNAc, N-acetylglucosamine; Neu5Ac, N-acetylneuraminic acid; Su, sulphate; SL, sialyl lactose; SLN, sialyllactosamine; DSL, disialylated lactose; LST, sialyl-lacto-N-tetraosese; LNTri, lacto-N-trisaccharide; R, C3H8O3; NH4FA, ammonium formate

* Corresponding authors

E-mail addresses: yanjingyu@dicp.ac.cn (J Yan), liangxm@dicp.ac.cn (X Liang)

1 Jiaqi Li and Maorong Jiang contributed equally to this work

Contents lists available at ScienceDirect Carbohydrate Polymers

journal homepage: www.elsevier.com/locate/carbpol

https://doi.org/10.1016/j.carbpol.2021.117734

Received 7 November 2020; Received in revised form 5 January 2021; Accepted 26 January 2021

Carbohydrate Polymers 259 (2021) 117734

& Carrington, 2014) Due to the much lower content of milk

oligosac-charides in non-human mammals than that in human and interference

from the large amount of lactose, the detection and analysis of SMOs

have not been straightforward Various methods have been developed to

overcome these problems (Mineguchi et al., 2018; Monti, Cattaneo,

Orlandi, & Curadi, 2015) We have recently established an online

solid-phase extraction with hydrophilic interaction chromatography

(HILIC) followed by negative-ion electrospray mass spectrometry

(ESI-MS) method for profiling SMOs in the human and other mammalian

milk (Yan et al., 2018) It showed great promise for detection and

sequence determination of acidic oligosaccharides in the milk,

espe-cially for the low content acidic oligosaccharides in the non-human

mammalian milk

Among the non-human mammals, rats produce much less milk This

poses considerable difficulty for the study of rat milk oligosaccharides

and there have been very limited reports on the oligosaccharides in rats

However, rats share 90 % of the genome with humans (Dvorak et al.,

2004) and have been a prevalent model in biomedical research Almost

all disease-related genes in human we currently know of have equivalent

ones within the rat genome, and this makes rat a suitable research tool

for human disease (Jacob & Kwitek, 2002) Well-established strains of

rats are currently used extensively in study of many human diseases The

rat has allowed us to build up an incredible wealth of knowledge about

basic biology and complex physiological interactions, and has served as

a model of human disease and learning, much of which has been

translated to greater knowledge about humans (Serikawa et al., 2014),

and used to answer many research questions (Melina, 2010)

Scientists can now breed genetically-altered transgenic rats or mice,

carrying genes similar to those that cause human diseases Likewise,

selected genes can be turned off or made inactive, creating “knockout”

rats or mice which can be used to evaluate the effects of cancer-causing

chemicals (carcinogens) and assess drug safety (Corpet & Pierre, 2005;

Vlaming et al., 2006) Rats and mice are very useful research animals

also because their anatomy, physiology, genetics, and all basic biology

and biochemistry are well-understood, making the changes of their

be-haviours and characteristics readily identifiable during specific

in-vestigations (Gosling, 2001)

Apart from directly affecting the survival and development of the

newborns, rat milk has other important biological functions (Briffa et al.,

2017; Dvorak et al., 2004; Egelrud, Helander, & Olivecrona, 1970;

Kariakin & Alekseev, 1991; Meng et al., 2013) However, rat milk

compositions, particularly the milk oligosaccharides, have not been

well-documented Rat milk oligosaccharides were reported half a

cen-tury ago Due to the difficulty in collection of sufficient amounts of milk

only three acidic oligosaccharides have been described so far:

3’-sia-lyllactose (3’-SL), 6’-sulphated lactose (6’-Su-L) and

6’-sulphate-3’-sia-lyllactose (6’-Su-3’-SL) (Carubelli, Ryan, Trucco, & Caputto, 1961; Choi

& Carubelli, 1968; Kuhn, 1972; Naccarato, Ray, & Wells, 1975)

