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.

Carbohydrate Polymers 259 (2021) 117734 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Characterization of rat and mouse acidic milk oligosaccharides based on hydrophilic interaction chromatography coupled with electrospray tandem mass spectrometry Jiaqi Li a, b, 1, Maorong Jiang c, 1, JiaoRui Zhou d, Junjie Ding a, Zhimou Guo a, b, Ming Li d, Fei Ding c, Wengang Chai e, Jingyu Yan a, b, *, Xinmiao Liang a, b, * a Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Key Laboratory of Separation Science for Analytical Chemistry, Dalian, 116023, China University of Chinese Academy of Sciences, Beijing, 100049, China c Key Laboratory of Neuroregeneration of Jiangsu and Ministry of Education, Co-Innovation Center of Neuroregeneration, Nantong University, Nantong, Jiangsu, 226001, China d Department of Microecology, College of Basic Medical Science, Dalian Medical University, Dalian, 116044, China e Glycosciences Laboratory, Faculty of Medicine, Imperial College London, Hammersmith Campus, London, W12 0NN, United Kingdom b A R T I C L E I N F O A B S T R A C T Keywords: Milk oligosaccharides Rat and mouse Structural characterization Electrospray 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 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 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) Jiaqi Li and Maorong Jiang contributed equally to this work https://doi.org/10.1016/j.carbpol.2021.117734 Received November 2020; Received in revised form January 2021; Accepted 26 January 2021 Available online 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/) J Li et al 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 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 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 at ◦ C After the removal of the top lipid layer, two volumes of ethanol were added to the mixture, and the mixture was stored at ◦ C for h The mixture was then centrifuged at 8000 rpm for 10 at ◦ 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 at ◦ 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 A/B (80/20) to A/B (40/60); 30.1–45 min, A/B (80/20) Gradient in XAmide column was 0− 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 (80/10/10) Both MS and MS/MS spectra were acquired in the negative-ion mode with an acquisition rate of 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 was set at 45 psi, gas 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 Carbohydrate Polymers 259 (2021) 117734 Fig Profiles of acidic oligosaccharides from rat milk (a) Total ion chromatogram, (b) Extracted single-ion chromatograms MS/MS spectra were acquired in the negative-ion mode with an acqui­ sition rate of 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 (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 Results and discussion As acidic oligosaccharides are the major components of oligosac­ 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 (-Galβ1-3GlcNAcβ1-) and type (-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 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 separated 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 J Li et al Carbohydrate Polymers 259 (2021) 117734 Table Acidic milk oligosaccharides identified in rat and mouse by LC-ESI-MS/MS Peak Noa RTb [M-H]− Compositionc Short namee Structured Found Calc’d 4.1 421.04 421.07 H2Su1 Gal(6Su)β1-4Glc 7.5 673.22 673.24 H1N1S1 7.7 9.2 9.2 10.5 10.8 632.21 673.22 632.21 794.25 835.27 632.21 673.24 632.21 794.27 835.29 11.9 835.27 12.6 10 Relative content (%)f Humang Bovineg References Rat Mouse 6’-Su-Lac 3.45 0.06 – – Neu5Acα2-3Galβ1-4GlcNAc 3’-SLN 0.08 0.02 – + H2S1 H1N1S1 H2S1 H3S1 H2N1S1 Neu5Acα2-3Galβ1-4Glc Neu5Acα2-6Galβ1-4GlcNAc Neu5Acα2-6Galβ1-4Glc Neu5Acα2-3Galβ1-3Galβ1-4Glc Neu5Acα2-3GlcNAcβ1-3Galβ1-4Glc 100 0.41 45.6 0.65 0.22 81.4 0.61 100 0.18 1.47 + + + + – + + + + – 835.29 H2N1S1 Neu5Acα2-6(GlcNAcβ1-3)Galβ1-4Glc 0.01 0.04 + – 997.33 997.34 H3N1S1 0.16 0.10 + – Chai et al (2006) 13.6 997.33 997.34 H3N1S1 LSTc 1.97 11.2 + + Chai et al (2006) 11 12 15.5 18.8 923.30 753.19 923.31 753.20 H2S2 H1N1S1Su1 Neu5Acα2-6(Galβ1-3)GlcNAcβ1-3 Galβ1-4Glc Neu5Acα2-6Galβ1-4GlcNAcβ1-3 Galβ1-4Glc Neu5Acα2-8Neu5Acα2-3Galβ1-4Glc Neu5Acα2-3Gal(6Su)β1-4GlcNAc 3’-SL 6’-SLN 6’-SL 3’’S-β3’-GL 3’-S-LNTriII 6’-S-LNTriII LSTb Barba & Caputto (1965) Albrecht et al (2014) Chai et al (2006) Chai et al (2006) Chai et al (2006) Yan et al (2018) Albrecht et al (2014) Yan et al (2018) 0.22 0.02 0.04 0.08 – – + – (Taufik, 2012) – 13 20.0 712.16 712.17 H2S1Su1 Neu5Acα2-3Gal(6Su)β1-4Glc 44.0 0.01 – – 14 24.6 1085.34 1085.36 H3S2 0.06 n.d – – 15 26.6 1077.30 1077.30 H3N1S1Su1 Neu5Acα2-3Galβ1-3(Neu5Acα2-6) Galβ1-4Glc Neu5Acα2-6Galβ1-4GlcNAcβ1-3 Gal (6Su)β1-4Glc DSL 3’-S-6’-SuLN 3’-S-6’-SuL DSβ3’-GL Su-6’-LSTc 0.06 0.01 – – Choi & Carubelli (1968) Albrecht et al (2014) – a b c d e f g HPLC peak numbers Retention time (in min) H, Hex; N, HexNAc; S, Neu5NAc, Su, SO3H Proposed structure based on MS/MS and comparison with literature data Trivial name is given based on MS/MS analysis and comparison with literature data S, Sialylated; Su, Sulphated Relative intensity to the most intense ion as 100 %, n.d.: not detected (relative content below 0.01 %) +, present; -, not present 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) 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, A3-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 3.2 Sequence determination of monosialylated oligosaccharide by ESICID-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 J Li et al Carbohydrate Polymers 259 (2021) 117734 Fig 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 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 B1α 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 B1α (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 disialylated 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 J Li et al Carbohydrate Polymers 259 (2021) 117734 Fig 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 Carbohydrate Polymers 259 (2021) 117734 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 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 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 Fig ESI-CID-MS/MS spectra of disialylated oligosaccharides (a) DSL, (b) DSβ3’-GL Structures are shown to indicate the proposed fragmentation (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-Oposition 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 Conclusions In this work we carried out a comprehensive analysis of oligosac­ charides using mL of rat milk or mouse gland tissue We detected and identified 15 acidic oligosaccharides and these include mono­ sialylated, disialylated, monosulphated, and both monosulphated and monosialylated Among these, 12 are reported here for the first time in rat milk and are novel structures As some of oligosaccharides are in very low concentrations this precludes fully sequence assignment The J Li et al Carbohydrate Polymers 259 (2021) 117734 Fig 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’-Osulphate Structures are shown to indicate the proposed fragmentation Fig Overlay extracted single-ion chromatograms of oligosaccharides in (a) rat milk, (b) mouse milk Peaks 4,6,7, 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, 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 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5027–5039 Yan, J., Ding, J., Jin, G., Yu, D., Yu, L., Long, Z., et al (2018) Profiling of sialylated oligosaccharides in mammalian milk using online solid phase extraction-hydrophilic interaction chromatography coupled with negative-ion electrospray mass spectrometry Analytical Chemistry, 90, 3174–3182 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 References Albrecht, S., Lane, J A., Marino, K., Al Busadah, K A., Carrington, S D., et al (2014) A comparative study of free oligosaccharides in the milk of domestic animals British Journal of Nutrition, 111, 1313–1328 Aparna, H S., & Salimath, P V (1995) Disialyl lactose from buffalo colostrum: Isolation and characterization Carbohydrate Research, 268, 313–318 Ballard, O., & Morrow, A L (2013) Human milk composition: Nutrients and bioactive factors Pediatric Clinics of North America, 60, 49–74 Barba, H S., & Caputto, R (1965) Isolation and identification of a lactose sulphate ester from rat mammary gland Biochimica et Biophysica Acta (BBA)-Mucoproteins and Mucopolysaccharides, 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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... using mL of rat milk or mouse gland tissue We detected and identified 15 acidic oligosaccharides and these include mono­ sialylated, disialylated, monosulphated, and both monosulphated and monosialylated... L., Long, Z., et al (2018) Profiling of sialylated oligosaccharides in mammalian milk using online solid phase extraction -hydrophilic interaction chromatography coupled with negative-ion electrospray