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Background: Prior studies suggested that glycans were differentially expressed in patients with ovarian cancer and controls. We hypothesized that glycan-based biomarkers might serve as a diagnostic test for ovarian cancer and evaluated the ability of glycans to distinguish ovarian cancer cases from matched controls., Methods: Serum samples were obtained from the tissue-banking repository of the Gynecologic Oncology Group, and included healthy female controls (n = 100), women diagnosed with low malignant potential (LMP) tumors (n = 52), and epithelial ovarian cancers (EOC) cases (n = 147). Cases and controls were matched on age at enrollment within ±5 years. Serum samples were analyzed by glycomics analysis to detect abundance differences in glycan expression levels. A two-stage procedure was carried out for biomarker discovery and validation. Candidate classifiers of glycans that separated cases from controls were developed using a training set in the discovery phase and the classification performance of the candidate classifiers was assessed using independent test samples that were not used in discovery., Results: The patterns of glycans showed discriminatory power for distinguishing EOC and LMP cases from controls. Candidate glycan-based biomarkers developed on a training set (sensitivity, 86% and specificity, 95.8% for distinguishing EOC from controls through leave-one-out cross-validation) confirmed their potential use as a detection test using an independent test set (sensitivity, 70% and specificity, 86.5%)., Conclusion: Formal investigations of glycan biomarkers that distinguish cases and controls show great promise for an ovarian cancer diagnostic test. Further validation of a glycan-based test for detection of ovarian cancer is warranted., Impact: An emerging diagnostic test based on the knowledge gained from understanding the glycobiology should lead to an assay that improves sensitivity and specificity and allows for early detection of ovarian cancer.

An absolute quantitation method for measuring free human milk oligosaccharides (HMOs) in milk samples was developed using multiple reaction monitoring (MRM). To obtain the best sensitivity, the instrument conditions were optimized to reduce the source and postsource fragmentation prior to the quadrupole transmission. Fragmentation spectra of HMOs using collision-induced dissociation were studied to obtain the best characteristic fragments. At least two MRM transitions were used to quantify and identify each structure in the same run. The fragment ions corresponded to the production of singly charged mono-, di-, and trisaccharide fragments. The sensitivity and accuracy of the quantitation using MRM were determined, with the detection limit in the femtomole level and the calibration range spanning over 5 orders of magnitude. Seven commercial HMO standards were used to create calibration curves and were used to determine a universal response for all HMOs. The universal response factor was used to estimate absolute amounts of other structures and the total oligosaccharide content in milk. The quantitation method was applied to 20 human milk samples to determine the variations in HMO concentrations from women classified as secretors and nonsecretors, a phenotype that can be identified by the concentration of 2'-fucosylation in their milk.

Objective: The aim of the study was to determine the impact of increasing doses of 2 prebiotic oligosaccharides and of an "all-human diet" on the intestinal microbiota of premature infants., Methods: Twelve premature infants receiving formula feedings were randomly assigned to receive either galacto-oligosaccharide (F+GOS) or a pooled concentrated donor human milk product containing human milk oligosaccharides (F+HMO) in increasing doses during a 5-week period. A second group of 15 premature infants received their mother's own milk fortified with either a concentrated donor human milk product (H+H) or a bovine powdered fortifier (H+B). Serial stool specimens from each infant were analyzed by terminal restriction fragment length polymorphism and quantitative polymerase chain reaction for bacterial composition., Results: All of the infants studied had relatively low levels of bifidobacteria and no measurable Lactobacilli. Infants from the F+GOS and F+HMO groups demonstrated an increase in relative numbers of Clostridia with increasing doses. Compared with the H+B group, the infants in the F+HMO and the H+H groups showed an unexpected trend toward an increase in γ-Proteobacteria over time/dose. Principal coordinate analyses and Shannon diversity scores were not significantly different among the 4 groups. Infants in the H+H group received more antibiotics during the study period than those in the other groups. Two of the infants receiving GOS developed feeding intolerance., Conclusions: None of the prebiotic interventions resulted in significant increases in bifidobacteria compared with baseline specimens or the H+B group; however, many of the infants did not receive the highest doses of GOS and HMO, and antibiotic use in the H+H group was high.

