Anal. Chem. 1999, 71, 3755-3762
Analysis of High-Molecular-Weight Oligosaccharides from Human Milk by Liquid Chromatography and MALDI-MS Berndt Finke,*,† Bernd Stahl,† Anja Pfenninger,‡ Michael Karas,‡ Hannelore Daniel,§ and Gu 1 nther Sawatzki†
Milupa Research, Milupa GmbH & Company KG, D-61381 Friedrichsdorf, Germany, Instrumentelle Analytische Chemie, Universita¨t Frankfurt, D-60590 Frankfurt/Main, Germany, and Institut der Erna¨hrungswissenschaft, Universita¨t Giessen, D-35392 Giessen, Germany
Pooled human milk oligosaccharides were fractionated by anion-exchange chromatography on AG 1-X2 and by an improved gel filtration procedure that allowed the separation of large oligosaccharides on Toyopearl HW 40 (S) and Bio-Gel P-6 columns, respectively. The analysis of the resulting nonderivatizated fractions by matrix-assisted laser desorption/ionization mass spectrometry (MALDIMS) revealed several neutral and acidic high-molecularweight oligosaccharides. So far unknown acidic oligosaccharides containing up to 20 monomers were detected in a molecular mass range of 2094-3626 Da. Furthermore, neutral structures containing up to 35 monosaccharides were identified after fractionation on Toyopearl HW 40 (S) and subsequent P-6 fractionation, demonstrating the suitability of the applied method for the preparation of oligosaccharides in this high-molecularmass range. The composition of the detected oligosaccharides was found to be the same as those previously identified in oligosaccharides of lower masses. However, an enormous structural heterogeneity was observed when acidic and neutral fractions were characterized by highpH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). From our analysis we may conclude that each molecular mass identified by MALDI-MS corresponds to a variety of isomeric structures. The total number of oligosaccharides occurring in human milk may consequently be much higher than estimated before. The oligosaccharide fraction of human milk comprises the third most quantitatively prominent constituent after lactose and lipids. Depending on the status and course of lactation as well as the Lewis blood group,1 the concentration of oligosaccharides varies between 6 and 12 g/L.2-5 Up to now, 84 different oligosaccharide * Corresponding author. Telephone: +49 6172 99/1321. Telefax: +49 6172 99/1862. E-mail:
[email protected]. † Milupa GmbH & Co. KG. ‡ Universita ¨t Frankfurt. § Universita ¨t Giessen. (1) Thurl, S.; Henker, J.; Siegel, M.; Tovar, K.; Sawatzki, G. Glycoconjugate J. 1997, 14, 795-799. (2) Egge, H. In New Perspectives in Infant Nutrition; Renner, B., Sawatzki, G., Eds.; Thieme, Stuttgart, Germany, 1994; pp 3-11. 10.1021/ac990094z CCC: $18.00 Published on Web 07/16/1999
© 1999 American Chemical Society
structures have been identified mainly by fast atom bombardement mass spectrometry (FAB-MS) and 1H nuclear magnetic resonance spectroscopy (1H NMR).6-8 Most of the compounds possess a lactose core unit and are built by addition of galactose (β1-3) N-acetylglucosamine (type 1) or galactose (β1-4) N-acetylglucosamine (type 2) sequences in either a linear or a branched fashion. In addition, fucosylation occurs at reducing glucose in the R1-3 position, nonreducing galactose (R1-2), and N-acetylglucosamine (R1-3, R1-4). Sialylation occurs on galactose in the R2-3 and R2-6 positions and on N-acetylglucosamine in the R2-6 position. The chain lengths of these oligosaccharides contain up to 50 monomer units. Previous studies have provided evidence of the presence of neutral oligosaccharides with molecular masses of up to 8000 Da in human milk. Those have been detected by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) applied to gel filtration fractions of pooled human milk oligosaccharides.9 MALDI-MS and high-pH anion exchange chromatography with pulsed amperometric detection (HPAECPAD) provide powerful tools for the analysis of human milk oligosaccharides with a high sensitivity and no need for derivatization.5,9,10 The biological functions of human milk oligosaccharides still have to be determined.11 However, there is some evidence that the oligosaccharides may protect breast-fed infants from pathogenic bacteria, viruses, toxins, protozoa, and fungi12,13 by acting as soluble receptor analogues that prevent the interaction of (3) Coppa, G. V.; Gabrielli, O.; Pierani, P.; Catassi, C.; Carlucci, A.; Giorgi, P. L. Pediatrics 1993, 91, 637-641. (4) Chatuvedi, P.; Warren, C. D.; Ruiz-Palacios, G. M.; Pickering, L. K.; Newburg, D. S. Anal. Biochem. 1997, 251, 89-97. (5) Thurl, S.; Mu ¨ ller-Werner, B.; Sawatzki, G. Anal. Biochem. 1996, 235, 202206. (6) Kobata, A.; Yamashita, K.; Tachibana, Y. Methods Enzymol. 1978, 50, 216220. (7) Bruntz, R.; Dabrowski, U.; Dabrowski, J.; Ebersold, A.; Peter-Katalanic, J.; Egge, H. Biol. Chem. Hoppe-Seyler 1988, 369, 257-273. (8) Strecker, G.; Wieruszeski, J.-M.; Michalski, J.-C.; Montreuil, J. Glycoconjugate J. 1989, 6, 169-182. (9) Stahl, B.; Thurl, S.; Zeng, J.; Karas, M.; Hillenkamp, F.; Steup, M.; Sawatzki, G. Anal. Biochem. 1994, 223, 218-226. (10) Kunz, C.; Rudloff, S.; Hintelmann, A.; Pohlentz, G.; Egge, H. J. Chromatogr., B 1996, 685, 211-221. (11) Varki, A. Glycobiology 1993, 3, 97-130. (12) Kunz, C.; Rudloff, S. Acta Paediatr. 1993, 82, 903-912. (13) Zopf, D.; Roth, S. Lancet 1996, 347, 1017-1021.
