Profiling with structural elucidation of the neutral and anionic O-linked oligosaccharides in the egg jelly coat of Xenopus laevis by Fourier transform mass spectrometry

A strategic method with high speed and sensitivity is outlined for the analysis of mucin-type oligosaccharide from the jelly coat of Xenopus laevis. The method relies primarily on mass spectrometric techniques, in this case matrix-assisted laser desorption/ionization Fourier-transform mass spectrometry (MALDI-FTMS) and collision-induced dissociation (CID). Separation with isolation of the oligosaccharides was streamlined to couple well with mass spectrometry allowing the rapid determination of all detectable components from both neutral and anionic species. Partial structures of anionic components, composed primarily of sulfate esters, were obtained with CID. For neutral species, a method that allowed the complete structural determination using mass spectrometry was used. The method builds on the structure of small number of known compounds to determine unknown structures from the same biological source. In this example, a small number of oligosaccharides, elucidated previously by NMR, were used to develop a set of substructural motifs that were characterized by CID. The presence of the motifs in the CID spectra were then used to determine the structures of unknown compounds that were in abundances too small for NMR analysis.     

Offline coupling of low-pressure anion-exchange chromatography with MALDI-MS to determine the elution order of human milk oligosaccharides

Pooled human milk oligosaccharides were separated into neutral and several acidic oligosaccharide fractions by preparative anion-exchange chromatography (AEC) using AG 1-X2. The oligosaccharides were eluted stepwise using deionized water and three different concentrations of ammonium acetate buffer, pH 6.8. The elution order of the compounds was determined directly by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of the AEC effluent without any cleanup or concentration steps. Up to a concentration of 500 mM ammonium acetate, the masses of acidic oligosaccharides could be detected by screening the fractions in an automated mode. The combination of the improved chromatographic procedure, the applied MALDI matrices, and operating parameters is suitable for the detection of neutral oligosaccharides as well as acidic oligosaccharides. The method provides high sensitivity and mass accuracy, including for the high-molecular-weight monosialylated oligosaccharides up to 2751.5 Da. The applied ionic strength of the anion-exchange eluents enables a rapid and an unambiguous composition assignment by MALDI-MS for neutral, monosialylated, and disialylated oligosaccharides from human milk. The acidic fractions have to be desalted by electrodialysis and were finally analyzed by HPAEC-PAD to get a high-resolution "fingerprint" of structures present in each fraction. From these analyses, it can be concluded that the isomeric variety of monosialylated oligosaccharides occurring in human milk is higher than estimated before.     

A relative and absolute quantification of neutral N-linked oligosaccharides using modification with carboxymethyl trimethylammonium hydrazide and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Quantification of oligosaccharides is of great importance to investigate variations or changes in the glycans of glycoconjugates. Mass spectrometry (MS) has been widely applied to identification and structural analysis of complex oligosaccharides. However, quantification using MS alone is still quite challenging due to heterogeneous charge states and different ionization efficiency of various types of oligosaccharides. To overcome such shortcomings, derivatization with carboxymethyl trimethylammonium hydrazide (Girard’s reagent T [GT]) was introduced to generate a permanent cationic charge at the reducing end of neutral oligosaccharides, resulting in mainly [M]⁺ ion using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), so that the ambiguities caused by metal adduct peaks such as [M+K]⁺ and [M + Na]⁺ were avoided. To verify our method, the relative and absolute quantification of neutral glycans from human immunoglobulin G (IgG) and ovalbumin with internal standards of dextran ladders using MALDI-TOF MS were compared with those performed by conventional normal-phase high-performance liquid chromatography (NP-HPLC) profiling. The quantification using GT derivatization and MALDI-TOF MS agreed well with the HPLC profiling data and showed excellent reliability and reproducibility with better resolution and sensitivity. This method was further applied to quantify the enzymatically desialylated N-glycans from miniature pig kidney membrane proteins. The results showed that the low-abundance structures that could not be resolved by NP-HPLC were quantified with high sensitivity. Thus, this novel method of using modification of neutral sugars with GT is quite powerful for neutral glycan analysis, especially to quantify minute glycan samples with undetectable levels using HPLC.     