In clinical settings, biopsies are routinely used to determine cancer type and grade based on tumor cell morphology, as determined via histochemical or immunohistochemical staining. Unfortunately, in a significant number of cases, traditional biopsy results are either inconclusive or do not provide full subtype differentiation, possibly leading to inefficient or ineffective treatment. Glycomic profiling of the cell membrane offers an alternate route toward cancer diagnosis. In this study, isomer-sensitive nano-LC/MS was used to directly obtain detailed profiles of the different N-glycan structures present on cancer cell membranes. Membrane N-glycans were extracted from cells representing various subtypes of breast, lung, cervical, ovarian, and lymphatic cancer. Chip-based porous graphitized carbon nano-LC/MS was used to separate, identify, and quantify the native N-glycans. Structure-sensitive N-glycan profiling identified hundreds of glycan peaks per cell line, including multiple isomers for most compositions. Hierarchical clusterings based on Pearson correlation coefficients were used to quickly compare and separate each cell line according to originating organ and disease subtype. Based simply on the relative abundances of broad glycan classes (e.g., high mannose, complex/hybrid fucosylated, complex/hybrid sialylated, etc.), most cell lines were readily differentiated. More closely related cell lines were differentiated based on several-fold differences in the abundances of individual glycans. Based on characteristic N-glycan profiles, primary cancer origins and molecular subtypes could be distinguished. These results demonstrate that stark differences in cancer cell membrane glycosylation can be exploited to create an MS-based biopsy, with potential applications toward cancer diagnosis and direction of treatment.

Many of the functional proteins and lipids in high density lipoprotein (HDL) particles are potentially glycosylated, yet very little is known about the glycoconjugates of HDL. In this study, HDL was isolated from plasma by sequential micro-ultracentrifugation, followed by glycoprotein and glycolipid analysis. N-Glycans, glycopeptides, and gangliosides were extracted and purified followed by analysis with nano-HPLC Chip quadrupole time of flight mass spectrometry and MS/MS. HDL particles were found to be highly sialylated. Most of the N-glycans (∼90%) from HDL glycoproteins were sialylated with one or two neuraminic acids (Neu5Ac). The most abundant N-glycan was a biantennary complex type glycan with two sialic acids (Hexose5HexNAc4Neu5Ac2) and was found in multiple glycoproteins using site-specific glycosylation analysis. The observed O-glycans were all sialylated, and most contained a core 1 structure with two Neu5Acs, including those that were associated with apolipoprotein CIII (ApoC-III) and fetuin A. GM3 (monosialoganglioside, NeuAc2-3Gal1-4Glc-Cer) and GD3 (disialoganglioside, NeuAc2-8NeuAc2-3Gal1-4Glc-Cer) were the major gangliosides in HDL. A 60% GM3 and 40% GD3 distribution was observed. Both GM3 and GD3 were composed of heterogeneous ceramide lipid tails, including d18:1/16:0 and d18:1/23:0. This report describes for the first time a glycomic approach for analyzing HDL, highlighting that HDL are highly sialylated particles.

Glycomics, a comprehensive study of glycans expressed in biologic systems, is emerging as a simple yet highly sensitive diagnostic tool for disease onset and progression. This study aimed to use glycomics to investigate glycan markers that would differentiate patients with gastric cancer from those with nonatrophic gastritis. Patients with duodenal ulcer were also included because they are thought to represent a biologically different response to infection with Helicobacter pylori, a bacterial infection that can cause either gastric cancer or duodenal ulcer. We collected 72 serum samples from patients in Mexico City that presented with nonatrophic gastritis, duodenal ulcer, or gastric cancer. N-glycans were released from serum samples using the generic method with PNGase F and were analyzed by matrix-assisted laser desorption/ionization Fourier transform-ion cyclotron resonance mass spectrometry. The corresponding glycan compositions were calculated based on accurate mass. ANOVA-based statistical analysis was performed to identify potential markers for each subgroup. Nineteen glycans were significantly different among the diagnostic groups. Generally, decreased levels of high-mannose-type glycans, glycans with one complex type antenna, bigalactosylated biantennary glycans, and increased levels of nongalactosylated biantennary glycans were observed in gastric cancer cases. Altered levels of serum glycans were also observed in duodenal ulcer, but differences were generally in the same direction as gastric cancer. Serum glycan profiles may provide biomarkers to differentiate gastric cancer cases from controls with nonatrophic gastritis. Further studies will be needed to validate these findings as biomarkers and identify the role of protein glycosylation in gastric cancer pathology.