In the present work, we aim to carry out a comprehensive study on

the acidic oligosaccharides in rat and mouse milk, by detection, profiling

and sequencing of acidic oligosaccharides For profiling, the online dual

functional HPLC coupled with ESI-MS (Yan et al., 2018) is used, in which

the SPE is for removal of the dominant lactose and enrichment of the

acidic oligosaccharides, while the subsequent HILIC is for their detailed

separation Collision-induced dissociation tandem ESI-MS

(ESI CID-MS/MS) is then used for sequence (Chai et al., 2006) and sialic acid

α2-3/α2-6 linkage analysis (Wheeler & Harvey, 2000) The complete list

of acidic oligosaccharides presents in the milk of rats and mice resulted

from this study can be considered as one of background information of

these animals and should be useful to future biological and biomedical

studies using rats and mice as models

2 Materials and methods

2.1 Reagents and materials

HPLC-grade ACN was obtained from Merck (Darmstadt, Germany) Ammonium formate and formic acid were from J&K Scientific (Beijing, China) All other reagents used in this work were of analytical grade or higher Water was purified by a Milli-Q water purification system (Billerica, USA) Rat milk sample was obtained from mature breast rats

in Experimental Animal Center of Nantong University (Nantong, China) Mouse mammary glands tissue extracts were prepared at Dalian Medical University (Dalian, China)

2.2 Preparation of milk oligosaccharides

Two lactating healthy rats were selected for breast milk collection three times a day with the help of manual squeezing over a period of one week The 1 mL rat milk sample collected was stored at − 40 ◦C before lyophilization The freeze-dried milk powder was then dissolved in water at a concentration of 30 mg/mL The resulting concentrated milk was centrifuged at 8000 rpm for 10 min at 4 ◦C After the removal of the top lipid layer, two volumes of ethanol were added to the mixture, and the mixture was stored at 4 ◦C for 6 h The mixture was then centrifuged

at 8000 rpm for 10 min at 4 ◦C The supernatant contains the oligo-saccharides and was used for analysis

As mouse milk was difficult to collect directly from lactating mouse

by squeesing, mammary tissue was used for extraction of milk oligo-saccharides After sacrifice, the entire mammary gland of maternal mouse was gently peeled off with a scalpel, and then immersed in phosphate-buffered saline (PBS) until the white milk was extracted completely After the removal of the top lipid layer by centrifugation at

8000 rpm for 10 min at 4 ◦C, two volumes of ethanol were added into the

200 μL supernatant to obtain an ethanol/water mixture and centrifuged

at 8000 rpm The supernatant was dried and redissolved in 50 μL 50 % ACN/H2O solution The mixture was then centrifuged again, and the supernatant was used for further analysis

2.3 Online SPE-HILIC and ESI-CID-MS/MS

Online SPE-HILIC-ESI-MS/MS was carried out according to the pre-vious report (Yan et al., 2018) The analysis platform was established by using an Ultimate 3000 UHPLC system (Thermo-Fisher Scientific, Milan, Italy) followed an SCIEX X500B QTOF (AB Sciex, Foster city, CA USA) or

an Agilent Q-TOF mass spectrometer (Agilent Technologies 6450 UHD) The UHPLC is consisted of a column compartment, an autosampler, a 10-port valve and a dual gradient pump system After injection the sialylated oligosaccharides in milk sample pass through “Click TE-GSH” column (5 μm, 2.1 × 50 mm), and separated by XAmide column (5 μm, 2.1 × 150 mm, Acchrom, Beijing, China) with a flow rate of 0.2 mL/min and the following mobile phase: solvent A, ACN; solvent B, NH4FA (100

mM, pH 3.2); solvent C, H2O Gradient in “Click TE-GSH” column was 0− 10 min, A/B (80/20); 10− 30 min A/B (80/20) to A/B (40/60); 30.1–45 min, A/B (80/20) Gradient in XAmide column was 0− 6 min, A/B/C (80/10/10); 6− 36 min, A/B/C (80/10/10) to A/B/C (50/40/10); 36.1–45 min (80/10/10) Both MS and MS/MS spectra were acquired in the negative-ion mode with an acquisition rate of 1 s per spectrum over a

mass range of m/z 300–2000 (for MS) and m/z 100–2000 (for MS/MS)