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pathogens with epithelial cells in the initial step of infections. Moreover, glycoconjugates with distinct epitopes, such as SialylLewisx, may act as ligands for E- and P-selectin that play key roles in inflammation and tissue injury.14 Furthermore, the N-acetylglucosamine-containing oligosaccharides have been reported to promote growth of Bifidobacterium bifidum while thereby suppressing growth of undesirable bacteria.15,16 For all biological activities of human milk oligosaccharides, knowledge of the number of compounds and their particular structure is a prerequisite for understanding their proposed protective roles. In this study, we describe a strategy for the preparation of both acidic and neutral high-molecular-weight oligosaccharides from pooled human milk samples using gel filtration. Some structures were isolated by reversed-phase highperformance liquid chromatography (rpHPLC). Isolated structures and gel filtration fractions were analyzed by HPAEC-PAD and MALDI-MS for determination of the molecular mass and possible compositions of the compounds. EXPERIMENTAL SECTION Preparation of Neutral and Acidic Oligosaccharides. Two liters of pooled human milk was delipidated by centrifugation at 4 °C, and the proteins were removed by ethanol precipitation (final concentration 66%), as described by Kobata.17 The subsequent fractionation into neutral and acidic oligosaccharides was done on an AG 1-X2 column (30 × 4.4 cm, 200-400 mesh) provided in acetate form (Bio-Rad, Mu¨nchen, Germany). One fraction containing the neutral compounds was sampled by eluting with deionized water and one total acidic fraction by elution with 250 mM ammonium acetate, pH 5.0 (Merck, Darmstadt, Germany). Carbohydrate-containing fractions were detected by spotting 10 µL of the fractions onto a silica gel 60 plate (Merck), monitored after treatment with 0.2% Orcin (Sigma) in 25% H2SO4 (Merck) and heating at 120 °C for 3 min. The neutral fractions were pooled and desalted using a mixed-bed AG 50W1-X8 (Bio-Rad) ionexchanger in OH-- and H+-forms, respectively. The total acidic fraction was desalted by passing through to Toyopearl HW 40 (S) columns, (TosoHaas, Stuttgart, Germany) using two 4.0 × 105 cm columns (Kronlab, Sinsheim, Germany), connected in series and equilibrated with deionized water at a flow rate of 1.5 mL min-1. The fractions were concentrated and freeze-dried. Gel Filtration. The total neutral oligosaccharide fraction was further separated by gel filtration on Toyopearl HW 40 (S) columns equilibrated with deionized water. During operation, the columns were maintained at 45 °C. The samples were applied to the columns in a concentration of 200 mg/mL in 5 mL of deionized water. The neutral high-molecular-weight fraction was further separated on a 2.6 × 90 cm column (Pharmacia, Freiburg, Germany) of Bio-Gel P-6 (400 mesh; Bio-Rad, Mu¨nchen, Germany) by elution with deionized water at a flow rate of 0.5 mL min-1 and kept at 60 °C during operation. Dextran polymers (T1 and T5; Pharmacia) were used as reference oligomers to calibrate the column for an estimation of the degree of polymerization (DP). (14) Wiederschain, G. Y.; Koul, O.; Aucoin, J. M.; Smith, F. I.; McCluer, R. H. Glycoconjugate J. 1998, 15, 379-388. (15) Gyo ¨rgy, P.; Norris, R.-F.; Rose, C.-S. Arch. Biochem. Biophys. 1954, 48, 193202. (16) Yoshioka, H.; Iseki, K.; Fujita, K. Paediatrics 1983, 72, 317-321. (17) Kobata, A. Methods Enzymol. 1973, 28, 262-271.