Bovine milk is known to contain naturally occurring peptides, but relatively few of their sequences have been determined. Human milk contains hundreds of endogenous peptides, and the ensemble has been documented for antimicrobial actions. Naturally occurring peptides from bovine milk were sequenced and compared with human milk peptides. Bovine milk samples from six cows in second-stage peak lactation at 78-121 days postpartum revealed 159 peptides. Most peptides (73%) were found in all six cows sampled, demonstrating the similarity of the intramammary peptide degradation across these cows. One peptide sequence, ALPIIQKLEPQIA from bovine perilipin 2, was identical to another found in human milk. Most peptides derived from β-casein, αs1-casein, and αs2-casein. No peptides derived from abundant bovine milk proteins such as lactoferrin, β-lactoglobulin, and secretory immunoglobulin A. The enzymatic cleavage analysis revealed that milk proteins were degraded by plasmin, cathepsins B and D, and elastase in all samples.

In addition to providing complete postnatal nutrition, breast milk is a complex biofluid that delivers bioactive components for the growth and development of the intestinal and immune systems. Lactation is a unique opportunity to understand the role of diet in shaping the intestinal environment including the infant microbiome. Of considerable interest is the diversity and abundance of milk glycans that are energetically costly for the mammary gland to produce yet indigestible by infants. Milk glycans comprise free oligosaccharides, glycoproteins, glycopeptides, and glycolipids. Emerging technological advances are enabling more comprehensive, sensitive, and rapid analyses of these different classes of milk glycans. Understanding the impact of inter- and intraindividual glycan diversity on function is an important step toward interventions aimed at improving health and preventing disease. This review discusses the state of technology for glycan analysis and how specific structure-function knowledge is enhancing our understanding of early nutrition in the neonate.

Prebiotics are nondigestible substrates that stimulate the growth of beneficial microbes in the human intestine. Galactooligosaccharides (GOS) are food ingredients that possess prebiotic properties, in particular, promoting the growth of bifidobacteria in situ. However, precise mechanistic details of GOS consumption by bifidobacteria remain poorly understood. Because GOS are mixtures of polymers of different lengths and linkages, there is interest in determining which specific structures provide prebiotic effects to potentially create better supplements. This paper presents a method comprising porous graphitic carbon separation, isotopic labeling, and mass spectrometry analysis for the structure-specific analysis of GOS isomers and their bacterial consumption rate. Using this strategy, the differential bacterial consumption of GOS by the bifidobacteria species Bifidobacterium longum subsp. infantis, Bifidobacterium animalis subsp. lactis, and Bifidobacterium adolescentis was determined, indicating that the use of specific GOS isomers in infant formula may provide enrichment of distinct species.

Three complementary experimental approaches for elucidating human milk oligosaccharide (HMOs) isomers by Fourier Transform Ion Cyclotron Resonance mass spectrometry (FT-ICR) are described: tandem-MS disruption by double resonance to distinguish different fragmentation pathways, examination of fragment intensity ratios arising from differential alkali metal ion affinities and monitoring competitive fragmentation rates. The interpretation of the fragmentation pattern from a mechanistic and thermochemical point of view permits the assignment of not only pure isomers but, in some cases, mixtures of them. Methodologically the procedures are simple, reliable and rapid making unnecessary both the use of previous separation techniques and tedious chemical modifications of the HMOs. In principle, the rationale can be expanded to resolve other isomeric mixtures of biological nature.