The ion source gas 1 was set at 45 psi, gas 2 at 50 psi, and source tem-perature at 450 ◦C detection using IDA survey Precursor-ion selection was carried out automatically by the data system based on ion abun-dance and dynamic background subtractions Seven precursors were selected from each MS spectrum and collision energy of − 65 V ± 20 V was used for collision-induced dissociation (CID) When using the Agi-lent Q-TOF mass spectrometer, the drying gas temperature was at 350 ◦C with a flow rate of 8.0 L/min The capillary was set at 3500 V and fragmentor 175 V The skimmer voltage was at ‒ 65 V Both MS and

J Li et al

MS/MS spectra were acquired in the negative-ion mode with an

acqui-sition rate of 1 s per spectrum Precursor-ion selection was made

auto-matically by the data system based on ion abundance Three precursors

were selected from each MS spectrum to carry out product-ion scanning

Collision energy of 40 V was used for CID

3 Results and discussion

As acidic oligosaccharides are the major components of

charides in animal milk, we focused on the analysis of acidic

oligosac-charides using the method developed for profiling sialylated

oligosaccharides (Yan et al., 2018) Based on the retention mechanism of

different oligosaccharides on the SPE and HILIC column, we considered

that the online SPE-HILIC method developed for sialylated

oligosac-charides could also be applicable to sulphated ones ESI-MS was used for

detection and the compositions of mammalian milk oligosaccharides can

be readily derived from the deprotonated molecules [M− H]− as their

biosynthetic pathways and the common backbone structures have been

well established Almost all human milk oligosaccharides contain a

lactose unit (Galβ1-4Glc) at their reducing end, while

N-acetyllactos-amine (Galβ1-4GlcNAc) can also be found in non-human mammalian

milk The disaccharide cores can be extended by type 1

(-Galβ1-3GlcNAcβ1-) and type 2 (-Galβ1-4GlcNAcβ1-) chains as linear or

branched sequences These are often terminated by a few α

-mono-saccharide residues including α-Gal, α-GalNAc, α-Fuc, and α-Neu5Ac

(and α-Neu5Gc in the case of non-human mammals) We here use negative-ion ESI-MS for detection and composition analysis of the acidic oligosaccharides as the native reducing sugars and ESI-CID-MS/MS for subsequent sequencing For the low quantity of milk oligosaccharides in rats and mice, their reduction by chemical methods can eliminate possible HPLC chromatographic peak splitting due to the separation of

α/β anomers and therefore increase ion signals Reducing terminal derivatization may also improve HPLC detection by UV or fluorescence However, after reduction or reducing-terminal tagging the fragmenta-tion patterns also change completely (Zhang et al., 2013), and the unique features established for sequence assignment (Chai et al., 2006) and sialic acid linkage determination (Wheeler & Harvey, 2000) are lost, and therefore reducing sugars without derivatization are used for negative-ion LC–MS

3.1 Profiling of acidic oligosaccharides in rat milk by SPE-HILIC-MS

After removal of lactose and the possible neutral oligosaccharide components by the SPE “Click TE-GSH” column, sialylated and sulph-ated oligosaccharides were eluted out and separsulph-ated by the HILIC amide column In the total ion chromatogram (TIC) shown in Fig 1a, three major components (#3, #5 and #13) were obtained The [M− H]− of

peaks #3 and #5 are identical at m/z 632.2, with the composition of

Hex2Neu5Ac1 (H2S1), and these two can be tentatively assigned as the two isomeric sialylated lactose (SL) widely found in mammalian milk as

Fig 1 Profiles of acidic oligosaccharides from rat milk (a) Total ion chromatogram, (b) Extracted single-ion chromatograms