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The total acidic fraction was further separated by gel filtration on two Toyopearl HW 40 (S) columns (4.4 × 90 cm) in line and eluted with 100 mM ammonium acetate at a flow rate of 1.8 mL min-1. The desalting of the obtained fractions was achieved by gel chromatography on HW 40 (S) columns as mentioned above. The neutral and acidic oligosaccharide fractions were monitored by refractive index (RI) detection (Knauer, Berlin, Germany). Reversed-Phase High-Performance Liquid Chromatography (rpHPLC). Single components of the oligosaccharide gel filtration fractions were isolated by rpHPLC using two 250 × 8 mm columns equipped with 30 × 8 mm guard columns and Eurospher 100 reversed-phase material (C18) of 5 µm particle size (Knauer, Berlin, Germany). Neutral structures were eluted isocratically with deionized water at a flow rate of 1.75 mL min-1 and monitored by UV detection (Kratos, Ramsey, NJ) at 215 nm; the acidic structures were eluted with 5 mM triethylamine hydrochloride (Merck), pH 5.0. The fractionation of high-molecular-mass acidic oligosaccharides (>1300 Da) required the addition of methanol at 4% (v/v) to reduce their retention time. High-pH Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD). HPAEC analyses were performed on a DX-300 Bio-LC system (Dionex, Idstein, Germany) with a pulsed electrochemical detector (PED 2, Dionex). Twenty-five microliter aliquots of samples were loaded on a CarboPac PA-100 (Dionex) pellicular anion-exchange column (4 × 250 mm) equipped with a guard column (4 × 50 mm) and separated at a flow rate of 1 mL min-1. The concentration of the oligosaccharide fractions applied was 1-2 g/L. Neutral and acidic oligosaccharides were analyzed using gradient conditions as described previously.5 Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS). MALDI-MS was performed by using a Voyager-DE STR and a Voyager RP system (Perseptives Biosystems, Framingham, MA) equipped with a nitrogen laser emitting at 337 nm wavelength. Both neutral and acidic oligosaccharides were analyzed in the linear mode. Voyager-DE STR: acceleration voltage applied was U ) 25 kV, with a two-stage ion source (first grid, 95% of U; second grid, ground potential). Voyager RP: U ) 20 kV (first grid, 90% of U; second grid, ground potential). A twopoint external calibration was used for mass assignment. Spectra were recorded in the positive-ion mode (neutral and acidic structures) or the negative-ion mode (acidic structures). Analysis of neutral oligosaccharides was performed with a twolayer matrix. One microliter of 5-chloro-2-mercaptobenzothiazole (CMBT) solution (10 g/L in tetrahydrofuran/ ethanol/deionized water; 1:1:1; v/v/v) was applied to the target and dried in a cold, strong stream of air. On top of the microcrystalline CMBT layer 1 µL of analyte (typically 1 g/L) and 1 µL of 2,5-dihydroxybenzoic acid (DHB) solution (20 g/L dissolved in deionized water) were placed and air-dried again.18 The matrix used for acidic oligosaccharides was 6-aza-2-thiothymine (ATT) (1 g/L dissolved in ethanol) with the addition (1:1; v/v) of an aqueous diammonium hydrogen citrate (DAHC) solution (20 mM) according of Papac et al.19 The analyte/matrix mixture was directly applied onto the stainless steel target and air-dried. (18) Pfenninger, A.; Bahr, U.; Karas, M.; Finke, B.; Stahl, B.; Sawatzki, G. J. Mass Spectrom. 1999, 34, 98-104. (19) Papac, D. I.; Wong, A.; Jones, A. J. S. Anal. Chem. 1996, 68, 3215-3223.
Figure 1. Gel filtration elution profile on Toyopearl HW 40 (S) columns of the total carbohydrate fraction from human milk. Elution was monitored by RI detection. Abbreviations: OS, oligosaccharides; F-L, fucosyllactoses; G-L, galactosyllactoses; DF-L, difucosyllactose; LNT, lacto-N-tetraoses; LNFP, lacto-N-fucopentaoses; LNDH, lactoN-difucohexaoses; LNH, lacto-N-hexaoses; F-LNH, fucosyllacto-Nhexaoses; DF-LNH, difucosyllacto-N-hexaoses; TF-LNH, trifucosyllactoN-hexaoses. For details, see the Experimental Section.
Nomenclature. The particular composition of certain highmolecular-mass oligosaccharides cannot be directly deduced from the molecular mass alone, since nearly identical masses can result if five fucoses for example are replaced by two galactose-Nacetylglucosamine (Gal-GlcNAc) units (average δm ) 0.042 Da) or one sialic acid replaced by two fucoses (average δm ) 1.028 Da). Consequently a number of different compositions are possible on the basis of one mass determined by MALDI analysis. For the clarity and simplicity regarding the possible configurations, we propose the following working nomenclature: Lx/y-z, where L refers to lactose, x to the number of Gal-GlcNAc units, y to the number of fucose residues, and z to the number of sialic acid residues. RESULTS AND DISCUSSION The carbohydrate fraction of human milk is dominated by lactose. For the analysis of the high-molecular-weight oligosaccharides, lactose and the predominant oligosaccharides have to be removed by chromatographic steps. Consequently anionexchange chromatography and gel filtration were applied for the purification of the carbohydrate fraction, allowing the detection of minor high-molecular-weight compounds. A representative pattern of a carbohydrate elution profile of pooled human milk is given in Figure 1. The main peaks in the chromatogram obtained on the HW 40 (S) column consist of fucosyllactoses, galactosyllactoses, difucosyllactose, lacto-N-tetraoses, lacto-N-fucopentaoses, lacto-N-difucohexaoses, and lacto-N-hexaoses with different degrees of fucosylation. Isolation and identification of the isomeric compounds of each major peak were achieved by rpHPLC, as previously described.20 The first fraction marked with an arrow in Figure 1 indicates the elution position of acidic as well as highermass neutral oligosaccharides eluting near the void volume. The partial coelution of large neutral oligosaccharides with the acidic fraction under these conditions required first a prefractionation (20) Thurl, S.; Offermanns, J.; Mu ¨ ller-Werner, B.; Sawatzki, G. J. Chromatogr. 1991, 568, 291-300.
