Altered linkage pattern of N-glycan sialic acids in pseudomyxoma peritonei.

Pseudomyxoma peritonei (PMP) is a highly mucinous adenocarcinoma growing in the peritoneal cavity and most commonly originating from the appendix. Glycans play an important role in carcinogenesis, and glycosylation is altered in malignant diseases, including PMP. We have previously demonstrated that fucosylation of N-glycans is increased in PMP, but we did not observe modulation of overall sialylation. As sialic acids can be attached to the rest of the glycan via α2,3- or α2,6-linkage, we have now analyzed the linkage patterns of sialic acids in tissue specimens of normal appendices, low-grade appendiceal mucinous neoplasms (LAMN), low-grade (LG) PMP and high-grade (HG) PMP. For the linkage analysis, the enzymatically released acidic N-glycans were first treated with ethyl esterification or α2,3-sialidase digestion followed by MALDI-TOF mass spectrometry. Significant increase in the relative abundance of α2,6-sialylated and decrease in α2,3-sialylated N-glycans was observed in PMP tumors as compared to the normal appendices (P < 0.025). More specifically, increased α2,6-sialylation (P < 0.05) and decreased α2,3-sialylation (P < 0.01) were detected in afucosylated and monofucosylated N-glycans of PMPs, whereas the less abundant multifucosylated glycans, containing terminal fucose, demonstrated increased α2,3-sialylation (P < 0.01). Importantly, the increase in α2,6-sialylation was also detected between PMP and the appendiceal precursor lesion LAMN (P < 0.01). The identified glycosylation alterations produce ligands for sialic acid-binding immunoglobulin-like lectins (Siglecs) and sialofucosylated glycans binding selectins, which play a role in the peritoneal dissemination and progression of the disease.

Recent advances in the analysis of polysialic acid from complex biological systems.

Polysialic acid (polySia) is a unique, well-characterised carbohydrate polymer highly-expressed on the cell surface of neurons in the early stages of mammalian brain development. Post-embryogenesis, it is also re-expressed in a number of tumours of neuroendocrine origin. It plays important roles in modulating cell-cell, and cell-matrix adhesion and migration, tumour invasion and metastasis. Techniques for structural and quantitative characterisation of polySia from tumours and cancer cells are thus essential in exploring the relationship between polySia expression levels and structural and functional changes associated with cancer progression and metastasis. A variety of techniques have been developed to structurally and quantitatively analyse polySia in clinical tissues and other biological samples. In this review, analytical approaches used for the determination of polySia in biological matrices in the past 20 years are discussed, with a particular focus on chemical approaches, and quantitative analysis.

Determination of the Structural Integrity and Stability of Polysialic Acid during Alkaline and Thermal Treatment.

Polysialic acid (polySia) is a linear homopolymer of varying chain lengths that exists mostly on the outer cell membrane surface of certain bacteria, such as Escherichia coli (E. coli) K1. PolySia, with an average degree of polymerization of 20 (polySia avDP20), possesses material properties that can be used for therapeutic applications to treat inflammatory neurodegenerative diseases. The fermentation of E. coli K1 enables the large-scale production of endogenous long-chain polySia (DP ≈ 130) (LC polySia), from which polySia avDP20 can be manufactured using thermal hydrolysis. To ensure adequate biopharmaceutical quality of the product, the removal of byproducts and contaminants, such as endotoxins, is essential. Recent studies have revealed that the long-term incubation in alkaline sodium hydroxide (NaOH) solutions reduces the endotoxin content down to 3 EU (endotoxin units) per mg, which is in the range of pharmaceutical applications. In this study, we analyzed interferences in the intramolecular structure of polySia caused by harsh NaOH treatment or thermal hydrolysis. Nuclear magnetic resonance (NMR) spectroscopy revealed that neither the incubation in an alkaline solution nor the thermal hydrolysis induced any chemical modification. In addition, HPLC analysis with a preceding 1,2-diamino-4,5-methylenedioxybenzene (DMB) derivatization demonstrated that the alkaline treatment did not induce any hydrolytic effects to reduce the maximum polymer length and that the controlled thermal hydrolysis reduced the maximum chain length effectively, while cost-effective incubation in alkaline solutions had no adverse effects on LC polySia. Therefore, both methods guarantee the production of high-purity, low-molecular-weight polySia without alterations in the structure, which is a prerequisite for the submission of a marketing authorization application as a medicinal product. However, a specific synthesis of low-molecular-weight polySia with defined chain lengths is only possible to a limited extent.

