Profiling of Human Milk Oligosaccharides for Lewis Epitopes and

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Profiling of Human Milk Oligosaccharides for Lewis Epitopes and Secretor Status by Electrostatic Repulsion Hydrophilic Interaction Chromatography Coupled with Negative-Ion Electrospray Tandem Mass Spectrometry Jingyu Yan, Junjie Ding, Gaowa Jin, Zhaojun Duan, Fan Yang, Dandi Li, Han Zhou, Ming Li, Zhimou Guo, Wengang Chai, and Xinmiao Liang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00687 • Publication Date (Web): 09 May 2019 Downloaded from http://pubs.acs.org on May 9, 2019

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Analytical Chemistry

Profiling of Human Milk Oligosaccharides for Lewis Epitopes and Secretor Status by Electrostatic Repulsion Hydrophilic Interaction Chromatography Coupled with Negative-Ion Electrospray Tandem Mass Spectrometry

Jingyu Yan1,a, Junjie Ding1,2,a, Gaowa Jin1, Zhaojun Duan3, Fan Yang1, Dandi Li3, Han Zhou1, Ming Li4, Zhimou Guo1,*, Wengang Chai5,*, Xinmiao Liang1,*

1 Dalian

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

2 University 3 National 4

of Chinese Academy of Sciences, Beijing 100049, China

Institute for Viral Disease Control and Prevention, Beijing 102206, China

College of Basic Medical Science, Dalian Medical University, Dalian, China

5 Glycosciences

Laboratory, Faculty of Medicine, Imperial College London, Hammersmith

Campus, Du Cane Road, London W12 0NN, United Kingdom

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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT

Human milk oligosaccharides (HMOs) are one of the most abundant ingredients in breast milk, and play a beneficial role for newborns and are important for infant health. The peripheral fucosylated sequences of HMOs, such as the histo-blood group ABH(O) and Lewis a, b, x, and y antigens, are determined by the expression of the secretor (Se) and Lewis (Le) genes in the mammary gland, and are often the recognition motifs and serve as decoy receptors for microbes. In this work, we developed a method for determination of Secretor status and Lewis blood phenotype and assignment of Lewis blood-group epitopes. The method was based on electrostatic repulsion/hydrophilic interaction chromatography coupled with tandem mass spectrometry (ERLIC-MS/MS). A specifically designed stationary phase, aspartic acid-bonded silica (ABS), was used to separate the acidic and neutral HMOs by electrostatic repulsion followed by HILIC. Negative-ion electrospray MS/MS was then used for analysis of Secretor status and Lewis blood phenotypes and assignment of important epitopes of HMOs from the lactating mothers by selecting a specific set of unique fragment ions.

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Introduction

Human milk oligosaccharides (HMOs) are the third most abundant ingredient in breast milk after lactose and fat, and are involved in numerous functions and play a beneficial role for newborns, as prebiotics, antiadhesives, antimicrobials and immune modulators, and as nutrients for brain development.1-3 Generally, a disaccharide, lactose, is located at the reducing end with extensions of N-acetyllactosamine units to form linear or branched backbone sequences. The backbone can be modified variously by sialic acid (N-acetylneuraminic acid, Neu5Ac) and/or fucose (Fuc) residues. The complexity of HMO structures make them unique compared with those from other mammalian species and confers a variety of functions.4-6 For example, sialylated HMOs were reported as effectively promoting healthy growth of children with malnutrition by regulating microbiota and had a protective effect from necrotizing enterocolitis for infants.7, 8 The peripheral sialylated and fucosylated sequences are frequently recognition motifs9, such as the histo-blood group ABH(O) and Lewis a, b, x, and y antigens. The fucosylated oligosaccharide sequences are determined by the expression of the secretor (Se) and Lewis (Le) genes in the mammary gland and are the minimal binding epitopes for lectins and are decoy receptors for microbes10. It has been reported that 2’-fucosylated HMOs produced by secretors are associated with protection of breast-fed infants from C. jejuni infection, one of the most common causes of bacterial diarrhea and infant mortality.11, 12 Lewis and secretor genes appear to mediate susceptibility to rotavirus infection, e.g. P[8] rotaviruses infect only persons who are Lewis-positive and Secretor.13 Therefore, profiling of HMOs from different mothers and lactation periods for specific structural motifs, such as the fucosylated sequences, is very important in understanding the activities of HMOs and their relationships to infant growth and health.14, 15 Various approaches have been developed for analysis of the complex HMOs.16-18 Thurl et al. reported different oligosaccharide patterns corresponding to the Lewis blood types in 50 milk samples.19 Lebrilla and colleagues have used HPLC-Chip-MS for profiling of HMOs, extracted 3