Figure 2. Gel filtration elution profile on a Bio-Gel P-6 column of the HW 40 (S) subfraction of the neutral carbohydrate fraction. Elution was monitored by RI detection. The labeled fractions A-G were analyzed by MALDI-MS; mass spectra of B, F, and G are depicted in Figure 3. The elution positions of glucose oligomers are indicated at the top; V0 indicates the position of void volume; for details, see the Experimental Section.
by anion-exchange chromatography on AG 1-X2 to obtain the total fraction of neutral compounds and the acidic fraction. Neutral Oligosaccharides. After the neutral fraction was desalted by anion-exchange chromatography with mixed-bed ionexchanger AG 50W1-X8, the oligosaccharides were further separated by gel filtration on the Toyopearl HW 40 (S) columns to remove lactose and to fractionate the neutral oligosacharides. The RI-detector signal for the acidic oligosaccharides was missing (data not shown). The higher-mass neutral fraction between the void volume of 900 mL and the elution volume of 1200 mL was collected and further separated on Bio-Gel P-6. The corresponding P-6 chromatogram is depicted in Figure 2. The degree of polymerization was estimated by comparison with reference oligosaccharides (Dextran polymers). A sufficient resolution of the separation was obtained between an elution volume of 200 and 400 mL. The peak near the void volume was found to consist of residual higher monosialylated oligosaccharides (data not shown). The neutral structures were at first fractionated by HW 40 (S), because its capacity for oligosaccharides is higher than that of the P-6 gel. Bio-Gel P-6, however, provides a decent separation of the highermass compounds, whereas HW 40 (S) has proven to be more suitable for fractionation of lower-mass oligosaccharides.7 The fractions labeled (A-G) in Figure 2 were submitted to analysis by MALDI-MS and HPAEC-PAD. The MALDI spectra of fractions labeled with the arrows are shown in Figure 3. The compounds were mainly detected as monosodium adducts [M + Na]+ in the positive-ion mode, whereas the adjacent peaks at higher m/z values of lower intensity were due to the formation of [M + K]+ molecular ions. In fraction B, m/z peaks at 1972-2410 are observed (Figure 3a). In general, the diversity of the observed masses increased with earlier elution of the fractions obtained from the Bio-Gel P-6 column. The MALDI spectrum of fraction F is dominated by main peaks between m/z 4383 and 5113 (Figure 3b). A 73 Da mass value represented the lowest peak-to-peak increment between neighboring [M + Na]+ ions observed in all of the MALDI spectra. In principle, each N-acetylglucosamine Analytical Chemistry, Vol. 71, No. 17, September 1, 1999
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Table 1. Calculated Molecular Weights of Neutral Oligosaccharides from Human Milk Found in Bio-Gel P-6 Fractions and Their Related Compositiona Bio-Gel P-6 fraction
Figure 3. Linear positive-ion MALDI mass spectra of Bio-Gel P-6 fractions B, F, and G (see Figure 2). Each spectrum is a sum of 50 single spectra (Voyager DE-STR): (a) section of the mass spectrum of fraction B; (b) section of the mass spectrum of fraction F; (c) section of the mass spectrum of fraction G. See Table 1 for mass assignments and compositions; for details, see the Experimental Section.
terminal galactoses and the glucose at the reducing end of the core oligosaccharide can be fucosylated. Each transfer results in a mass increment of 146 Da. Typical for human milk oligosaccharides is the core building unit Gal-GlcNAc leading to a mass increment of 365 Da. Thus, the lowest molecular mass difference occurs for one Gal-GlcNAc unit and two fucose residues (292 Da) in 73 Da. Despite the weak RI-detector signal in the P-6 fraction designated G, a diversity of neutral oligosaccharides, all containing lactose at their reducing end, in a mass range of m/z 4749-6065 with the maximum of intensity at m/z 5187 was detected (Figure 3c). On the basis of the MALDI spectra of the P-6 fractions and the characteristic mass differences, the possible compositions of human milk oligosaccharides were calculated and are shown in Table 1. Although many chromatographic methods have been applied to isolate neutral oligosaccharides;21-25 the largest distinct neutral (21) Tachibana, Y.; Yamashita, K.; Kobata, A. Arch. Biochem. Biophys. 1978, 188, 83-89.