The use of gas chromatography and gas chromatography-mass spectrometry for the characterization of permethylated oligosaccharides with molecular mass up to 2300

Gas chromatography and gas chromatography-mass spectrometry were adapted for the analysis of large permethylated oligosaccharides of different types. Permethylated isomaltooligosaccharides with up to 11 sugar residues and a mass of 2291 Da and two branched blood group H-type decasaccharides derived from the corresponding glycosphingolipids with masses of 2150 Da were successfully analyzed. The capillary columns used have extremely good resolution exemplified by the separation of the two decasaccharides which only differed by one internal linkage position and by the separation of four isomeric tetrasaccharides. The combined information of retention times and mass spectra gave detailed information of 22 neutral oligosaccharides from porcine intestinal mucin and the approach thus allow quick screening of O-linked-type glycans. The procedure for permethylation of oligosaccharides using solid NaOH has been investigated and adapted for structures having a glucose alditol as in reduced oligosaccharides derived from milk and glycosphingolipids.     

Strategy for the investigation of O-linked oligosaccharides from mucins based on the separation into neutral, sialic acid- and sulfate-containing species

A method for the separation of O-linked oligosaccharides into neutral, sialic acid-containing and sulfated species was applied to oligosaccharides released by alkaline borohydride from mucin glycopeptides from porcine small intestine. The released mixture of reduced oligosaccharides was applied to an anion exchange column, and the neutral oligosaccharides were collected as the unretarded fraction. A mixture of dimethyl sulfoxide and iodomethane was passed through the column to convert the sialic acid-containing oligosaccharides into methyl esters that were eluted and converted to methyl amides by methyl amine. Finally the sulfated oligosaccharide fraction was eluted with salt. The neutral and the derivatized sialic acid-containing oligosaccharides were analysed by gas chromatography-mass spectrometry after permethylation and the sulfated oligosaccharide fraction was analysed by high performance anion exchange chromatography.Abbreviations GC gas chromatography - GC/MS gas chromatography-mass spectrometry - HPAEC-PAD high performance anion exchange chromatography-pulsed amperometric detection - Hex hexose - HexNAc N-acetyl hexosamine - HexNAcol N-acetyl hexosaminitol - Fuc Fucose - NeuAc N-acetyl neuraminic acid - NeuGc N-glycolyl neuraminic acid     

Fucose-containing oligosaccharides from human milk from a donor of blood group 0 Le(a) nonsecretor

The neutral oligosaccharides from the milk of a single donor with blood group 0, Lewis(a+b-) nonsecretor were separated into 18 fractions essentially according to the number of carbohydrate constituents using gel permeation chromatography on Biogel P-4 and Fractogel TSK HW-40. Further separation was achieved by HPLC and by HPTLC after reduction and peracetylation. The fractions obtained were analysed by FAB mass spectrometry with and without derivatization and by one- and two-dimensional proton NMR. Besides the already described 3-fucosidolactose, fucopentaose II (2), fucopentaose III (6) and difucohexaose II (4) the following fucosylated oligosaccharides could be identified. Among the higher oligosaccharides a branched lacto-N-decaose (12) was obtained in pure form after removal of the fucose residues by mild acid treatment.     

Fast-atom-bombardment mass spectrometry and electron-impact direct-probe mass spectrometry in the structural analysis of oligosaccharides with special reference to the poly(N-acetyl-lactosamine) series.

A comparison has been made of positive- and negative-ion fast-atom-bombardment (FAB) and electron-impact (EI) mass spectrometry for analysis of oligosaccharides and alditols containing alternating and consecutive sequences of neutral and acetamido sugars. Among these were novel chemically synthesized tetrasaccharides with Ii antigen activities. FAB ionization has the advantage that it is applicable to non-derivatized oligosaccharides and it can determine Mr. However, the abundance of fragment ions providing structural information and the amount of material required for analysis (1-50 nmol) varied from sample to sample. In contrast, EI mass spectrometry of 5 nmol of permethylated or peracetylated oligosaccharides reliably gives all the fragment ions formed by cleavage across the glycosidic bonds.     