from the milk of 60 women from The Gambia, as oligosaccharide alditols after chemical reduction and proposed some intact glycan molecules as markers for rapid determination of Secretor status without the need for serological testing.20 Kunz and colleagues have developed an automated tandem MALDI-MS with laser-induced dissociation (LID-MS/MS) for correlation of HMO structures with Lewis blood-groups on the basis of selected fragment ion spectra of oligosaccharides even as a mixture of isomeric structures without chromatographic separation.21 Kamerling and colleagues have used 1H-NMR for milk group classification based on Le and H epitopes.22 Recently, Hofmann et al. have established ion mobility-tandem MS fingerprinting for identification of Lewis and blood-group epitopes.23 In the present report, we developed a LC-MS method based on mixed-mode electrostatic repulsion/hydrophilic interaction liquid chromatography (ERLIC)24 coupled with negative-ion electrospray ionization tandem MS with collision-induced dissociation (ESI-CID-MS/MS) for profiling and assignment of Lewis blood-group and Secretor status of HMOs. As ESI-CID-MS/MS in the negative-ion mode has only shown unique fragmentation features useful for structural assignment of neutral oligosaccharides but not for sialylated ones25-31, it is important to use electrostatic repulsion to separate the complex HMOs into acidic and neutral groups while the oligosaccharide components in each group can be further fractionated by hydrophilic interaction32,33. For this, a new aspartic acid-based ERLIC stationary phase, aspartic acid-bonded silica (ABS) gel, was designed and prepared for chromatographic separation of HMOs. By linking the two method and using LC-ESI-MS/MS, we explore the possibility of a convenient method for assignment of Secretor status and Lewis blood-group phenotype by selecting some characteristic fragment ions rather than intact molecules. Fourteen milk samples from different mothers and at different lactation periods were used to establish the principle and demonstrate the utility.

Experimental Section

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Materials Oligosaccharide standards, 3’-sialyllactose (3’-SL), 6’-sialyllactose (6’-SL), 2′-fucosyllactose (2’FL), difucosyllactose (DF-L), lacto-N-neotetraose (LNnT), lacto-N-fucopentaose I, II, and III (LNFP-I, -II, and -III, respectively), lacto-N-hexaose (LNnH), lacto-N-difucohexaose I and II (LNDFH-I and -II, respectively), lacto-N-neodifucohexaose I (LNnDFH-I) and lactose were purchased from Elicityl (Crenoble, France). Aspartic acid, ammonium formate and formic acid were obtained from J&K Scientific (Beijing, China) and (3-glycidyloxypropyl) triethoxysilane (98%) was from TCI (Tokyo, Japan). HPLC-grade acetonitrile and methanol were obtained from Merck (Darmstadt, Germany). All other reagents used were of analytical grade or higher. Spherical silica (5 m, 100 Å pore size, 300 m2g-1 surface area) was purchased from Fuji Silysia Chemical (Kasugai, Japan). Water was purified by a Milli-Q purification system (Billerica, MA, USA).

Preparation of ABS gel Silica gel (240 g, approximately 570 mmol silanol groups) was dried at 120 oC overnight. After it cooled to room temperature, 1,000 mL of sodium acetate/acetic acid solution (0.1 M, pH 4.0) was added to the gel under N2, followed by 240 mL of (3-glycidyloxypropyl) triethoxysilane (860 mmol) with agitation. The solution was then heated to 90 oC and stirred for 24 h. The silanized silica was ﬁltered and washed successively with anhydrous toluene (1,000 mL), methanol (1,000 mL), water (500 mL) and methanol (1,000 mL) before drying at 80 oC overnight to obtain the epoxy-silica. Aspartic acid solution (20 g in 300 mL of water) was then prepared and the pH was adjusted to about 8.5 by adding 16.96 g of Na2CO3 and 5.88 g of NaHCO3. Finally, 20 g of epoxy silica was added to the aspartic acid solution and the mixture was stirred at 65 oC for 24 h. The reaction mixture was ﬁltered and washed with water (300 mL) and methanol (300 mL) in succession. The final product was dried at 80 oC overnight to obtain the ABS gel.