3758 Analytical Chemistry, Vol. 71, No. 17, September 1, 1999
A A A A; B A; B A; B A; B B; C B; C B; C C; D C; D C; D C; D C; D D D D; E D; E D; E D; E D; E D; E D; E D; E D; E D; E D; E D; E E E E E; F E; F E; F E; F E; F E; F E; F F; G F; G F; G F; G F; G F; G F; G F; G F; G G G G G G G G G G G G
[M + Na]+ 1753.6 1826.6 1900.7 1972.8 2045.9 2118.9 2192.0 2265.1 2338.2 2411.2 2484.3 2557.4 2630.4 2703.5 2776.6 2849.6 2922.7 2995.8 3068.8 3141.9 3215.0 3288.0 3361.1 3434.1 3507.2 3580.3 3653.4 3726.4 3799.5 3872.6 3945.7 4018.7 4091.8 4164.9 4237.9 4311.0 4384.1 4457.2 4603.3 4676.3 4749.4 4822.5 4895.6 4968.7 5041.7 5114.8 5187.9 5260.9 5334.0 5407.1 5480.2 5553.2 5626.3 5699.4 5772.4 5845.5 5918.5 5991.6 6064.6
Lx/y-z L3/2-0 L4/0-0 L3/3-0 L4/1-0 L3/4-0 L4/2-0 L3/5-0; L5/0-0 L4/3-0 L3/6-0; L5/0-0 L4/4-0 L5/2-0 L4/5-0; L6/0-0 L5/3-0 L4/6-0; L6/1-0 L5/4-0 L4/7-0; L6/2-0 L5/5-0; L7/0-0 L4/8-0; L6/3-0 L5/6-0; L7/1-0 L6/4-0 L5/7-0; L7/2-0 L6/5-0; L8/0-0 L5/8-0; L7/3-0 L6/6-0; L8/1-0 L5/9-0; L7/4-0 L6/7-0; L8/2-0 L7/5-0; L9/0-0 L6/8-0; L8/3-0 L7/6-0; L9/1-0 L6/9-0; L8/4-0 L7/7-0; L9/2-0 L8/5-0; L10/0-0 L7/8-0; L9/3-0 L8/6-0; L10/1-0 L7/9-0; L9/4-0 L8/7-0; L10/2-0 L7/10-0; L9/5-0; L11/0-0 L8/8-0; L10/3-0 L8/9-0; L10/4-0 L9/7-0; L11/2-0 L8/10-0; L10/5-0; L12/0-0 L9/8-0; L11/3-0 L8/11-0; L10/6-0; L12/1-0 L9/9-0; L11/4-0 L8/12-0; L10/7-0; L12/2-0 L9/10-0; L11/5-0; L13/0-0 L10/8-0; L12/3-0 L9/11-0; L11/6-0; L13/1-0 L10/9-0; L12/4-0 L9/12-0; L11/7-0; L13/2-0 L10/10-0; L12/5-0; L14/0-0 L11/8-0; L13/3-0 L10/11-0; L12/6-0; L14/1-0 L11/9-0; L13/4-0 L10/12-0; L12/7-0; L14/2-0 L11/10-0; L13/5-0 L10/13-0; L12/8-0; L14/3-0 L11/11-0; L13/6-0; L15/1-0 L12/9-0; L14/4-0
a MALDI-MS of neutral oligosaccharides from human milk found in Bio-Gel P-6 fractions and their related composition formulas. The average molecular weights are provided. Explanation for Lx/y-z; L ) lactose; x ) number of Gal-GlcNAc units; y ) number of fucoses; z ) number of sialic acids.
structure identified until to date was a 2169 Da pentafucosylisolacto-N-octaose.26 We revealed at least 50 different neutral oligosaccharide compositions with masses from 1754 Da (L3/2-0)
Table 2. Structures and Their Molecular Weights of the Used Human Milk Oligosaccharidesa
Figure 4. HPAEC profiles of neutral oligosaccharide fractions from human milk: (a) Lactose-deprived total neutral fraction; (b) Bio-Gel P-6 fraction F (see Figure 2). For used abbreviations and structures, see Table 2.
to 6065 Da (L12/9-0 or/and L14/4-0). On the basis of the ambiguity of masses with five fucoses equal to two Gal-GlcNAc units (δm: 0.025 Da), the following two compositions could be attributed to the mass of 5187: L10/8-0 and L12/3-0. These compounds, however, may exist in a large number of isomers. Since low-molecular-mass oligosaccharides with the mass of 853 Da (lacto-N-fucopentaoses) have already been shown to represent four isomeric structures, it can be assumed that the oligosaccharides with high molecular weight exist in a huge isomeric variety. The total neutral oligosaccharide fraction after removal of lactose by gel filtration was submitted to HPAEC-PAD chromatography. Figure 4a shows the chromatogram obtained. The abbreviations and the quantitatively main structures are given in Table 2. The neutral high-molecular-weight oligosaccharide fractions as obtained from the P-6 column were separated under identical gradient conditions, were strongly retained, and were eluted between 45 and 55 min. As an example, the elution pattern of the P-6 fraction F is depicted in Figure 4b. The resolution was found to be insufficient. Only the nonfucosylated LNT and LNH structures already known have a similar retention. The composition of these structures, as revealed by MALDI-MS, consist of 23-31 monosaccharide units; most of the molecules are multifucosylated. In constrast, the (22) Anderson, A.; Donald, A. S. R. J. Chromatogr. 1981, 211, 170-174. (23) Donald, A. S. R.; Feeney, J. Carbohydr. Res. 1988, 178, 79-91. (24) Dakour, J.; Lundblad, A.; Zopf D. Arch. Biochem. Biophys. 1988, 264, 203213. (25) Strecker, G.; Wieruszeski, J.-M.; Michalski, J.-C.; Montreuil, J. Glycoconjugate J. 1988, 5, 385-396. (26) Haeuw-Fivre, S.; Wieruszeski, J.-M.; Plancke, Y.; Michalski, J.-C.; Montreuil, J.; Strecker, G. Eur. J. Biochem. 1993, 215, 361-371.
a Abbreviations: Fuc, fucose; Gal, galactose; Glc, glucose; GlcNAc, N-acetylglucosamine; NeuAc, sialic acid. The average molecular weights are provided.
difucosylated neutral oligosaccharides LNDH I and LNDH II (Table 2) show nearly identical retention times of about 10 min (Figure 4a). The HPAEC retention of the higher neutral structures after Bio-Gel P-6 separation is evidently higher than any of known structures. This strongly suggests that the separation conditions usually applied are not particularly suitable for resolving the higher-mass neutral structures. The elution order, for example, on a CarboPac column depends on charge, acidity of hydroxyl groups, overall polarity, and conformation of the glycans.27-31 Several effects concerning (27) Lee, Y. C. Anal. Biochem. 1990, 189, 151-162. (28) Hermentin, P.; Witzel, R.; Vliegenthardt, J. F. G.; Kamerling, J. P.; Nimtz, M.; Conradt, H. S. Anal. Biochem. 1992, 203, 281-289. (29) Townsend, R. R.; Hardy, M. R.; Hindsgaul, O.; Lee, Y. C. Anal. Biochem. 1988, 174, 459-470. (30) Hardy, M. R.; Townsend, R. R. Carbohydr. Res. 1989, 188, 1-7.