Intact Human Alpha-Acid Glycoprotein Analyzed by ESI-qTOF-MS: Simultaneous Determination of the Glycan Composition of Multiple Glycosylation Sites.

Alpha-1-acid glycoprotein (AGP) is a highly glycosylated protein from human plasma with five N-type glycosylation sites carrying dominantly higher antennary structures and thus represents a challenging target for characterization of glycan heterogeneity. Here, we show that glycan composition over all five glycosylation sites can be determined quantitatively by ESI-qTOF-MS of the intact glycoprotein in negative ion mode. We find numerous glycan species extending the mass range of the glycoprotein species from 35.0 to 38.5 kDa. The dominant glycan compositions contain tri- and tetraantennary structures on all glycosylation sites. The mass degeneracy of two fucosyl units versus one sialic acid was resolved by treating the sample with sialidase and analyzing the resulting desialylated AGP by electrospray ionization-mass spectrometry in positive ion mode. The pattern of nonsialylated oligosaccharides was used for interpretation of the fully sialylated species using bioinformatics tools. From pooled human plasma, we find 90, 101, and 64 different glycan compositions for genetic variants ORM1*F1, ORM1*S, and ORM2, respectively. Glycan structures carry dominantly between 15 and 16 sialic acids indicating an almost complete termination of all antenae with sialic acid. AGP from human plasma samples of single individuals was analyzed as desialylated glycoproteins and showed variations in fucosylation and in the amount of antennary structures between individuals.

Identification of sialyl oligosaccharides including an oligosaccharide nucleotide in colostrum of an addax (Addax nasomaculatus) (Subfamily Antelopinae).

Mammalian milk/colostrum usually contains milk oligosaccharides along with the predominant lactose. Although milk oligosaccharides of a variety of Bovidae species including cow, sheep and goat have been characterized, those of the addax, an Antelopinae species of the Bovidae, have not as yet been clarified. In this study, several sialyl oligosaccharides were purified from a sample of addax colostrum and characterized as follows: Neu5Ac(α2-8)Neu5Ac(α2-3)Gal(ß1-4)Glc, Neu5Gc(α2-8)Neu5Gc(α2-3)Gal(ß1-4)Glc, Neu5Ac(α2-3)Gal(ß1-4)Glc, Neu5Ac(α2-6)Gal(ß1-4)GlcNAc, Neu5Gc(α2-3)Gal(ß1-4)Glc, Neu5Gc(α2-6)Gal(ß1-4)Glc, Neu5Gc(α2-6)Gal(ß1-4)GlcNAc. In addition, an oligosaccharide nucleotide Neu5Gc(α2-6)Gal(ß1-4)GlcNAcα1-UDP was characterized. Molecular species of a variety of sialyl oligosaccharides found in milk and colostrum of these Bovidae were compared.

Different properties of polysialic acids synthesized by the polysialyltransferases ST8SIA2 and ST8SIA4.

Polysialic acid (polySia) is mainly found as a modification of neural cell adhesion molecule (NCAM) in whole embryonic brains, as well as restricted areas of adult vertebrate brains, including the hippocampus. PolySia shows not only repulsive effects on NCAM-involved cell-cell interactions due to its bulky and hydrated properties, but also attractive effects on the interaction with neurologically active molecules, which exerts a reservoir function. Two different polysialyltransferases, ST8SIA2 and ST8SIA4, are involved in the synthesis of polySia chains; however, to date, the differences of the properties between polySia chains synthesized by these two enzymes remain unknown. In this study, to clarify this point, we first prepared polySia-NCAMs from HEK293 cells stably expressing ST8SIA4 and ST8SIA2, or ST8SIA2 (SNP-7), a mutant ST8SIA2 derived from a schizophrenia patient. The conventional sensitive chemical and immunological characterizations showed that the quantity and quality (structural features) of polySia are not so much different between ST8SIA4- and ST8SIA2-synthesized ones, apart from those of ST8SIA2 (SNP-7). Then, we assessed the homophilic and heterophilic interactions mediated by polySia-NCAM by adopting a surface plasmon resonance measurement as an in vitro analytical method. Our novel findings are as follows: (i) the ST8SIA2- and ST8SIA4-synthesized polySia-NCAMs exhibited different attractive and repulsive effects than each other; (ii) both polySia- and oligoSia-NCAMs synthesized by ST8SIA2 were able to bind polySia-NCAMs; (iii) the polySia-NCAM synthesized by a ST8SIA2 (SNP-7) showed markedly altered attractive and repulsive properties. Collectively, polySia-NCAM is suggested to simultaneously possess both attractive and repulsive properties that are highly regulated by the two polysialyltransferases.