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Characterization of the ABS gel and evaluation of the ABS column The ABS gel was characterized by elemental analysis and -potential. The elemental analysis was performed on a Vario EL III elemental analysis system (Elementar, Hanau, Germany). The -potential was measured on a Malvern Zetasizer Nano-ZS90 instrument (Malvern, UK). With 20 mL of methanol as slurry solvent and 40 mL of methanol as propulsive solvent under a pressure of 35 MPa, 1.0 g of ABS gel was slurry-packed into a stainless-steel column (150 mm×2.1 mm I.D.). The column evaluation was performed on a conventional HPLC system, Thermo Dionex Ultimate 3000 equipped with a charged aerosol detector (CAD) and operated by Chromeleon 7 software. Oligosaccharide standard solutions of 2’FL, LNnT, LNFP-I, LNnH, 3’-SL, 6’-SL and lactose were individually prepared at a concentration of 1 mg/mL in acetonitrile/water (50:50, v/v), and the mixture solution was then prepared by mixing the individual solutions in equal volume. The solvent gradient was performed with acetonitrile and water at a flow rate of 0.2 mL/min from 20% to 50% water in 40 min. The injection volume was 1 μL.

Preparation of milk oligosaccharides Fourteen human milk samples were collected from 12 healthy mothers who gave birth to healthy term infants and enrolled in hospitals. One of the mothers provided three milk samples at different lactation days (7th, 14th and 60th day). All milk samples were stored at 40oC before use. Collected milk (200 L) was centrifuged at 9,000 rpm for 20 min at 4 oC to remove lipid. Ethanol (400 L) was then added to the skim milk before centrifugation at 9,000 rpm for 10 min at 4 oC to remove protein. The obtained supernatant was used for analysis.

LC-MS analysis Oligosaccharide analysis was carried out on an LC-ESI-CID-MS/MS system, in which an UPLC unit Agilent 1290 LC was coupled with an Agilent 6540 TOF mass spectrometer. The 6


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mobile phase conditions were the same as those used in chromatographic evaluation of ASP described above and 1 L of sample solution was typically injected. The drying gas temperature was 350 °C with a flow rate of 8.0 L/min. 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 50-2,000 (for MS/MS). Precursor-ion selection during LC-MS was made automatically by the data system based on ion abundance, and a cutoff threshold was set at 0.01% of relative intensity and 200 counts of absolute intensity. Three precursors were selected from each MS spectrum to carry out product-ion scanning. Collision energy of 15V was used for collision-induced dissociation (CID). HMO identification and quantitation were performed via Agilent Mass Hunter Qualitative Analysis software (version B.03.01).

Results and Discussion

Preparation of ABS stationary phase Aspartic acid is a typical acidic amino acid and has hydrophilic property, making it suitable for use in an ERLIC matrix. Polyaspartic acid-bonded silica has been developed and commercialized as PolyCAT A. It has been used as zwitterionic phase for simultaneous separation of inorganic anions and cations34,35, due to the presence of both the unreacted protonated propylamine and the dissociated carboxyl functionality of the polyaspartic acid attached to the silica. PolyCAT A has also been used as a weak cation-exchange material for protein separation36 and in the ERLIC mode for removal of SDS from a peptide digest before fractionation of the peptides.37 In order to develop an aspartic acid-based ERLIC matrix for fractionation of both acidic sialylated and neutral oligosaccharides in human milk using electrostatic repulsion mechanism for the former and HILIC for the latter, we used a different synthetic strategy for preparation of ABS with the aim of eliminating the residual positively charged silica-bonded propylamine groups. Silica gel was first functionalized by epoxy groups and the aspartic acid was bonded to silica via the ring-opening reaction between the epoxy-silica 7



and the amino group of the aspartic acid, leaving the two carboxyl groups for electrostatic interaction (Fig. S1). To characterize the ABS gel, elemental analysis was carried out to determine the surface coverage of aspartic acid. Compared with bare silica and epoxy bonded silica, the increase of nitrogen content of ABS (from 0 to 0.41%, Table S1) indicated that aspartic acid was successfully bonded onto the silica surface with the surface coverage of 1.02 mol/m2. Its surface charge state was also assessed by measuring the  potential. The charge status of ASP in solutions with different pH is shown in Figure S2. As expected, ABS exhibited electro-negativity even at a low pH, enabling the ABS column to work at a suitable pH condition for acidic compounds by repulsive force, which is very important in the separation of sialylated HMOs.

Evaluation of ABS column for fractionation of milk oligosaccharides Before the ABS column was used for separation of HMO samples, its chromatographic properties were assessed with a standard mixture which contains lactose, several acidic and neutral oligosaccharides mimicking the HMOs (Table S2), as shown in Figure 1. The acidic 3’and 6’-SL peaks were eluted early close to the void time, and lactose and other neutral oligosaccharides were retained and separated. The results indicated that the ABS stationary phase could separate the acidic from the neutral oligosaccharides, while the latter can be eluted and resolved by the hydrophilic interaction with the elution order of di-, tri-, tetra-, penta- and hexasaccharides, following the order of the degree of polymerization (DP) of the oligosaccharides.