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Figure 5. Gel filtration elution profile on Toyopearl HW 40 (S) column of the total acidic oligosaccharide fraction from human milk. Elution was monitored by RI detection. Abbreviations: S-L, sialyllactoses; LST, sialyllacto-N-tetraoses; S-LNH, sialyllacto-N-hexaoses; S-LNH-F, fucosylsialyllacto-N-hexaoses; DS-LNT, disialyllacto-Ntetraose. The labeled fractions 1-3 were analyzed by MALDI-MS (see Figure 6); for details, see the Experimental Section.
oligosaccharide structure and their retention are described: GalGlcNAc units, sialic acids, fucoses, and branching have shown different effects.32 Although most of the found higher-mass neutral structures are multifucosylated, they are strongly retained and not sufficiently resolved under the conditions used for the total neutral oligosaccharid fraction. Increasing chain length of neutral structures alters their elution behavior by prolonging retention, whereas multiple fucosylation or branching shortens it. Acidic Oligosaccharides. For the detection of high-molecularweight oligosaccharides, it was found that a prefractionation into a total acidic and a neutral fraction by employing anion-exchange chromatography increased the quality and sensitivity for detection of individual compounds significantly. Fractionation by gel filtration on Toyopearl HW 40 (S) with ammonium acetate as the eluent resulted in an elution profile as shown in Figure 5. Further characterization by HPAEC-PAD and rpHPLC of the main peaks allowed an identification of the compounds as given in this figure. Only a few studies have so far described gel filtration for separation of charged oligosaccharides.10,20,33 By using gel filtration, we obtained an enrichment without separating the highmolecular-mass mono-, di-, and trisialylated oligosaccharides, as opposed to conventional anion-exchange chromatography.34 Gel filtration fractions 1-3 were analyzed by MALDI-MS using the negative-ion mode (Figure 6), resulting in m/z values between 1654 and 3627. The observed masses represent the deprotonated molecular ions. The composition of sialylated oligosaccharides can be directly derived from the MALDI-MS signal when the negativeion mode is applied. Here the corresponding signal-to-noise ratio is higher than that measured in the positive-ion mode (data not shown) and each oligosaccharide, consisting of the identical composition, delivers only one mass signal, whereas in the positive-ion mode sodium salt formations can occur, giving multiple but lower peak intensities for mono-, di-, and trisialylated oligosaccharides. We observed no significant loss of sialic acid or COOH as a result of prompt fragmentation. This was proven by isolated standards, which were measured under identical (31) Reddy, G. P.; Bush, C. A. Anal. Biochem. 1991, 198, 278-284 (32) Wang, W. T.; Erlanson, K.; Lindh, F.; Lundgren, T.; Zopf, D. Anal. Biochem. 1990, 190, 182-187. (33) Thibault, J. F. J. Chromatogr. 1980, 194, 315-322. (34) Veh, R. W.; Michalski, J.-C.; Corfield, A. P.; Sander-Wewer, M.; Gies, D.; Schauer, R. J. Chromatogr. 1981, 212, 313-322.
3760 Analytical Chemistry, Vol. 71, No. 17, September 1, 1999
Figure 6. Linear negative-ion MALDI mass spectra of Toyopearl HW 40 (S) fractions 1-3 (see Figure 5). Each spectrum is a sum of 50 single spectra (Voyager RP): (a) section of the mass spectrum of fraction 1; (b) section of the mass spectrum of fraction 2; (c) section of the mass spectrum of fraction 3. Major peaks are labeled with m/z values; all peaks are mentioned in Table 3.
conditions. The results of the MALDI-MS analysis of the first three fractions derived the possible compositions of acidic lactosecontaining human milk oligosaccharides given in Table 3. The acidic structures found in gel filtration fraction 3 (Figure 5) represent the first identified compounds with free reducing ends in this molecular mass range in human milk. Several human milk oligosaccharides were isolated by gel filtration and affinity chromatography followed by HPLC separation.35-38 The largest compound consisted of 12 monosaccharide units. The corresponding mass of this structure could be identified in our gel filtration fraction by a prominent signal (Figure 6b). A first distinct trisialylated oligosaccharide structure from human milk was also (35) Kitagawa, H.; Nakada, H.; Numata, Y.; Kurosaka, A.; Fukui, S.; Funakoshi, I.; Kawasaki, T.; Shimada, K.; Inagaki, F.; Yamashina, I. J. Biochem. 1988, 104, 591-594. (36) Kitagawa, H.; Nakada, H.; Kurosaka, A.; Hiraiwa, N.; Numata, Y.; Fukui, S.; Funakoshi, I.; Kawasaki, T.; Yamashina, I. Biochemistry 1989, 28, 88918897. (37) Kitagawa, H.; Nakada, H.; Numata, Y.; Kurosaka, A.; Fukui, S.; Funakoshi, I.; Kawasaki, T.; Shimada, I.; Inagaki, F.; Yamshina, I. J. Biol. Chem. 1990, 265, 4859-4862. (38) Kitagawa, H.; Nakada, H.; Fukui, S.; Funakoshi, I.; Kawasaki, T.; Yamashina, I.; Tate, S.; Inagaki, F. Biochemistry 1991, 30, 2869-2876.