Carbohydrate Polymers 259 (2021) 117734

the main components The broad peak at 20 min, #13, with a [M− H]− at

m/z 712.2, an increase of 80 Da m/z 632.2, was deduced as the

sulph-ated SL with a composition of H2S1Su1 (Su: sulphate) previously found

in rat milk (Choi & Carubelli, 1968; Sturman, Lin, Higuchi, & Fellman,

1985)

Additional minor components can be found by extracted ion

chro-matograms (EICs) using different m/z values observed during MS

scanning (Fig 1b) EIC of m/z 421.0 showed a single peak (Peak #1)

which was considered as the sulphated lactose H2Su1 (Barba & Caputto,

1965; Choi & Carubelli, 1968) EIC of m/z 673.2 exhibited two peaks,

#2 and #4, and from the composition of H1N1S1 (N: HexNAc) these can

be considered as the sialyllactosamine (SLN) isomers The peak split of

both #2 and #4 indicated that a GlcNAc is at the reducing end as the

separation of the α and β anomers of HexNAc tends to be more

promi-nent Peaks #6–#10 were all identified as mono-sialylated

oligosac-charides (Fig 1b and Table 1) while #11–#15 each contain two acidic

groups either di-sialylated (#11 and #14) or mono-sialylated and

mono-sulphated (#12, #13 and #15) Clearly sulphate is similar to sialic

acid to have stronger electrostatic interaction with the amide stationary

phase and increased retention time The largest oligosaccharides found

are pentasaccharides but there was no fucose detected in any of the rat

milk oligosaccharides Apart from SLN (#2 and #4) and SL (#3 and #5)

discussed above, two more well resolved isomeric pairs were detected:

#7/#8 (H2N1S1), and #9/#10 (H3N1S1)

3.2 Sequence determination of monosialylated oligosaccharide by ESI-

CID-MS/MS

Different fragmentation patterns in negative ion ESI-CID-MS/MS

(Chai et al., 2006) was then used to determine the sequence and

par-tial linkages of the detected milk oligosaccharides and to differentiation

of the isomeric structures

SL with α2-3 or α2-6 linkages (peaks #3 and #5, respectively) were identified by their different fragmentations Consistent to literature data (Chai et al., 2006), characteristic fragments C2 (m/z 470), 0,2A2 (m/z

410) and 0,2A2-CO2 (m/z 306) in the spectrum of #5 (Fig 2b) indicated a Neu5Ac α2-6-linked lactose (6’-SL), whereas, the unique fragments 2,

4A3-CO2 (m/z 468) and B2-CO2 (m/z 408) identified a

Neu5-Acα2-3-linked lactose (3’-SL) 3’-SL and 6’-SL are most common acidic oligosaccharides in mammalian milk In human, the content of 6’-SL is usually higher than that of 3’-SL, but in non-human mammals, 3’-SL is often of higher concentration than 6’-SL The presence of 6’-SL in rat milk has not been previously reported and this was likely due to the low abundance of 6’-SL and insufficient resolving power during oligosac-charide separation Here, we identified both 3’-SL and 6’-SL in similar concentrations as those found in other non-human mammals A pair of

sialylated N-acetyllactosamine isomers, 6’-SLN (peak #4) and 3’-SLN

(peak #2), were also found in rat milk Similar characteristic fragment ions to those of 6’-SL and 3’-SL were observed Again, the 2-6 linkage specific fragment 0,4A2-CO2 (m/z 306) (Wheeler & Harvey, 2000) was only present in the spectrum of 6’-SLN (Fig 2d) but not in the 2-3 linked 3’-SLN (Fig 2c), and therefore the isomers could be readily differentiated

Only one peak (#6) was found to have the composition of H3S1 Three possible structures including Neu5Acα2-3Galβ1-3 Galβ1-4Glc, Neu5Acα2-3(Galβ1-6)Galβ1-4Glc and Galβ1-3(Neu5Acα2-6)Galβ1-4Glc have been reported in non-human mammalian milk(Urashima et al.,