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in mammals. However, humans cannot oxidize cytidine monophosphate-N-acetylneuraminic acid to cytidine monophosphate-N-glycolylneuraminic acid due to a primary exon deletion of the CMAH gene. To understand the effects and implications of the lack of CMAH activity in more detail, a Cmah knock-out model in mice is of keen interest in basic and applied research. The analysis method to determine the phenotype of this mouse model is herein described in detail, and is based on the detection of both N-acetylneuraminic acid and N-glycolylenuraminic acid in the liver and milk of wild-type and Cmah knock-out mice. Endogenous sialic acids are released and derivatized with o-phenylenediamine to generate fluorogenic derivatives, which can be subsequently analyzed by HPLC. The presented protocol can be also applied for the analysis of milk and tissue samples from various other origins, and may be of use to investigate the nutritional and health effects of N-glycolylneuraminic acid.

Improved sensitivity by post-column chemical environment modification of CE-ESI-MS using a flow-through microvial interface.

Post-column chemical environment modification can affect detection sensitivity and signal appearance when capillary electrophoresis is coupled through electrospray ionization to mass spectrometry (CE-ESI-MS). In this study, changes in the signal intensity and peak shape of N-Acetylneuraminic acid (Neu5Ac) were examined when the modifier solution used in a flow-through microvial interface for CE-ESI-MS was prepared using an acidic or basic background electrolyte (BGE) composition. The use of a basic modifier resulted in improved detection compared to the results obtained when an acidic modifier was used in negative ion mode. Increased sensitivity and more symmetrical peak shape were obtained. Using an acidic modifier, the LOD of Neu5Ac was 47.7 nM, whereas for a basic modifier, the LOD of Neu5Ac was 5.20 nM. The calculated asymmetry factor at 100 nM of Neu5Ac ranged from 0.71 to 1.5 when an acidic modifier was used, while the factor ranged from 1.0 to 1.1 when a basic modifier was used. Properly chosen post-column chemical modification can have a significant effect on the performance of the CE-MS system.

Sialic Acid Derivatization for the Rapid Subclass- and Sialic Acid Linkage-Specific MALDI-TOF-MS Analysis of IgG Fc-Glycopeptides.

Matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF)-mass spectrometry (MS) is a highly suitable method for the rapid analysis of IgG glycopeptides, providing a wealth of structural information. A limitation of this approach is that it generates a bias when analyzing sialylated species due to the labile nature of sialic acid glycosidic linkages. One way to overcome this problem is by chemical derivatization of the sialic acids. The method presented here results in both the stabilization of the sialic acids, as well as the differentiation of α2,3- and α2,6-linked sialic acids by mass. Described in this chapter are the isolation of IgG from plasma or serum, tryptic digestion of the samples, derivatization, and finally MALDI-TOF-MS measurement and data analysis.

Advances in analytical methodologies to guide bioprocess engineering for bio-therapeutics.

This study was performed to monitor the glycoform distribution of a recombinant antibody fusion protein expressed in CHO cells over the course of fed-batch bioreactor runs using high-throughput methods to accurately determine the glycosylation status of the cell culture and its product. Three different bioreactors running similar conditions were analysed at the same five time-points using the advanced methods described here. N-glycans from cell and secreted glycoproteins from CHO cells were analysed by HILIC-UPLC and MS, and the total glycosylation (both N- and O-linked glycans) secreted from the CHO cells were analysed by lectin microarrays. Cell glycoproteins contained mostly high mannose type N-linked glycans with some complex glycans; sialic acid was α-(2,3)-linked, galactose ß-(1,4)-linked, with core fucose. Glycans attached to secreted glycoproteins were mostly complex with sialic acid α-(2,3)-linked, galactose ß-(1,4)-linked, with mostly core fucose. There were no significant differences noted among the bioreactors in either the cell pellets or supernatants using the HILIC-UPLC method and only minor differences at the early time-points of days 1 and 3 by the lectin microarray method. In comparing different time-points, significant decreases in sialylation and branching with time were observed for glycans attached to both cell and secreted glycoproteins. Additionally, there was a significant decrease over time in high mannose type N-glycans from the cell glycoproteins. A combination of the complementary methods HILIC-UPLC and lectin microarrays could provide a powerful and rapid HTP profiling tool capable of yielding qualitative and quantitative data for a defined biopharmaceutical process, which would allow valuable near 'real-time' monitoring of the biopharmaceutical product.