Holothurians are sedentary marine organisms known to produce saponins (triterpene glycosides), secondary metabolites exhibiting a wide range of biological activities. In this paper, we investigated the saponin contents of semi-purified and membranolytic HPLC fractionated extracts from the body wall of three species of Holothuriidae as an attempt to examine its chemical diversity in relation to phylogenetic data. MALDI-FTICR MS and nano-HPLC-chip Q-TOF MS were used for mass profiling and isomer separation, respectively giving a unique chemical saponin fingerprint. Moreover, the methods used yield the highest number of congeners. However, saponin concentration, bioactivity and chemical diversity had no apparent relationship. MS fingerprint showed the presence of holothurinosides, which was observed for the first time in other Holothuria genera besides the basally positioned Holothuria forskali. This congener is proposed to be a primitive character that could be used for taxonomic purposes. The phylogenetic mapping also showed that the glycone part of the compound evolved from non-sulfated hexaosides to sulfated tetraosides, which have higher membranolytic activity and hydrophilicity, the two factors affecting the total ecological activity (i.e. chemical defense) of these compounds. This might be an adaptation to increase the fitness of the organism., (© 2013 Elsevier Inc. All rights reserved.)

Milk gangliosides have gained considerable attention because they participate in diverse biological processes, including neural development, pathogen binding, and activation of the immune system. Herein, we present a quantitative measurement of the gangliosides present in bovine milk and other dairy products and byproducts. Ultrahigh performance liquid chromatography separation was used for high-throughput analysis and achieved a short running time without sacrificing chromatographic resolution. Dynamic multiple reaction monitoring was conducted for 12 transitions for GM3 and 12 transitions for GD3. Transitions to sialic acid fragments (m/z 290.1) were chosen for the quantitation. There was a considerable amount of gangliosides in day 2 milk (GM3, 0.98 mg/L; GD3, 15.2 mg/L) which dramatically decreased at day 15 and day 90. GM3 and GD3 were also analyzed in pooled colostrum, colostrum cream, colostrum butter, and colostrum buttermilk. The separation and analytical approaches here proposed could be integrated into the dairy industry processing adding value to side-streams.

Despite recent advances, site-specific profiling of protein glycosylation remains a significant analytical challenge for conventional proteomic methodology. To alleviate the issue, we propose glyco-analytical multispecific proteolysis (Glyco-AMP) as a strategy for glycoproteomic characterization. Glyco-AMP consists of rapid, in-solution digestion of an analyte glycoprotein (or glycoprotein mixture) by a multispecific protease (or protease cocktail). Resulting glycopeptides are chromatographically separated by isomer-specific porous graphitized carbon nano-LC, quantified by high-resolution MS, and structurally elucidated by MS/MS. To demonstrate the consistency and customizability of Glyco-AMP methodology, the glyco-analytical performances of multispecific proteases subtilisin, pronase, and proteinase K were characterized in terms of quantitative accuracy, sensitivity, and digestion kinetics. Glyco-AMP was shown be effective on glycoprotein mixtures as well as glycoproteins with multiple glycosylation sites, providing detailed, quantitative, site- and structure-specific information about protein glycosylation.