2001) Apart from the 2-3/2-6 linkage of the Neu5Ac, the position of the extra Gal is the main point of assignment Although a branched Gal can produce fragment ion B1 at m/z 161, a decarboxylated B2 ion (B2-CO2) at

m/z 408 suggested the Neu5Ac linked to a Gal (Fig 3c) The C3 ion at

m/z 632 further identified a Neu5Ac-Gal-Gal- sequence The D-ion m/z

Table 1

Acidic milk oligosaccharides identified in rat and mouse by LC-ESI-MS/MS

Peak

No a RT b

[M-H] −

name e

Relative content (%) f

Human g Bovine g References

(1965)

(2014)

7 10.8 835.27 835.29 H2N1S1 Neu5Acα2-3GlcNAcβ1-3Galβ1-4Glc 3’-S-LNTri-

II 0.22 1.47 – – Albrecht et al (2014)

8 11.9 835.27 835.29 H2N1S1 Neu5Acα2-6(GlcNAcβ1-3)Galβ1-4Glc 6’-S-LNTri-

9 12.6 997.33 997.34 H3N1S1 Neu5Acα2-6(Galβ1-3)GlcNAcβ1-3

10 13.6 997.33 997.34 H3N1S1 Neu5Acα2-6Galβ1-4GlcNAcβ1-3

12 18.8 753.19 753.20 H1N1S1Su1 Neu5Acα2-3Gal(6Su)β1-4GlcNAc 3’-S-6’-Su-

13 20.0 712.16 712.17 H2S1Su1 Neu5Acα2-3Gal(6Su)β1-4Glc 3’-S-6’-Su-

L 44.0 0.01 – – Choi & Carubelli (1968)

14 24.6 1085.34 1085.36 H3S2 Neu5Acα2-3Galβ1-3(Neu5Acα2-6)

15 26.6 1077.30 1077.30 H3N1S1Su1 Neu5Acα2-6Galβ1-4GlcNAcβ1-3 Gal

aHPLC peak numbers

b Retention time (in min)

cH, Hex; N, HexNAc; S, Neu5NAc, Su, SO3H

dProposed structure based on MS/MS and comparison with literature data

eTrivial name is given based on MS/MS analysis and comparison with literature data S, Sialylated; Su, Sulphated

fRelative intensity to the most intense ion as 100 %, n.d.: not detected (relative content below 0.01 %)

g+, present; -, not present

J Li et al

161 produced by the Gal is typical for a 3-linked residue Finally, the

lack of Neu5Acα2-6 specific fragment m/z 306 indicated a α2-3-linked

sialic acid Therefore 3”S-β3’-GL with the sequence of

Neu5-Acα2-3Galβ1-3Galβ1-4Glc (Table 1) can be tentatively proposed

Two peaks, #7 and #8, were found with the composition of H2N1S1

([M− H]− at m/z 835) Although the spectral signals of peak #8 is very

weak (Fig 1b), from the product-ion spectra the isomeric pair can still be

assigned based on some important ions observed The branched

sequence of #8 is apparent from the C1 at m/z 202 and B1 α at m/z 290

(Fig 3b) Further glycosidic cleavage at B2 (m/z 655) and its

desialy-lated ion B2-S (m/z 364) identified the branching point at the Gal as the

tetrasaccharide structure 6’-S-LNTri-II, GlcNAcβ1-3(Neu5Acα2-6)

Galβ1-4Glc, which was found previously in other reports (Albrecht et al.,

2014; Yan et al., 2018) (Table 1) The linear sequence of #7 can be

deduced by the B1 at m/z 290 and B2 at m/z 493 The double glycosidic

D-type ion D1-2 at m/z 202 indicated the internal GlcNAc 3-linked to the

Gal, and therefore 3’-S-LNTri-II (Neu5Acα2-3GlcNAcβ1-3Galβ1-4Glc)

can be proposed (Table 1)