Oligosaccharides from feces of preterm infants fed on breast milk

Nine neutral and five acidic oligosaccharides were isolated from feces of a preterm (30th postmenstrual week) blood group A nonsecretor infant fed on pooled breast milk. Structural analyses were carried out using sugar and methylation analyses, fast atom bombardment mass spectrometry, and 1H NMR. The acidic oligosaccharides are well-known components of human milk. The neutral oligosaccharides are characteristic of nonsecretor milk. Surprisingly, no secretor gene-dependent oligosaccharides were present in the feces. Another preterm (27th postmenstrual week) blood group A, secretor infant fed on pooled breast milk showed the same fecal oligosaccharide pattern as above during the first week after birth, despite being a secretor individual. Also notable was the absence of blood group A-active oligosaccharides in this sample. Another sample of feces collected 8 weeks later from the latter infant contained the expected blood group A-active oligosaccharides. Furthermore, free sialic acid was present at the cost of the sialyl oligosaccharides seen earlier. Thus, infants born prematurely do not show the same degree of development of oligosaccharide metabolism as their more mature counterparts.     

Analysis of O-linked reducing oligosaccharides released by an in-line flow system

Reducing O-linked oligosaccharides from bovine submaxillary mucin, bovine fetuin, and porcine gastric mucin were recovered by nonreductive alkaline beta-elimination from an in-line flow system. Glycoproteins where attached to a solid support using hydrophobic interaction with alkali-resistant Poros reversed phase beads and a flow of alkali released the oligosaccharides. The alkali was subsequently neutralized by a continuous flow through cation exchange resin. The released oligosaccharides in the flow were trapped in a cartridge filled with graphitized carbon. Salt-free oligosaccharides could be recovered as a concentrated solution by elution with organic solvents from the cartridge. The glycosylation pattern of the released oligosaccharides was compared with the conventionally released and reduced oligosaccharides recovered from alkaline beta-elimination in the presence of borohydride. In general, the recovery from the in-line release was sometimes lower than from the reductive elimination method, but it was shown that alkaline degradation of reducing oligosaccharides was limited in this system. Liquid chromatography using graphitized carbon packing and high pH mobile phases together with negative ion electrospray mass spectrometry showed that both neutral and acidic reducing oligosaccharides could be analyzed in a single run. Reducing O-linked oligosaccharides could also be recovered in this way from human glycophorin separated by SDS-PAGE. The polyacrylamide was sufficient to retain the glycoprotein in the gel while the flow of alkali released the oligosaccharides. It was also shown that the alkaline conditions for releasing O-linked oligosaccharides from fetuin would partially release some N-linked oligosaccharides, particularly in the presence of reducing agent.     

Neutral oligosaccharides in the urine of a patient with glycogen storage disease type II

Neutral oligosaccharides were isolated from urine of an adult patient with glycogen storage disease type II, a deficiency of lysosomal acid alpha-glucosidase, by chromatography on columns of activated charcoal, Dowex 50 X 2 and Dowex 1 X 2. Total neutral oligosaccharides in the urine of the patient were increased about 5-fold as compared with those in normal controls. The most accumulated oligosaccharide was separated by Bio-Gel P-2 column chromatography, and finally purified by paper chromatography. Based on various studies, including carbohydrate analysis, chemical ionization mass spectrometry, fast atom bombardment mass spectrometry, degradation by glucoamylase and isopullulanase, and methylation analysis, the structure of this oligosaccharide was deduced to be Glc alpha 1----6Glc alpha 1----4Glc alpha 1----4Glc. This oligosaccharide appears to be accumulated in urine of the patient with acid alpha-glucosidase deficiency as an end product of the hydrolysis of glycogen.     

Investigations of the in vitro transport of human milk oligosaccharides by a Caco-2 monolayer using a novel high performance liquid chromatography-mass spectrometry technique.