Structure of the K12 capsule containing 5,7-di-N-acetylacinetaminic acid from Acinetobacter baumannii isolate D36.

The repeat unit of the K12 capsular polysaccharide isolated from the Acinetobacter baumannii global clone 1 clinical isolate, D36, was elucidated by means of chemical and spectroscopical methods. The structure was shown to contain N-acetyl-D-galactosamine (D-GalpNAc), N-acetyl-D-fucosamine and N-acetyl-L-fucosamine linked together in the main chain, with the novel sugar, 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-altro-non-2-ulosonic acid (5,7-di-N-acetylacinetaminic acid or Aci5Ac7Ac), attached to D-GalpNAc as a side branch. This matched the sugar composition of the K12 capsule and the genetic content of the KL12 capsule gene cluster reported previously. D-FucpNAc was predicted to be the substrate for the initiating transferase, ItrB3, with the Wzy polymerase making a α-D-FucpNAc-(1 → 3)-D-GalpNAc linkage between the repeat units. The three glycosyltransferases encoded by KL12 are all retaining glycosyltransferases and were predicted to form specific linkages between the sugars in the K12 repeat unit.

Phosphoneurofilament heavy chain and N-glycomics from the cerebrospinal fluid in amyotrophic lateral sclerosis.

BACKGROUND: Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease of the motor neuron for which no clinically validated biomarkers have been identified. METHODS: We have quantified by ELISA the biomarker phosphoneurofilament heavy chain (pNFH) in the cerebrospinal fluid (CSF) of ALS patients (n=29) and age-matched control patients with other diseases (n=19) by ELISA. Furthermore, we compared protein N-glycosylation of the CSF in ALS patients and controls, by applying a glycomics approach based on liquid chromatography and mass spectrometry. RESULTS: pNFH levels were significantly higher in ALS patients in comparison with controls (P<0.0001) in particular in fast progressors. The N-glycans found in the CSF were predominantly complex diantennary with sialic acid in α2,3- and α2,6-linkage, and bisecting N-acetylglucosamine-containing structures as well as peripherally fucosylated structures were found. As compared with controls the ALS group had a significant increase of a peak composed of the monosialylated diantennary glycans A2G2S(6)1 and FA2G2S(3)1 (P=0.0348). CONCLUSIONS: Our results underscore the value of pNFH as a biomarker in ALS. In addition, we identified a variation of the N-glycosylation pattern in ALS, suggesting that this change should be explored in future studies as potential biomarker.

Therapeutic antibody glycosylation analysis: a contract research organization perspective in the frame of batch release or comparability support.

Glycosylation of the Fc moiety of a monoclonal antibody is a heterogeneous posttranslational process considered as a critical quality attribute of the purified drug substance due to its major impact on safety and efficacy (i.e., immunogenicity, CDC or ADCC effector functions, etc.). Glycosylation should thus be addressed for batch-to-batch comparability and for drug substance characterization, in terms of identity and/or purity testing. We present below a set of efficient, performing and complementary analytical tests that can be used alone or in combination, depending on the information needed and available laboratory instrumentation. The results obtained using these techniques for "global" glycosylation profile, N-glycans profiling, monosaccharides, and sialic acids determination are presented for the Trastuzumab (Herceptin)-humanized mAb produced in CHO.

Capillary electrophoresis-mass spectrometry of carbohydrates.

The development of methods for capillary electrophoresis (CE) with on-line mass spectrometric detection (CE/MS) is driven by the need for accurate, robust, and sensitive glycomics analysis for basic biomedicine, biomarker discovery, and analysis of recombinant protein therapeutics. One important capability is to profile glycan mixtures with respect to the patterns of substituents including sialic acids, acetate, sulfate, phosphate, and other groups. There is additional need for an MS-compatible separation system capable of resolving carbohydrate isomers. This chapter summarizes applications of CS/MS to analysis of carbohydrates, glycoproteins, and glycopeptides that have appeared since 2008. Readers are referred to recent comprehensive reviews covering earlier publications.

Ultrasound-assisted hydrolysis and chemical derivatization combined to lab-on-valve solid-phase extraction for the determination of sialic acids in human biofluids by µ-liquid chromatography-laser induced fluorescence.