Analysis of HMOs by ERLIC-MS. The ABS column was then used for UPLC in conjunction with negative-ion ESI-MS to characterize oligosaccharides in human milk from a mother at her 14th-day of lactation. As human milk is dominated by lactose and the extremely high concentration of lactose can overload both the chromatograph and mass spectrometer, lactose was removed by switching off 8


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the valve and linking mass spectrometer to waste. From negative-ion mass spectra, 27 HMOs were identified based on the molecular anions. A typical extracted ion chromatogram (EIC) and the identities of HMOs are shown in Figure 2. A 4-bit code was used to define the monosaccharide compositions in terms of hexose (H), N-acetyl-hexosamine (N), fucose (F) and sialic acid (A), e.g. the peak marked as 4-2-1-1 at 6 min represents H4 (4 hexose residues), N2 (two N-acetyl-hexosamine residues), F1(one fucose residue) and A1 (one sialic acid residue) with a nominal molecular mass 1509. From the compositions marked on the chromatogram, it is apparent that the acidic HMOs eluted before 8 min and the neutral ones after 11 min. Within both the acidic and neutral groups, the oligosaccharides were separated according to electronegativity and hydrophilicity. For the neutral oligosaccharides, the retention times became longer with their increasing DPs. For acidic HMOs, the retention times of HMOs were additionally affected by the numbers of sialic acid residues. For example, the hexasaccharide 3-1-0-2 eluted earlier than the pentasaccharide 3-1-0-1 because the hexasaccharide, although larger in molecular size, contains two sialic acids and therefore eluted faster due to the stronger repulsive force from the ABS matrix. Among the 27 HMOs detected, 10 were acidic and 17 were neutral. These represent some 90% of the oligosaccharide content in human milk38, and the molecular anions of these 27 were used to generate EIC profiles of HMOs from milk samples of 12 mothers (Figure 3). The apparent differences among these are the diversity and relative intensities of the HMOs detected. Milk samples #1#6, #8 and #9 are similar in patterns, in which sialylated oligosaccharide 2-0-0-1 was the most intensive and the peaks of neutral HMOs 2-0-1-0, 2-0-2-0, 3-1-0-0, 3-1-1-0, and 3-1-2-0 were also very prominent with similar intensities. The EIC profiles of samples #7, and #10#12 appeared different as these showed fewer peaks and lower total ion intensities. Sample #7 showed low intensity peaks of sialylated HMOs and less fucosylated HMOs of higher DPs. In samples #10#12, sialylated HMOs and trisaccharide 2-0-1-0 were of lower intensities while the peak of tetrasaccharide 2-0-2-0 was absent and the double peak with the composition of 3-1-1-0 became single. The reason for the latter was that among the two 9



incompletely resolved peaks the first one which contains LNFP-I was absent. As ionization efficiency is different between neutral and sialylated oligosaccharides, the ion intensity is not a direct reflection of the relative abundance of each HMO in the milk sample. We introduced a detection sensitivity factor 3.5 (sensitivity ratio of sialylated:neutral) to recalculate the relative abundance of each HMO component based on the ion intensity and the results were shown as a heatmap in Figure S3. The factor was obtained by measuring and comparing ion signals of the sialylated and neutral oligosaccharide standards, 3’-SL, 6’-SL, 2’FL and LNFP-I which were present in abundance in various samples (data now shown).

Assignment of Lewis blood-group epitopes by ERLIC-MS/MS. Milk samples from different individual donors exhibit significant qualitative and quantitative difference in their neutral HMO profiles and different Lewis blood-group antigens are present in the milks of different donors.14 Lewis A or X (Lea or Lex, respectively) are formed by addition of a 1,4-linked Fuc to subterminal GlcNAc on type 1 chain or a 1,3-linked Fuc to the GlcNAc on type 2 chain, respectively, while Leb or Ley are formed by addition of these two differently linked Fuc to 1,2-fucosylated HMOs. As shown in Table 1 and Figure S4, some fragment ions under negative-ion ESI-CID/MS/MS can be considered unique to specific structural epitopes of Lewis blood-group antigens, e.g. the double glycosidic D-ion produced by C- and Z-type cleavage at a 3-linked GlcNAc or Glc residue25. The D-ion m/z 348 is specific to a Lea while the D-ion m/z 364 is unique to a Lex epitope. To characterize a Leb, two fragments are required, and these include the m/z 348 and an additional C2-ion at m/z 325. For assignment of a Ley, the single D-ion at m/z 510 is sufficient although a C2-ion at m/z 325 may also be useful to corroborate its identification. Using milk sample #1 as the example we assessed the usage of these fragment ions in identifying Lewis antigens present in human milk. The EICs from the negative-ion ESI-CID-MS/MS clearly showed that all four Lewis blood-group types were present in the HMOs of sample #1 (Figure 4). Together with the molecular ions detected, LNFP-III of Lex was 10


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identified by EIC of m/z 364 (Figure 4a), and similarly LNnDFH-I of Ley was identified via the fragment of m/z 510 (Figure 4b) and LNFP-II of Lea by m/z 348 (Figure 4c). LNDFH-I of Leb was assigned by its fragment ions of both m/z 348 and m/z 325 (Figure 4d).