Peaks #9 and #10 can be readily assigned as LSTb and LSTc

(Table 1), respectively, by comparison of the product-ion spectra

(Fig 3d and e) with literature data (Chai et al., 2006), and by the

fragment ions obtained In the spectrum of #9, the full set of sequence

ions B1 α (m/z 290), C1 (m/z 179), C2 (m/z 673) and C3 (m/z 835) defined

the sequence, and 0,4A2-CO2 indicated the Neu5Ac2-6 linkage and D1-2

(m/z 493) suggested a 3-linked GlcNAc Peak #10 was similarly assigned

as LSTc (Table 1) The assignment was confirmed by comparison with

both retention times and product-ion spectra of standard LSTb and LSTc

(Figs S-1 and S-2)

The nine monosialylated oligosaccharides described above are

common acidic oligosaccharides in mammalian milk

3.3 Sequence determination of disialyated and sulphated oligosaccharides by ESI-CID-MS/MS

The oligosaccharides in peak #11 and #14 are both disialylated Peak #11 has a composition of H2S2 and is considered as disialylated lactose (DSL) As shown in Fig 4a, the Neu5Ac-Neu5Ac- sequence can unambiguously assigned by B1 m/z 290 and B2 m/z 581, the latter

accompanied by a decaboxylated ion m/z 537 with an α2-8 linkage Y1 at

m/z 632 can further confirm this sequence The lack of Neu5Acα2-6

specific ion at m/z 597 (306 + 291), equivalent to m/z 306 in the case of

monosialylated oligosaccharides (see above for discussion), highly indicated an α2-3 linkage between the Neu5Ac and Gal The linkage between the two sialic acid residues was tentatively assigned as α2-8 as those found in bovine milk (Veh et al., 1981) and buffalo colostrum (Aparna & Salimath, 1995) Therefore, peak #11 can be identified as Neu5Acα2-8Neu5Acα2-3Galβ1-4Glc

The disialylated oligosaccharide in peak #14 contains an additional hexose (Fig 4b) The absence of the characteristic fragments m/z 581

and 537 for Neu5Acα2-8Neu5Ac- (as shown in Fig 4a for DSL) and the

presence of the mono-desialylated ion m/z 794 indicate the two sialic acids at different positions A weak ion at m/z 161 from a double

glycosidic D-type cleavage indicated a 3-linked Gal in the penta-saccharide Although the product-ion spectrum was very weak and insufficient fragment ions to give a full assignment, a sequence of Neu5Acα2-3Galβ1-3(α2-6Neu5Ac)Galβ1-4Glc (Fig 4b) can be specu-lated which was previously named as DSβ3’-GL These two disialyspecu-lated oligosaccharides have been found in the milk of domestic animals (Albrecht et al., 2014)

The remaining four oligosaccharides are all sulfated Peak #1 was identified as the 6’-sulfated lactose (Table 1) as the sulfate on the Gal is apparent by the presence of strong ion pair of B1 m/z 241 and C1 m/z 259

Fig 2 ESI-CID-MS/MS spectra of sialyllactose and sialyl-N-acetyllactosamine (a) 3’-SL, (b) 6’-SL, (c) 3’-SLN, (d) 6’-SLN Structures are shown to indicate the

proposed fragmentation

Carbohydrate Polymers 259 (2021) 117734

Fig 3 ESI-CID-MS/MS spectra of sialylated oligosaccharides (a) 3′-S-LNTri-II, (b) 6′-S-LNTri-II, (c) 3′ ′-S-β3′-GL, (d) LSTb, (e) LSTc Structures are shown to indicate the proposed fragmentation