Complex lactose-derived oligosaccharides belong to the main components of human milk and are believed to exert multiple functions in the breast-fed infant. Therefore, we investigated the transepithelial transport of human milk oligosaccharides over Caco-2 monolayers. Main human milk oligosaccharides (HMOs) in the apical, basolateral, or intracellular compartment were separated by high performance liquid chromatography using a Hypercarb(TM) column and analyzed on line by mass spectrometry. This method allowed the identification and quantification of these components in intra- and extracellular fractions without prior purification. Using this technique we were able to show that acidic and neutral HMOs cross the epithelial barrier. The transepithelial flux of neutral, but not acidic, oligosaccharides was temperature-sensitive and partly inhibited by brefeldin A and bafilomycin A. Furthermore, net flux from the apical to the basolateral compartment was only observed for the neutral components. Similarly, apical cellular uptake was only found for neutral components but not for acidic oligosaccharides. Intracellular concentrations of neutral HMOs were significantly increased by inhibitors of transcytosis such as brefeldin A, N-ethylmaleimide, or bafilomycin A. The cellular uptake was saturable, and an apparent K(m) for lacto-N-fucopentaose I of 1.7 +/- 0.1 mmol/liter and for lacto-N-tetraose of 1.8 +/- 0.4 mmol/liter was determined. Furthermore, the uptake of lacto-N-fucopentaose I could be inhibited by the addition of the stereoisomer lacto-N-fucopentaose II but not by lacto-N-tetraose. These findings suggest that neutral HMOs are transported across the intestinal epithelium by receptor-mediated transcytosis as well as via paracellular pathways, whereas translocation of acidic HMOs solely represents paracellular flux.     

A general approach to desalting oligosaccharides released from glycoproteins

Desalting of sugar samples is essential for the success of many techniques of carbohydrate analysis such as mass spectrometry, capillary electrophoresis, anion exchange chromatography, enzyme degradation and chemical derivatization. All desalting methods which are currently used have limitations: for example, mixed-bed ion-exchange columns risk the loss of charged sugars, precipitation of salt by a non-aqueous solvent can result in co-precipitation of oligosaccharides, and gel chromatography uses highly crosslinked packings in which separation of small oligosaccharides is difficult to achieve. We demonstrate that graphitized carbon as a solid phase extraction cartridge can be used for the purification of oligosaccharides (or their derivatives) from solutions containing one or more of the following contaminants: salts (including salts of hydroxide, acetate, phosphate), monosaccharides, detergents (sodium dodecyl sulfate and Triton X-100), protein (including enzymes) and reagents for the release of oligosaccharides from glycoconjugates (such as hydrazine and sodium borohydride). There is complete recovery of the oligosaccharides from the adsorbent which can also be used to fractionate acidic and neutral glycans. Specific applications such as clean-up of N-linked oligosaccharides after removal by PNGase F and hydrazine, desalting of O-linked glycans after removal by alkali, on-line desalting of HPAEC-separated oligosaccharides and -eliminated alditols prior to electrospray mass spectrometry, and purification of oligosaccharides from urine are described.     

Separation of oligosaccharides by capillary supercritical fluid chromatography and analysis by direct coupling to high-resolution mass spectrometer: application to analysis of oligomannosidic N-glycans

Supercritical fluid chromatography separations and supercritical fluid chromatography chemical ionization mass spectrometry analysis of permethylated and pertrimethylsilylated oligosaccharides are reported. Supercritical fluid chromatography was carried out using a DB-5 coated capillary column with carbon dioxide as a mobile phase. Peralkylated oligosaccharides were detected by flame ionization and by chemical ionization mass spectrometry using the GC interface. Analysis of permethylated malto-oligosaccharides, as well as oligomannosides from mannosidosis, was achieved by chemical ionization mass spectrometry with ammonia and provided the pseudo-molecular ions (M+H)+ and (M+NH4)+, in addition to some other fragments which allow interpretations of the structure of different oligosaccharides. The good resolution and sensitivity obtained emphasize the potential of supercritical fluid chromatography mass spectrometry for rapid separations and analysis of complex glycan mixtures.     

High resolution and high sensitivity methods for oligosaccharide mapping and characterization by normal phase high performance liquid chromatography following derivatization with highly fluorescent anthranilic acid