Determination of Secretor status and Lewis blood-group phenotype by ERLIC-MS/MS of HMOs. The milk of Secretor woman is abundant in 2’FL, LNFP-I and other 1,2-fucosylated HMOs. In contrast, non-Secretors lack a functional FUT2 enzyme and their milk does not contain 1,2-fucosylated HMOs3. Therefore, Fuc1-2Gal can be considered as unique to a Secretor. We attempted to use m/z 325 as the fragment ion marker to assign the Secretor’s status. As shown in Figure 4d, the ion chromatogram of m/z 325 of sample #1 gave 5 peaks, 2’FL, DF-L, LNFP-I, LNnDFH-I and LNDFH-I. All five of these HMOs are indeed produced by individual donors with Secretor gene FUT2. As sample #1 also contains Lea and Leb blood-group antigens, this donor mother is therefore classified as a Lewis-positive Secretor (SeLe) with blood-group type Lewis(ab). We further analyzed three additional milk samples, # 5, #7 and #10 (Figure 5). Clearly, samples #5 and #7 were from Secretors while sample #10 from a non-Secretor, as the former two contained oligosaccharide components with fragment ion m/z 325 (Figure 5a and 5b) albeit with different numbers of peaks and different abundances, whereas the latter showed lack of oligosaccharide with fragment m/z 325 (Figure 5c). Further Lewis blood-group phenotyping using m/z 348 for either Lea or Leb can be achieved. Two peaks in the m/z 348 EIC of sample #5 (Figure 5d) were from two Lea oligosaccharides (LNFP-III and LNDFH-II) and one from Leb (LNDFH-I) as it also produced m/z 325 (Figure 5a and Figure S4b). Therefore, sample #5 was from a SeLe individual with blood-group phenotype Lewis(ab). Sample #7 contains m/z 325 components only (Figure 5b) without any oligosaccharide producing fragment m/z 348 (Figure 5e), indicating a Secretor SeLe and blood-group phenotype Lewis(ab). Finally, sample #10 showed no component with fragment ion m/z 325 but had two components with fragment m/z 348, indicating a Lea structural epitope and defining a non-Secretor SeLe with 11



blood-group phenotype Lewis(ab). Lewis-positive women have an active FUT3 allele and their milk contains 1,4-fucosylated HMOs and therefore, m/z 348 can be used as an additional fragment ion marker to assign a Lewis positive phenotype. Based on these characteristic chromatograms, other women were assigned to different milk groups. As shown in Table 2, samples #1#9 were from the Secretor mothers and #10#12 from non-Secretors. Samples #16, 8, and 9 were from women with the Lewis (ab) phenotype and samples #1012 were from women with Lewis (ab). Sample #7 was from a Lewis-negative woman. The differences in Secretor and Lewis blood-group phenotypes of samples could explain the different chromatograms in Figure 3. Among the 12 mothers, three (25%) were Le(ab) phenotypes, eight (67%) were Le(ab) and one (8%) was Lewis negative. Compared with the literature21, the high ratios of both non-secretors and Le(ab) may be due to the small sample size and/or ethnicity. After assignments of the Secretor status and Lewis phenotypes of samples #5, #7 and #10 using the fragment marker ions m/z 325 and m/z 348, the presence of oligosaccharide Lex/y antigens was also determined using characteristic fragments m/z 364 and m/z 510 (Table 1). As shown in Figure S5, samples #5, #7 and #10 all contained oligosaccharide with the Lex epitope (LNFP-III). Ley antigen (LNnDFH-I) was present in samples #5 and #7, but not in #10. Together with the results obtained above using m/z 325 and m/z 348, it is apparent that the fragment m/z 510 for a Ley determinant can be used to corroborate the assignment of Secretor status (Figure S6 and Table 1) whereas Lex epitope assigned by m/z 364 is independent of Secretor status and Lewis phenotypes.