J Li et al

(Fig 5a) Peak #13 was the 3’-sialyl-6’-sulfated lactose (3’-S-6’-Su-L,

Fig 5b and Table 1) B1 at m/z 290 and lack of 0,4A2-CO2 at m/z 306

indicated a 3-linked Neu5Ac Y1 at m/z 421 is indicative of the sulfate on

the lactose moiety Extensive decarboxylation and desulphation made it

impossible to assign exactly the position of the sulphate group The two

sulfated oligosaccharides have been reported previously, and the

sul-phate group was identified by elemental composition and the Gal-6-O-

position assigned by methylation analysis (Barba & Caputto, 1965;

Choi & Carubelli, 1968; Michael et al., 2013)

Peak #12 can be readily assigned by comparison with the spectrum

of peak #13 (3’-S-6’-Su-L (Fig 5b) The reducing terminal disaccharide

N-acetyllactosamine rather than lactose is apparent from their

compo-sitions (H1N1S1Su1and H2S1Su1, respectively) and the Y1 ion at m/z

462 (compared with Y1 at m/z 421 in the spectrum of #13, Fig 5b)

Although very weak signal due to the extremely low content (0.06 %,

Table 1) NeuAcα2-3Galβ(6Su)1-4GlcNAc

With the composition of H3N1S1Su1, peak #15 was predicted to be

either the sulphated LSTb or LSTc which are present in rat milk (peak #9

and #10, Table 1) As LSTc is more abundant (1.97 %) compared with

LSTb (0.16 %), sulphated LSTc was the most possible structure As

shown in Fig 5d, the glycosidic ions C1 at m/z 308 and B2 at m/z 452

clearly identified the sialic acid at the non-reducing end while the

sul-phate is not at this Gal The 0,2A3-h at m/z 554 also indicated the absence

of sulphate on the GlcNAc Therefore, the sulphate at the lactose site

could be assigned Due to very low concentration and extremely weak

signals in addition to the facile loss of the sulphate, it is difficult to have a definitive assignment from the mass spectral fragmentation but the likely sulphation at the 6-position of the Gal is assumed and sulphated LSTc, Neu5Ac2-6Gal1-4GlcNAc1-3Gal(6Su)1-4Glc, is proposed Although a phosphate group is also of 80 Da and phosphorylated oligosaccharides have been found in animal milk (Urashima et al.,

2001), #12 and #15 were assigned as sulphated This is because sul-phation has been identified in rat milk oligosaccharides (#1 and #13) (Choi & Carubelli, 1968; Sturman et al., 1985) and it is unlikely sul-phation and phosphorylation can occur in the milk of the same animals

It has been recently recognized that human milk oligosaccharides play important roles in shaping up the infant’s intestinal microbiota composition and in serving as soluble decoy receptors preventing pathogen attachment to infant mucosal surfaces and lower the risk for viral, bacterial and protozoan parasite infections Although milk oligo-saccharides in general do not have nutritional value, early work spec-ulated for the possible nutritional importance of sulphate in oligosaccharides present in rat milk Sulphate is not considered as an essential nutrient in mature mammals but it could be a nutrient in the neonate In an experiment using 35S, sulphated SL was found to be hydrolysed in the gut of rat neonates, and the sulphur absorbed as inorganic sulphate (Sturman et al., 1985) The presence of this may permit the simultaneous delivery of two essential nutrients, sulphate and calcium, in early life, avoiding the precipitation of insoluble calcium sulphate in the milk (Sturman et al., 1985) However, in human infants the function of milk oligosaccharides is primarily protective rather than nutritional (Newburg, 2000)

3.4 Comparison of acidic oligosaccharides in rat, mouse and human milk

Analysis of oligosaccharides in mouse milk is more challenging due

to the very small amount of mouse milk available and the difficulty for collection directly from lactating mouse A single mouse mammary tis-sue was used for extraction of milk oligosaccharides Fourteen acidic oligosaccharides were similarly detected (Table 1) but DSβ3’-GL (#14) was not found For comparison, Fig 6 shows the two acidic oligosac-charide profiles from rat and mouse milk To make the low abundant peaks more visible different magnifying factors were applied (please note the different colours representing different magnifying factors) There is an apparent difference in relative abundances of oligosaccha-rides in rat and mouse milk (Table 1) In rat, 3’-SL is most abundant, whereas in mouse it is 6’-SL The content of sulphated oligosaccharides

in rat milk was much higher than those in mouse milk Apart from 6’-SL and 3’-SL, sulphated 3’-SL is the most abundant with a relative intensity

of 44.0 %, but it is less than 0.01 % in mouse milk In mouse milk, LSTc is the third most abundant one