Facile labeling of oligosaccharides (acidic and neutral) in a nonselective manner was achieved with highly fluorescent anthranilic acid (AA, 2-aminobenzoic acid) (more than twice the intensity of 2- aminobenzamide, AB) for specific detection at very high sensitivity. Quantitative labeling in acetate-borate buffered methanol (approximately pH 5.0) at 80 degreesC for 60 min resulted in negligible or no desialylation of the oligosaccharides. A high resolution high performance liquid chromatographic method was developed for quantitative oligosaccharide mapping on a polymeric-NH2bonded (Astec) column operating under normal phase and anion exchange (NP-HPAEC) conditions. For isolation of oligosaccharides from the map by simple evaporation, the chromatographic conditions developed use volatile acetic acid-triethylamine buffer (approximately pH 4.0) systems. The mapping and characterization technology was developed using well characterized standard glycoproteins. The fluorescent oligosaccharide maps were similar to the maps obtained by the high pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), except that the fluorescent maps contained more defined peaks. In the map, the oligosaccharides separated into groups based on charge, size, linkage, and overall structure in a manner similar to HPAEC-PAD with contribution of -COOH function from the label, anthranilic acid. However, selectivity of the column for sialic acid linkages was different. A second dimension normal phase HPLC (NP-HPLC) method was developed on an amide column (TSK Gel amide-80) for separation of the AA labeled neutral complex type and isomeric structures of high mannose type oligosaccharides. The oligosaccharides labeled with AA are compatible with biochemical and biophysical techniques, and use of matrix assisted laser desorption mass spectrometry for rapid determination of oligosaccharide mass map of glycoproteins is demonstrated. High resolution of NP-HPAEC and NP-HPLC methods combined with mass spectrometry (MALDI-TOF) can provide an effective technology for analyzing a wide repertoire of oligosaccharide structures and for determining the action of both transferases and glycosidases.      

荧光标记电泳对透明质酸寡糖片段鉴定的方法建立

近年来,透明质酸寡糖片段（hyaluronan oligosaccharides, o-HA）的生物学活性引起国外学者的重视,因为o-HA具有一定的生物学活性,如参与免疫调节、刺激新生血管形成等.本研究建立一种经济、简便的ANTS（8-氨基奈-1,3,6-三磺酸）荧光标记电泳对透明质酸寡糖片段大小鉴定的新实验方法.实验原理为,ANTS能与糖分子发生还原反应,在反应时提供3个电子和1个荧光基团,通过高浓度PAGE分离,在特定波长下呈现颜色反应.采用酶消化法得到不同分子量大小的o-HA片段,测得不同片段大小的o-HA聚合度,分别与高效液相色谱（high-performance liquid chromatography, HPLC）和静电喷雾电离质谱（electrospray ionization mass spectrometry, ESI-MS）进行比较,结果吻合.研究提示,用荧光标记电泳法分析寡糖分子量,操作简单、设备低廉、灵敏度较高且检测速度快,是一种检测鉴定寡糖分子的较好方法.     

Transfer of glycerol by Endo-beta-N-acetylglucosaminidase F to oligosaccharides during chitobiose core cleavage

N-Linked oligosaccharides, when hydrolyzed by glycerol-containing preparations of endo-beta-N-acetylglucosaminidase (Endo) F from Flavobacterium meningosepticum were found to have glycerol attached to their reducing ends. The absence of a reducing end was confirmed by high-field 1H NMR spectroscopy, and the incorporated glycerol was verified through mass spectrometry and collisionally activated decomposition fast atom bombardment/mass spectrometry/mass spectrometry techniques. Periodate oxidation of [1(3)-14C]glycerol-labeled oligosaccharides indicated glycerol was glycosidically linked via its 1(3) carbon to the C1 of the reducing end N-acetylglucosamine. In a second, less favored reaction, the glycerol glycoside was hydrolyzed by Endo F using water as the terminal nucleophile, thus regenerating the N-acetylglucosamine reducing end. Glycerol could be removed from Endo F preparations without affecting enzyme stability, and chitobiosyl core hydrolysis in its absence provided intact oligosaccharides with normal N-acetylglucosamine reducing ends. The incorporation of labeled glycerol may provide a useful method for monitoring of Endo F release of oligosaccharides.     

Structure determination by MALDI-IRMPD mass spectrometry and exoglycosidase digestions of O-linked oligosaccharides from Xenopus borealis egg jelly