Analysis of HMOs of different lactation periods. Lactation periods are often divided into three stages: colostrum (0-7th day), transition (7-14th day) and mature (after 14th day). It has been reported that the content of HMOs decreases and the Lewis antigens also change with lactation periods.14 Evaluation of HMOs in each period is important to understand the functions of HMOs. Three milk samples from the 12


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three different stages donated by mother #1 were analyzed. As mentioned above, 27 typical oligosaccharides were used to characterize HMOs, and the sum of their peak areas was used to reflect the total content of HMOs. As ionization efficiency and therefore the detection sensitivity in MS are different between neutral and sialylated oligosaccharides, the content changes were measured separately for the neutral and acidic HMOs. As shown in Figure 6a, all the oligosaccharides decreased with progression of lactation. Compared with the sialylated group, the content of the neutral oligosaccharides dropped more quickly, especially from the transition to mature period. The contents of fucosylated and non-fucosylated HMOs were also assessed separately for neutral and acidic HMOs during the three lactation periods (Figure 6b). In this Lewis(ab+) phenotype mother’s milk, neutral HMOs were dominated by fucosylated HMOs, while the acidic were mainly non-fucosylated. As a result, the decrease of neutral HMOs was due mainly to the reduction in fucosylated HMOs. By contrast, the decrease in acidic ones involved non-fucosylated HMOs. Further analysis was carried out to evaluate possible concentration changes in the Lewis blood-group epitopes during lactation. As shown in Figure 6c, the content of m/z 325, representing the H-epitope, decreased apparently with lactation while a moderate decline of m/z 348 of Lea epitope was shown. The reduction of Ley between Day 7 and Day 14 was significant. Only the Lex epitope (m/z 364) remained primarily the same and seemed to increase moderately in the mature lactation. It has been known that the compositions of human milk were well-matched with the requirements of infant growth. It was reasonable to assume that the high content of 1,2-fucosylgalactose and Lea epitope in colostrum could be important in provision of more protection against pathogens for newborns.

Conclusions

In this study, we developed a novel ERLIC-MS/MS based strategy for profiling HMOs using ABS gel as the HPLC matrix for initial group separation followed by detailed resolution of 13



oligosaccharides within each of the acidic and neutral fraction. A carefully selected fragment ion set in negative-ion ESI-CID-MS/MS was used for determination of Secretor status and Lewis blood-group phenotype. HMOs are complex mixtures and are believed to contain more than 200 components. Initial fractionation of HMOs by ERLIC into neutral and acidic groups is important for ESI-MS/MS to focus on the neutral oligosaccharides and also useful to partially reduce the complexity to facilitate the subsequent resolution of the sialylated and neutral oligosaccharides separately. By selecting several characteristic fragment ions to construct overlaid extracted ion chromatograms we were able to profile HMOs in human milk samples for Secretor status and the Lewis blood-group phenotypes by using m/z 325 and 348 as the diagnostic fragment ions. The method can also assign some structural epitopes, e.g. blood-group H and Lea/b/x/y, which are important recognition motifs of HMOs. Although the high resolution PGC column has been used for LC-MS, the HMOs had to be reduced to minimize the complexity caused by anomeric separation39 and therefore, the special feature of product-ion spectra in negative-ion detection is no longer available after alditol formation.30 These unique fragmentations do not occur in positive-ion detection with either MALDI21 or ESI30 of reducing sugars, highlighting the importance of the reducing terminal hemiacetal functionality to produce deprotonated molecule [MH] which can be used as the precursor ion for structure-informative fragmentation. The method we provide here is an alternative approach for large-scale profiling of oligosaccharides in human milk. AUTHOR INFORMATION Corresponding Author Email: [email protected] (Z. Guo); [email protected] (W. Chai) and [email protected] (X. Liang) ORCID Wengang Chai: 0000-0003-2977-5347 Xinmiao Liang: 0000-0001-5802-1961 14


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Author Contributions aThese

authors contributed equally to this work.

Notes The authors declare no competing financial interest.

Acknowledgments This work was supported in part by the National Key Research and Development Program of China (2017YFD0400600, 2016YFB0301502), the Joint Funds of the National Natural Science Foundation of China (U1608255), the Nutrition and Care of Maternal & Child Research Fund Project of Guangzhou Biostime Institute of Nutrition & Care (2016BINCMCF1102), and by the March of Dimes Prematurity Research Center grant (22-FY18-821).

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Figure captions

Figure 1. Chromatogram of a standard HMO mixture using an ABS column (2.1 x 150 mm) on a HPLC system. Running conditions: see Experimental.

Figure 2. Overlaid EICs of 27 HMOs in sample #1. The 4-bit code marked on each peak showed their compositions (sequentially the numbers of hexose, N-acetylhexosamine, fucose and sialic acid residues). For online LC-MS experiment, an UPLC system is used.

Figure 3. Overlaid EIC HMO profiles of 12 milk samples from mothers with different Secretor and Lewis status.