Human milk contains a high concentration of complex oligosaccharides that influence the composition of the intestinal microbiota in breast-fed infants. Previous studies have indicated that select species such as Bifidobacterium longum subsp. infantis and Bifidobacterium bifidum can utilize human milk oligosaccharides (HMO) in vitro as the sole carbon source, while the relatively few B. longum subsp. longum and Bifidobacterium breve isolates tested appear less adapted to these substrates. Considering the high frequency at which B. breve is isolated from breast-fed infant feces, we postulated that some B. breve strains can more vigorously consume HMO and thus are enriched in the breast-fed infant gastrointestinal tract. To examine this, a number of B. breve isolates from breast-fed infant feces were characterized for the presence of different glycosyl hydrolases that participate in HMO utilization, as well as by their ability to grow on HMO or specific HMO species such as lacto-N-tetraose (LNT) and fucosyllactose. All B. breve strains showed high levels of growth on LNT and lacto-N-neotetraose (LNnT), and, in general, growth on total HMO was moderate for most of the strains, with several strain differences. Growth and consumption of fucosylated HMO were strain dependent, mostly in isolates possessing a glycosyl hydrolase family 29 α-fucosidase. Glycoprofiling of the spent supernatant after HMO fermentation by select strains revealed that all B. breve strains can utilize sialylated HMO to a certain extent, especially sialyl-lacto-N-tetraose. Interestingly, this specific oligosaccharide was depleted before neutral LNT by strain SC95. In aggregate, this work indicates that the HMO consumption phenotype in B. breve is variable; however, some strains display specific adaptations to these substrates, enabling more vigorous consumption of fucosylated and sialylated HMO. These results provide a rationale for the predominance of this species in breast-fed infant feces and contribute to a more accurate picture of the ecology of the developing infant intestinal microbiota.

Purpose: There is a need to identify better glycan biomarkers for diagnosis, early detection, and treatment monitoring in lung cancer using biofluids such as blood. Biofluids are complex mixtures of proteins dominated by a few high abundance proteins that may not have specificity for lung cancer. Therefore, two methods for protein enrichment were evaluated; affinity capturing of IgG and enrichment of medium abundance proteins, thus allowing us to determine which method yields the best candidate glycan biomarkers for lung cancer., Experimental Design: N-glycans isolated from plasma samples from 20 cases of lung adenocarcinoma and 20 matched controls were analyzed using nLC-PGC-chip-TOF-MS (where PGC is porous-graphitized carbon). N-glycan profiles were obtained for five different fractions: total plasma, isolated IgG, IgG-depleted plasma, and the bound and flow-through fractions of protein enrichment., Results: Four glycans differed significantly (false discovery rate, FDR < 0.05) between cases and controls in whole unfractionated plasma, while four other glycans differed significantly by cancer status in the IgG fraction. No significant glycan differences were observed in the other fractions., Conclusions and Clinical Relevance: These results confirm that the N-glycan profile in plasma of lung cancer patients is different from healthy controls and appears to be dominated by alterations in relatively abundant proteins., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Studies aimed toward glycan biomarker discovery have focused on glycan characterization by the global profiling of released glycans. Site-specific glycosylation analysis is less developed but may provide new types of biomarkers with higher sensitivity and specificity. Quantitation of peptide-conjugated glycans directly facilitates the differential analysis of distinct glycoforms associated with specific proteins at distinct sites. We have developed a method using MRM to monitor protein glycosylation normalized to absolute protein concentrations to examine quantitative changes in glycosylation at a site-specific level. This new approach provides information regarding both the absolute amount of protein and the site-specific glycosylation profile and will thus be useful to determine if altered glycosylation profiles in serum/plasma are due to a change in protein glycosylation or a change in protein concentration. The remarkable sensitivity and selectivity of MRM enable the detection of low abundance IgG glycopeptides, even when IgG was digested directly in serum with no cleanup prior to the liquid chromatography. Our results show a low limit of detection of 60 amol and a wide dynamic range of 3 orders magnitude for IgG protein quantitation. The results show that IgG glycopeptides can be analyzed directly from serum (without enrichment) and yield more accurate abundances when normalized to the protein content. This report represents the most comprehensive study so far of the use of multiple reaction monitoring for the quantitation of glycoproteins and their glycosylation patterns in biofluids.