Differences in the fertilization behavior of Xenopus borealis from X. laevis and X. tropicalis suggest differences in the glycosylation of the egg jellies. To test this assumption, O-linked glycans were chemically released from the egg jelly coat glycoproteins of X. borealis. Over 50 major neutral glycans were observed, and no anionic glycans were detected from the released O-glycan pool. Preliminary structures of ～30 neutral oligosaccharides were determined using matrix-assisted laser desorption/ionization (MALDI) infrared multiphoton dissociation tandem mass spectrometry (MS). The mass fingerprint of a group of peaks for the core-2 structure of O-glycans was conserved in the tandem mass spectra and was instrumental in rapid and efficient structure determination. Among the 29 O-glycans, 22 glycans contain the typical core-2 structure, 3 glycans have the core-1 structure and 2 glycans contained a previously unobserved core structure with hexose at the reducing end. There were seven pairs of structural isomers observed in the major O-linked oligosaccharides. To further elucidate the structures of a dozen O-linked glycans, specific and targeted exoglycosidase digestions were carried out and the products were monitored with MALDI-MS. Reported here are the elucidated structures of O-linked oligosaccharides from glycoproteins of X. borealis egg jelly coats. The structural differences in O-glycans from jelly coats of X. borealis and its close relatives may provide a better understanding of the structure-function relationships and the role of glycans in the fertilization process within Xenopodinae.     

Structural characterization of novel oligosaccharides of cell-surface glycoproteins of Trypanosoma cruzi

Affinity-purified glycopeptides were prepared from Trypanosomacruzi using the carbohydrate-specific monoclonal antibody WIC29.26.These glycopeptides contain rhamnose, fucose, xylose, and galactose,in the ratio 1:1:2:3. A series of oligosaccharides was releasedfrom the glycopeptides by mild acid hydrolysis, while, in contrast,no oligosaccharides were released by either peptide N-glycosidaseF or conventional base-catalyzed ß-elimination andreduction. This suggested the presence of a phosphodiester linkagebetween the carbohydrate and peptide, which was further supportedby the detection of phosphothreonine in the glycopeptides. Themild acid liberated (MAL) fraction was resolved into two majoracidic oligosaccharides (MAL-P1 and MAL-P2), two minor neutraloligosaccharides (MAL P1b and MAL-P2b) and a neutral fraction(MAL-N1), consisting of Gal and Xyl monosaccharides. The MAL-P1and MAL-P2 oligosaccharides proved to be hexa- and heptasaccharidesthat shared a common xylose reducing terminus, but differedby one galactofuranose residue, and their negative charge wasshown to be due to the presence of cyclic-phosphate attachedto nonreducing terminal galactofuranose residues. The MAL-P1band MAL-P2b oligosaccharides appeared to be nonphosphorylatedversions of MAL-P1 and MAL-P2. Partial structures of MAL-P1and MAL-P2 are suggested, based on compositional analyses, electrospraymass spectrometry, and tandem mass spectrometry before and afterpermethylation. The origin and significance of these uniquetrypanosomatid glycoconjugates is discussed. glycoprotein monoclonal antibody oligosaccharide structure Trapanosoma cruzi     

Fucosylated human milk oligosaccharides vary between individuals and over the course of lactation.

Specific human milk oligosaccharides, especially fucosylated neutral oligosaccharides, protect infants against specific microbial pathogens. To study the concentrations of individual neutral oligosaccharides during lactation, a total of 84 milk samples were obtained from 12 women at 7 time periods during weeks 1-49 postpartum. The neutral oligosaccharides from each sample were isolated, perbenzoylated, resolved, and quantified by reversed-phase high-performance liquid chromatography. The resultant oligosaccharide peaks, identified by co-elution with authentic standards and mass spectrometry, ranged in size from tri- to octasaccharides. The total concentration of oligosaccharides declined over the course of lactation; the mean concentration at 1 year was less than half that in the first few weeks postpartum. One of the 12 donors produced milk fucosyloligosaccharides that were essentially devoid of alpha1,2 linkages (but contained alpha1,3- and alpha1,4-linked fucose) until late in lactation, consistent with the nonsecretor phenotype. In milk samples from the remaining 11 donors, fucosyloligosaccharides containing alpha1,2-linked fucose were prevalent, and their profiles were distinct from those of fucosyloligosaccharides devoid of alpha1,2-linked fucose. The ratio of alpha1,2-linked oligosaccharide concentrations to oligosaccharides devoid of alpha1,2-linked fucose changed during the first year of lactation from 5:1 to 1:1. Furthermore, the absolute and the relative concentrations of individual oligosaccharides varied substantially, both between individual donors and over the course of lactation for each individual. The patterns of milk oligosaccharides among individuals suggest the existence of many genotype subpopulations. This variation in individual oligosaccharide concentrations suggests that the protective activities of human milk could also vary among individuals and during lactation.