Table 3. Molecular Weights of Acidic Oligosaccharides from Human Milk Found in Toyopearl HW 40 (S) Fractions and Their Related Compositionsa HW MW: [M - 1]40 (S) fraction obsd calcd 1
1654.4
1; 2
1800.8
1; 2 1
1874.9 1946.9
1; 2
2021.3
1
2093.9
1; 2; 3
2166.6
1; 2; 3 1; 2; 3
2240.7 2312.4
2; 3
2386.4
2; 3
2458.7
2; 3
2532.0
2; 3
2606.1
2; 3
2678.7
3
2749.1
1654.5 1655.5 1800.6 1801.7 1874.7 1945.8 1946.8 1947.8 2019.8 2020.8 2091.9 2092.9 2093.9 2166.0 2167.0 2240.0 2311.1 2312.1 2313.1 2385.2 2386.2 2457.2 2458.2 2459.2 2459.3 2531.3 2532.3 2605.4 2605.4 2677.4 2678.5 2750.5 2751.5 3628.3
Lx/y-z L 2/0-2 L 2/2-1 L 2/1-2 L 2/3-1 L 3/1-1 L 2/0-3 L 2/2-2 L 2/4-1 L 3/0-2 L 3/2-1 L 2/1-3 L 2/3-2 L 4/0-1 L 3/1-2 L 3/3-1 L 4/1-1 L 3/0-3 L 3/2-2 L 3/4-1 L 4/0-2 L 4/2-1 L 3/1-3 L 3/3-2 L 5/0-1 L 3/5-1 L 4/1-2 L 4/3-1 L 3/6-1 L 5/1-1 L 4/2-2 L 4/4-1 L 5/0-2 L 5/2-1 L 7/3-1
HW MW: [M - 1]40 (S) fraction obsd calcd 2; 3
2823.2
3
2896.4
3
2970.5
3
3041.9
3
3114.8
3
3188.0
3
3261.8
3
3331.6
3
3406.0
3
3552.0
3
3626.6
2823.6 2824.6 2824.6 2896.6 2897.7 2968.7 2969.7 2970.8 3041.8 3042.8 3043.8 3113.8 3114.8 3115.9 3115.8 3116.9 3187.9 3188.9 3189.9 3190.0 3262.0 3263.0 3333.0 3334.0 3335.1 3407.1 3408.1 3553.2 3554.3 3626.3 3627.4 3627.3 3628.4
Lx/y-z L 4/3-2 L 6/0-1 L 4/5-1 L 5/1-2 L 5/3-1 L 4/2-3 L 4/4-2 L 4/6-1 L 5/0-3 L 5/2-2 L 5/4-1 L 4/1-4 L 4/3-3 L 4/5-2 L 6/0-2 L 6/2-1 L 5/1-3 L 5/3-2 L 7/0-1 L 5/5-1 L 6/1-2 L 6/3-1 L 5/0-4 L 5/2-3 L 5/4-2 L 6/0-3 L 6/2-2 L 6/1-3 L 6/3-2 L 5/4-3 L 5/6-2 L 7/1-2 L 5/8-1
a MALDI-MS of acidic oligosaccharides from human milk found in Toyopearl HW 40 (S) fractions 1-3 and their related composition formulas. The calculated molecular weights are average values. Explanation for Lx/y-z; L ) lactose; x ) Number of Gal-GlcNAc units; y ) number of fucoses; z ) number of sialic acids.
isolated previously by combining anion-exchange chromatography, preparative paper chromatography and HPLC.39 Moreover, 11 different sialylated oligosaccharides were purified and characterized from a pool of 100 L human milk.40-42 The largest structures found in this study possessed a molecular mass of 1655 Da. The corresponding mass was also detected (Figure 6(a)) in the present analysis. Two high-molecular-weight acidic oligosaccharides were isolated from milk of the tammar wallaby (Macropus eugenii). Their molecular masses were approximately 2500 Da.43 This is the only mammalian milk up to now for which acidic structures in this mass range have been identified. The HPAEC analyses of the total acidic oligosaccharides and HW 40 (S) fraction 3 are depicted in Figure 7. The major (39) Fievre, S.; Wieruszeski, J.-M.; Michalski, J.-C.; Lemoine, J.; Montreuil, J.; Strecker, G. Biochem. Biophys. Res. Commun. 1991, 177, 720-725. (40) Gro ¨nberg, G.; Lipniunas, P.; Lundgren, T.; Erlansson, K.; Lindh, F.; Nilsson, B. Carbohydr. Res. 1989, 191, 261-278. (41) Gro¨nberg, G.; Lipniunas, P.; Lundgren, T.; Lindh, F.; Nilsson, B. Arch. Biochem. Biophys. 1990, 278, 297-311. (42) Gro¨nberg, G.; Lipiniunas, P.; Lundgren, T.; Lindh, F.; Nilsson, B. Arch. Biochem. Biophys. 1992, 296, 597-610. (43) Urashima, T.; Saito, T.; Tsuji, Y.; Taneda, Y.; Takasawa, T.; Messer, M. Biochim. Biophys. Acta 1994, 1200, 64-72.