The determination of sialic acids (SIAs) has recently gained interest because of their potential role as markers of inflammatory disorders or chronic diseases. Hydrolysis of conjugated derivatives, solid-phase extraction (SPE) and derivatization steps constitute sample preparation prior to insertion of the analytical sample into a µ-liquid chromatograph-laser induced fluorescence (µ-LC-LIF) detector in the present method for the determination of two representative SIAs of human metabolism. Ultrasound-accelerated hydrolysis released free SIAs, which were efficiently concentrated in a dynamic manner using a lab-on-valve (LOV) module that allows automation of SPE for preconcentration and cleanup. This step was on-line connected with DMB-labeling of SIAs (derivatization), which was shortened from 180 min required with the conventional heating method to 20min with ultrasound assistance. Individual separation of the target analytes was achieved within 20 min by µ-LC, while LIF detection endowed the overall method with high sensitivity. The LODs and LOQs provided by the method ranged 0.1-0.8 ng mL(-1) and 0.4-1.0 ng mL(-1) (between 0.1-0.8 pg and 0.4-1.0 pg expressed as on-column amount), respectively. High efficiency for interferents removal by SPE enabled the application of the method to four different biofluids-serum, urine, saliva and breast milk-for the determination of the target metabolites.

Occurrence of free deaminoneuraminic acid (KDN)-containing complex-type N-glycans in human prostate cancers.

We previously reported on the accumulation of a substantial amount of free N-acetylneuraminic acid (Neu5Ac)-containing complex-type N-glycans in human pancreatic cancer cells (Yabu M, Korekane H, Takahashi H, Ohigashi H, Ishikawa O, Miyamoto Y. 2013. Accumulation of free Neu5Ac-containing complex-type N-glycans in human pancreatic cancers. Glycoconj J, 30(3):247-256). In the present paper, we further extend our cancer glycomic study of human prostate cancer. Specifically, we demonstrate that, in addition to the free Neu5Ac-containing N-glycans, significant amounts of free deaminoneuraminic acid (KDN, 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid)-containing N-glycans had accumulated in the prostate cancer tissues from four of five patients. Indeed, in one of the four cases, the free KDN glycans accumulated as major components in prostate cancer tissue. The structures of the KDN-containing free oligosaccharides were analyzed by a variety of methods. Specifically, we used fluorescent labeling with aminopyridine combined with two-dimensional mapping, KDNase digestion and mass spectrometry to facilitate identification. The analysis also utilized newly synthesized KDN-linked oligosaccharides as standards. The prostate-specific glycans were composed of five species having the following sequence, KDN-Gal-GlcNAc-Man-Man-GlcNAc (α2,6-KDN-linked glycans being the dominant form). The most abundant free KDN-containing N-glycan was KDNα2-6Galß1-4GlcNAcß1-2Manα1-3Manß1-4GlcNAc followed by KDNα2-6Galß1-4GlcNAcß1-2Manα1-6Manß1-4GlcNAc. This is the first study to show unequivocal chemical evidence for the occurrence of KDN glycoconjugates in human tissues together with their detailed structures. These oligosaccharides might be developed as tumor markers, especially for prostate cancer.

Sialome analysis of the cephalochordate Branchiostoma belcheri, a key organism for vertebrate evolution.

Sialic acid, a common terminal substitution of glycoconjugates, has been so far consistently identified in all vertebrates as well as in a growing number of bacterial species. It is assumed to be widely distributed among animal species of the deuterostome phylum, based on its identification in few echinoderm and all vertebrate species. However, whole sections of deuterostome, especially those intermediate species between invertebrates and vertebrates including cephalochordates, urochordates and hemichordates, are still unexplored in term of sialylation capacities. The discovery of functional sialic acid machinery in some of these species may shed new light onto the evolution of glycosylation capacities in deuterostome lineage. In a first approach, we investigated the sialylation pattern of a cephalocordate species, Branchiostoma belcheri, which occupies a strategic phylogenetic position to understand the transition of invertebrates toward vertebrates. Structural analysis of B. belcheri glycoconjugates established that this organism synthesizes large quantities of various sialic acids, some of which present rare or novel structures such as methylated sialic acids. These sialic acids were shown to be mainly associated with mono- and disialylated core 1-type O-glycans. Moreover, screening of the animal organs revealed the existence of exquisite tissue specificity in the distribution of sialic acids. Description of sialylation profiles was then correlated with the expression patterns of key enzymes involved in the biosynthesis of major forms of sialic acids, which provides the first complete overview of the sialylation patterns in cephalochordates.

Titanium preferential binding sites in human serum transferrin at physiological concentrations.