Figure 4. EICs of different fragment ions from negative-ion ESI-CID-MS/MS product-ion spectra and the assigned HMOs in sample #1. (a) m/z 325; (b) m/z 348; (c) m/z 364; (d) m/z (510). Symbols:

glucose (Glc),

galactose (Gal),

N-acetylglucosamine (GlcNAc),

fucose (Fuc).

Figure 5. EICs of m/z 325 (a, b, c) and m/z 348 (d, e, f) from negative-ion ESI-CID-MS/MS product-ion spectra of three milk samples and the assigned Secretor status and Lewis blood-group phenotypes. Sample #5: Secretor, Lewis (ab+) (a, d); Sample #7: Secretor, Lewis (ab) (b, e); and Sample #10: non-Secretor, Lewis (a+b) (c, f).

Figure 6. Relative contents (mean relative peak areas) of neutral and sialylated HMOs (a), fucosylated and non-fucosylated HMOS in neutral and sialylated fractions (b), and different Lewis blood-group antigens in milk from three lactation periods of the contributor of sample #1.

16


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2011, 401, 2495-2510. [22] van Leeuwen, S. S.; Schoemaker, R. J. W.; Gerwig, G. J.; van Leusen-van Kan, E. J. M.; Dijkhuizen, L.; Kamerling, J. P. Rapid milk group classification by H-1 NMR analysis of Le and H epitopes in human milk oligosaccharide donor samples. Glycobiology 2014, 24, 728-739. [23] Hofmann, J.; Stuckmann, A.; Crispin, M.; Harvey, D. J.; Page, K.; Struwe, W. B. Identification of Lewis and blood group carbohydrate epitopes by ion mobility-tandem-mass spectrometry fingerprinting. Anal. Chem. 2017, 89, 2318-2325. [24] Alpert, A. J. Electrostatic repulsion hydrophilic interaction chromatography for isocratic separation of charged solutes and selective isolation of phosphopeptides. Anal. Chem. 2008, 80, 62-76. [25] Chai, W. G.; Piskarev, V.; Lawson, A. M. Negative ion electrospray mass spectrometry of neutral underivatized oligosaccharides. Anal. Chem. 2001, 73, 651-657. [26] Pfenninger, A.; Karas, M.; Finke, B.; Stahl, B. Structural analysis of underivatized neutral human milk oligosaccharides in the negative ion mode by nano-electrospray MSn (Part 1: Methodology). J. Am. Soc. Mass Spectrom. 2002, 13, 1331-1340. [27] Pfenninger, A.; Karas, M.; Finke, B.; Stahl, B. Structural analysis of underivatized neutral human milk oligosaccharides in the negative ion mode by nano-electrospray MSn (Part 2: Application to isomeric mixtures). J. Am. Soc. Mass Spectrom. 2002, 13, 1341-1348. [28] Chai, W.; Piskarev, V. E.; Mulloy, B.; Liu, Y.; Evans, P. G.; Osborn, H. M. I.; Lawson, A. M. Analysis of chain and blood group type and branching pattern of sialylated oligosaccharides by negative ion electrospray tandem mass spectrometry. Anal. Chem. 2006, 78, 1581-1592. [29] Ninonuevo, M. R.; Lebrilla, C. B. Mass spectrometric methods for analysis of oligosaccharides in human milk. Nutri. Rev. 2009, 67 Suppl 2, S216-226. [30] Zhang, H.; Zhang, S.; Tao, G.; Zhang, Y.; Mulloy, B.; Zhan, X.; Chai, W. Typing of blood-group antigens on neutral oligosaccharides by negative-ion electrospray ionization tandem mass spectrometry. Anal. Chem. 2013, 85, 5940-5949. [31] Mank, M.; Welsch, P.; Heck, A. J. R.; Stahl, B. Label-free targeted LC-ESI-MS2 analysis of human milk oligosaccharides (HMOS) and related human milk groups with enhanced structural selectivity. Anal. Bioanal. 19