Figure 7. HPAEC profiles of acidic oligosaccharide fractions from human milk: (a) total acidic fraction (L2/1-1, L2/1-2, and L2/0-2 were determined by MALDI-MS and rpHPLC); (b) Toyopearl HW 40 (S) fraction 1 (see Figure 5). For abbreviations used and structures, see Table 2.
components 3’-sialyllactose, 6’-sialyllactose, lacto-N-sialyltetraoses a-c, and disialyllacto-N-tetraose were previously isolated and identified by FAB-MS.9 The compounds L2/1-1, L2/1-2, and L2/ 0-2 (Figure 7a) were identified after fractionation by gel filtration on Toyopearl HW 40 (S) and isolation by rpHPLC followed by MALDI-MS analysis (data not shown). The molecular masses of the major acidic structures (see Table 2) were found to range between 634 Da (sialyllactoses) and 1289 Da (disialyllacto-N-tetraose). Most of the structures of the HW 40 (S) fractionation eluted between 30 and 45 min, and the corresponding molecular masses, as determined by MALDI-MS, were found to range between 2314 and 3627 Da. The absence of signals in the lower mass range indicated the successful removal of the major compounds but also the lack of fragmentation of the oligosaccharides during MALDI-MS analysis. The HPAE chromatogram of HW 40 (S) fraction 3, as given in Figure 7b, shows a much higher complexity than that of the total acidic fraction. This is due to the high concentration of the low-molecular-mass structures in the total fraction interfering with the weakly abundant complex structures. In contrast to the HPAEC of P-6 fractions of the high-molecular-mass neutral oligosaccharides, HPAE chromatography allowed the high-mass acidic structures to be analyzed with an applied gradient in high resolution. Consequently identical or similar gradient conditions can be used to isolate distinct structures by HPAEC for a further structural analysis. Apart from a few exceptions, it is concluded that the composition of the high-molecular-weight acidic oligosaccharides based on MALDI-MS analysis consists mainly of monoand disialylated compounds (Table 3). The main influence on their elution on a CarboPac column is given by the number of charged Analytical Chemistry, Vol. 71, No. 17, September 1, 1999
3761
groups, i.e., their sialic acid content. An HPAEC-PAD method for the mapping of N-glycans was previously established, using a linear gradient for sialylated N-glycans.28 This method enables structural analysis, e.g. of composition and antennae, by mere comparison of retention times. The classification according to the number of sialic acids should be applicable for acidic human milk oligosaccharides but will probably result in much more complex chromatograms. The highest PED response for HW 40 (S) fraction 3 was observed between 30 and 45 min (Figure 7b), which corresponds to disialylated oligosaccharides containing a core of lacto-N-hexaose and larger units. The separation of acidic structures depends mainly on the number of sialic acid residues. CONCLUSION This study demonstrates that there are significantly more free oligosaccharides in human milk than previously found. It was shown that the applied gel filtration methods for the fractionation of higher neutral as well as higher acidic oligosaccharides exemplified for human milk oligosaccharides are a prerequisite for the following HPAEC-PAD and MALDI-MS analyses. The matrix preparation and the employed operation parameters of the MALDI-MS analyses are suitable for the detection of highmolecular-weight oligosaccharides from human milk with high resolution and without a significant prompt fragmentation to lose sialic acids and fucoses, respectively. On the basis of the results (44) Stahl, B.; Klabunde, T.; Witzel, H.; Krebs, B.; Steup, M.; Karas, M.; Hillenkamp, F. Eur. J. Biochem. 1994, 220, 321-330 (45) Harvey, D. J.; Ku ¨ ster, B.; Naven, T. J. P. Glycoconjugate J. 1998, 15, 333338. (46) Pfenninger, A.; Bahr, U.; Karas, M.; Finke, B.; Stahl, B.; Sawatzki, G. In preparation.
3762 Analytical Chemistry, Vol. 71, No. 17, September 1, 1999
of HPAEC-PAD analyses, it can be suggested that the total number of structures on the basis of isomers is much higher than estimated before. The HPAEC gradient used for acidic oligosaccharides enables the isolation of distinct higher structures, which can be completely elucidated by applying different methods: FABMS and NMR are established and widespread techniques for this purpose. Furthermore, sequence and linkage information was obtained by using exoglycosidases in combination with MALDIMS, as realized for N-glycans.44,45 The same holds for a combination with HPAEC-PAD. Recently, sequence and structure analyses using the electrospray ionization mass spectrometry ion trap (ESI ion trap MS) of a complex mixture of nonderivatizated human milk oligosaccharides were demonstrated.46 A combination of liquid chromatography and MALDI-MS applied in this study with one or more of the above-mentioned methods will allow for the unambiguous assignment of each of the oligosaccharide structures detected. ACKNOWLEDGMENT We thank B. Mu¨ller-Werner and M. Mank, Milupa GmbH & Co. KG, for their excellent technical assistance. Financial support of the Bundesministerium fu¨r Bildung und Forschung under Grant BEO/22 0311229 is gratefully acknowledged. This work was performed in partial fulfillment of the requirements of B.F. for the degree of a Dr. oec. troph. at Justus-Liebig-Universita¨t, Giessen, Germany, and A.P. for the degree of Dr. phil. nat. at Johann-Wolfgang-Goethe-Universita¨t, Frankfurt, Germany. Received for review January 29, 1999. Accepted May 27, 1999. AC990094Z