Serum transferrin (Tf) is an iron binding glycoprotein that plays a central role in the metabolism of this essential metal but it also binds other metal ions. Four main transferrin forms containing different iron binding states can be distinguished in human serum samples: monoferric (C-site or N-site), holotransferrin (with two Fe atoms) and apotransferrin (with no metal). Recently, it has been reported that Tf binds also Ti even more tightly than does Fe, in artificially Ti(iv) spiked solutions. However, very limited work has been done on the Ti binding to Tf at physiological concentrations in patients carrying intramedullary Ti nails. Here we report the chemical association of Ti to Tf "in vivo" under different chromatographic conditions by elemental mass spectrometry using double focusing inductively coupled plasma (DF-ICP-MS) as detector. For the separation of the Ti/Fe-Tf forms different gradient conditions have been explored. The observed results reveal that human serum Ti (from patients carrying intramedullary Ti nails) is uniquely associated to the N-lobe of Tf. The investigation of the influence of sialic acid in the carbohydrate chain of human serum Tf, studied by incubating the protein with neuraminidase (sialidase) to obtain the monosialilated species, revealed that the binding affinity of Ti was similar for monosialo-Tf and for native-Tf and occurs in the N-lobe. These results suggest that the species Fe(C)Ti(N)-TF might provide a route for Ti entry into cells via the transferrin receptors after the release of the metal from its implants.

Lectin affinity chromatography of articular cartilage fibromodulin: Some molecules have keratan sulphate chains exclusively capped by α(2-3)-linked sialic acid.

Fibromodulin from bovine articular cartilage has been subjected to lectin affinity chromatography by Sambucus nigra lectin which binds α(2-6)- linked N-acetylneuraminic acid, and the structure of the keratan sulphate in the binding and non-binding fractions examined by keratanase II digestion and subsequent high pH anion exchange chromatography. It has been confirmed that the keratan sulphate chains attached to fibromodulin isolated from bovine articular cartilage may have the chain terminating N-acetylneuraminic acid residue α(2-3)- or α(2-6)-linked to the adjacent galactose residue. Although the abundance of α(2-6)-linked N-acetylneuraminic acid (ca. 22%) is such that this could cap one of the four chains in almost all fibromodulin molecules, it was found that ca. 34% of the fibromodulin proteoglycan molecules from bovine articular cartilage were capped exclusively with α(2-3)-linked N-acetylneuraminic acid. The remainder of the fibromodulin proteoglycans, which bound to the lectin had a mixture of α(2-3)- and α(2-6)-linked N-acetylneuraminic acid capping structures. The keratan sulphates attached to fibromodulin molecules capped exclusively with α(2-3)- linked N-acetylneuraminic acid were found to have a higher level of galactose sulphation than those from fibromodulin with both α(2-3)- and α(2-6)-linked N-acetylneuraminic acid caps, which bound to the Sambucus nigra lectin. In addition, both pools contained chains of similar length (ca. 8-9 disaccharides). Both also contained α(1-3)-linked fucose, showing that this feature does not co-distribute with α(2-6)-linked N-acetylneuraminic acid, although these two features are present only in mature articular cartilage. These data show that there are discrete populations of fibromodulin within articular cartilage, which may have differing impacts upon tissue processes.

Neutral and acidic oligosaccharides in Holstein-Friesian colostrum during the first 3 days of lactation measured by high performance liquid chromatography on a microfluidic chip and time-of-flight mass spectrometry.

Oligosaccharides (OS) from bovine milk are a class of bioactive molecules that are receiving increasing commercial attention for their potential health benefits. In the present work we measured, comprehensively and systematically, free milk OS in the colostrum of 7 Holstein-Friesian cows during the first 3 d of lactation in 12-h intervals by HPLC-chip/time-of-flight mass spectrometry to determine the biological variation of free milk OS in early lactation. The high sensitivity and resolution of the analytical technique made it possible to monitor all OS species, thus providing a comprehensive and quantitative analysis of OS variations during colostrum production. This study confirmed that although sialyllactose is the major OS in bovine colostrum, several neutral OS species are present in significant abundance even at the third day of lactation. Furthermore, variation in terms of OS species and relative abundances of OS between cows suggest individual animal variation. These variations are likely due to genetic factors because environmental factors such as nutrition, lactation number, and accommodation were the same for all cows. This investigation revealed that colostrum milk from Holstein-Friesian cows is a rich source of neutral and acidic OS for the food and pharmaceutical industries.