Chem. 2019, 411, 231-250. [32] Hemstrom, P.; Irgum, K. Hydrophilic interaction chromatography. J. Sep. Sci. 2006, 29, 1784-1821. [33] Alpert, A. J.; Shukla, M.; Shukla, A. K.; Zieske, L. R.; Yuen, S. W.; Ferguson, M. A. J.; Mehlert, A.; Pauly, M.; Orlando, R., Hydrophilic-interaction chromatography of complex carbohydrates. J. Chromatogr. A 1994, 676, 191-202. [34] Kiseleva, M. G.; Kebets, P. A.; Nesterenko, P. N. Simultaneous ion chromatographic separation of anions and cations on poly(aspartic acid) functionalized silica. The Analyst 2001, 126, 2119-2123. [35] Kebets, P. A.; Nesterenko, E. P.; Nesterenko, P. N.; Alpert, A. J. Ion-chromatography performance of poly(aspartic) acid bonded silica with various pore sizes at different ionic strengths and column temperatures. Microchim. Acta 2004, 146, 103-110. [36] Papazyan, R.; Taverna, S. D. Separation and purification of multiply acetylated proteins using cation-exchange chromatography. Methods Mol. Biol. (Clifton, N.J.) 2013, 981, 103-113. [37] Serra, A.; Gallart-Palau, X.; Dutta, B.; Sze, S. K., Online removal of sodium dodecyl sulfate via weak cation exchange in liquid chromatography-mass spectrometry based proteomics. J. Proteome Res. 2018, 17, 2390-2400. [38] Hong, Q.; Ruhaak, L. R.; Totten, S. M.; Smilowitz, J. T.; German, J. B.; Lebrilla, C. B., Label-free absolute quantitation of oligosaccharides using multiple reaction monitoring. Anal. Chem. 2014, 86, 2640-2647. [39] Ninonuevo, M. R.; Park, Y.; Yin, H.; Zhang, J.; Ward, R. E.; Clowers, B. H.; German, J. B.; Freeman, S. L.; Killeen, K.; Grimm, R.; Lebrilla, C. B. A strategy for annotating the human milk glycome. J. Agric. Food Chem. 2006, 54, 7471-7480.

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Table 1. Characteristic fragment ions in negative-ion ESI-CID-MS/MS for assignment of Secretor status and Lewis blood-group phenotypes

Characteristic

Glycosidic

Carbohydrate

Lewis

Secretor

Representative

fragment ion

cleavage

epitope

phenotype

status

HMOs

m/z 325

C-type

secretor

2’-FL, DF-L,

H(O)

LNFP-I, LNDFH-I m/z 348

D-type

Lea

Lewis (ab)

non-secretor

LNFP-II

Lewis (ab)

secretor

LNDFH-II

Lewis (ab)

secretor or

LNFP-III

Lewis (ab)

non-secretor

LNnDFH-II

secretor

LNnDF-I

Leb m/z 364

D-type

Lex

Lewis(ab) m/z 510

D-type

Ley

Lewis (ab) Lewis(ab)

Symbols:

galactose (Gal), N-acetylglucosamine (GlcNAc),

fucose (Fuc).

21



Page 22 of 29

Table 2. The Secretor status and Lewis blood-group phenotypes of 12 samples analyzed Sample

#1

#2

#3

#4

#5

#6

#7

#8

#9

#10

#11

#12

Se

























Le

ab

ab

ab

ab

ab

ab

ab

ab

ab

ab

ab

ab

22


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For TOC only

23



Lactose 2’FL

LNFP I

pA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 29

3’-SL

LNnT

6’-SL

Figure 1


LNnH

Figure 2

ACS Paragon Plus Environment 5-3-2-0 6-4-1-0 5-3-3-0 6-4-2-0 5-3-4-0 6-4-3-0

5-3-1-0 4-2-3-0

4-2-2-0

4-2-1-0

4-2-0-0

3-0-0-0

To waste

4-2-1-1 4-2-2-1 5-3-0-1

4-2-0-1

4-2-0-2 3-1-1-1

3-1-0-2 4-2-1-2

2-0-2-0

2-0-1-0

3-1-2-0

3-1-1-0

3-1-0-1

3-1-0-0

2-0-0-1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Ion counts

Page 25 of 29 Analytical Chemistry


(b) #2

(c) #3

(d) #4

(e) #5

(f) #6

(g) #7

(h) #8

(i) #9

(k) #11

(l) #12

Ion counts

Ion counts

Ion counts

(a) #1

(j) #10 Ion counts

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 29

Figure 3


Page 27 of 29

(a) m/z 364

(b) m/z 510

ion counts

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(c) m/z 348

(d) m/z 325

Figure 4



#5 (Lewis (a-b+), Se+) (a) m/z 325

2’FL Ion counts

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

DF-L

#7 (Lewis (a-b-), Se+) (b) m/z 325

LNFP-I LNnDFH-I

LNDFH-I & -II

#10 (Lewis (a+b-), Se-) (c) m/z 325

2’FL DF-L LNnDFH-I

LNDFH-I

(d) m/z 348

LNFP-I

Page 28 of 29

(e) m/z 348

(f) m/z 348

LNFP-II

LNFP-II

LNDFH-II

Figure 5


Page 29 of 29

Ion counts

(a)

Neutral HMOs

Ion counts

(b)

Ion counts

Sialylated HMOs

(c) percentage (%) of antigens

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6


Profiling of Human Milk Oligosaccharides for Lewis Epitopes and

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