The 15 acidic oligosaccharides detected in rat and mouse milk can be compared with the 30 sialylated oligosaccharides in human milk iden-tified in a previous study (Yan et al., 2018) As shown in Table 1, seven oligosaccharides are common in both rat and human and these include 3’-SL, 6’-SL, 6’-SLN, 3’’-β3’-GL, 6’-S-LNTri-II, LSTb and LSTc The other eight oligosaccharides are absent in human milk Compared with do-mestic animals (such as cow, goat and sheep), rat and mouse share more common oligosaccharides with human The acidic oligosaccharides in mouse milk are more similar to human milk due to the higher contents of 6’-SL and LSTc

4 Conclusions

In this work we carried out a comprehensive analysis of oligosac-charides using 1 mL of rat milk or 1 mouse gland tissue We detected and identified 15 acidic oligosaccharides and these include 9 mono-sialylated, 2 dimono-sialylated, 1 monosulphated, and 3 both monosulphated and monosialylated Among these, 12 are reported here for the first time

in rat milk and 2 are novel structures As some of oligosaccharides are in very low concentrations this precludes fully sequence assignment The

Fig 4 ESI-CID-MS/MS spectra of disialylated oligosaccharides (a) DSL, (b)

DSβ3’-GL Structures are shown to indicate the proposed fragmentation

Carbohydrate Polymers 259 (2021) 117734

Fig 5 ESI-CID-MS/MS spectra of sulphate and sialylated oligosaccharides (a) 6’-L-O-sulphate, (b) 3’-SL-6’-O-sulphate, (c) 3’-SLN-6’-O-sulphate, (d) LSTc-6’-O-

sulphate Structures are shown to indicate the proposed fragmentation

Fig 6 Overlay extracted single-ion chromatograms of oligosaccharides in (a) rat milk, (b) mouse milk Peaks 4,6,7, 9 and 11 were magnified by a factor of 20, peaks

2, 8, 12, 14 and 15 were magnified by a factor of 200 in rat milk Peaks 4, 6, 7, 9 and 11 were magnified by a factor of 40, peaks 2, 8, 13, 14 and 15 were magnified by

a factor of 1000 in mouse milk Legends: yellow circle, galactose; purple diamond, N-acetylneuraminic acid; blue square, N-acetylglucosamine; and blue

cir-cle, glucose

J Li et al

sulphation is likely at the 6-O-position of the Gal at the reducing side

When this position is occupied by sialic acid sulphation does not seem to

take place

CRediT authorship contribution statement

Jiaqi Li: Data curation, Formal analysis, Writing - original draft

Maorong Jiang: Methodology JiaoRui Zhou: Data curation Junjie

Ding: Data curation Zhimou Guo: Conceptualization, Project

admin-istration Ming Li: Funding acquisition Fei Ding: Methodology

Wen-gang Chai: Funding acquisition, Writing - review & editing Jingyu

Yan: Funding acquisition, Writing - review & editing Xinmiao Liang:

Funding acquisition, Project administration

Declaration of Competing Interest

The authors report no declarations of interest

Acknowledgments

The work is supported in part by the National Natural Science

Foundation of China (21934005, 22074143, and 31900920), and by the

March of Dimes Prematurity Research Center grant (22-FY18-821) and

the Wellcome Trust Biomedical Resource grant (WT 218304/Z/19/Z)

Appendix A Supplementary data

Supplementary material related to this article can be found, in the

online version, at doi:https://doi.org/10.1016/j.carbpol.2021.117734

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