Site-specific glycosylation (SSG) of glycoproteins remains a considerable challenge and limits further progress in the areas of proteomics and glycomics. Effective methods require new approaches in sample preparation, detection, and data analysis. While the field has advanced in sample preparation and detection, automated data analysis remains an important goal. A new bioinformatics approach implemented in software called GP Finder automatically distinguishes correct assignments from random matches and complements experimental techniques that are optimal for glycopeptides, including nonspecific proteolysis and high mass resolution liquid chromatography/tandem mass spectrometry (LC/MS/MS). SSG for multiple N- and O-glycosylation sites, including extensive glycan heterogeneity, was annotated for single proteins and protein mixtures with a 5% false-discovery rate, generating hundreds of nonrandom glycopeptide matches and demonstrating the proof-of-concept for a self-consistency scoring algorithm shown to be compliant with the target-decoy approach (TDA). The approach was further applied to a mixture of N-glycoproteins from unprocessed human milk and O-glycoproteins from very-low-density-lipoprotein (vLDL) particles.

Bovine milk oligosaccharides (BMOs) are recognized by the dairy and food industries, as well as by infant formula manufacturers, as novel, high-potential bioactive food ingredients. Recent studies revealed that bovine milk contains complex oligosaccharides structurally related to those previously thought to be present in only human milk. These BMOs are microbiotic modulators involved in important biological activities, including preventing pathogen binding to the intestinal epithelium and serving as nutrients for a selected class of beneficial bacteria. Only a small number of BMO structures are fully elucidated. To better understand the potential of BMOs as a class of biotherapeutics, their detailed structure analysis is needed. This study initiated the development of a structure library of BMOs and a comprehensive evaluation of structure-related specificity. The bovine milk glycome was profiled by high-performance mass spectrometry and advanced separation techniques to obtain a comprehensive catalog of BMOs, including several novel, lower abundant neutral and fucosylated oligosaccharides that are often overlooked during analysis. Structures were identified using isomer-specific tandem mass spectroscopy and targeted exoglycosidase digestions to produce a BMO library detailing retention time, accurate mass and structure to allow their rapid identification in future studies.

Mice are the premier mammalian models for studies of human physiology and disease, bearing extensive biological similarity to humans with far fewer ethical, economic, or logistic complications. To facilitate glycomic studies based on the mouse model, we comprehensively profiled the mouse serum N-glycome using isomer-specific nano-LC/MS and -LC/MS/MS. N-Glycans were identified by accurate mass MS and structurally elucidated by MS/MS. Porous graphitized carbon nano-LC was able to separate out nearly 300 N-linked glycan compounds (including isomers) from just over 100 distinct N-linked glycan compositions. Additional MS/MS structural analysis was performed on a number of novel N-glycans, revealing the structural characteristics of modifications such as dehydration, O-acetylation, and lactylation. Experimental findings were combined with known glycobiology to generate a theoretical library of all biologically possible mouse serum N-glycan compositions. The library may be used for automated identification of complex mixtures of mouse N-glycans, with possible applications to a wide range of mouse-related research endeavors, including pharmaceutical drug development and biomarker discovery.

Milk is traditionally considered an ideal source of the basic elemental nutrients required by infants. More detailed examination is revealing that milk represents a more functional ensemble of components with benefits to both infants and mothers. A comprehensive peptidomics method was developed and used to analyze human milk yielding an extensive array of protein products present in the fluid. Over 300 milk peptides were identified originating from major and many minor protein components of milk. As expected, the majority of peptides derived from β-casein, however no peptide fragments from the major milk proteins lactoferrin, α-lactalbumin, and secretory immunoglobulin A were identified. Proteolysis in the mammary gland is selective-released peptides were drawn only from specific proteins and typically from only select parts of the parent sequence. A large number of the peptides showed significant sequence overlap with peptides with known antimicrobial or immunomodulatory functions. Antibacterial assays showed the milk peptide mixtures inhibited the growth of Escherichia coli and Staphylococcus aureus . The predigestion of milk proteins and the consequent release of antibacterial peptides may provide a selective advantage through evolution by protecting both the mother's mammary gland and her nursing offspring from infection.

Human milk oligosaccharides (HMOs), though non-nutritive to the infant, shape the intestinal microbiota and protect against pathogens during early growth and development. Infant formulas with added galacto-oligosaccharides have been developed to mimic the beneficial effects of HMOs. Premature infants have an immature immune system and a leaky gut and are thus highly susceptible to opportunistic infections. A method employing nanoflow liquid chromatography time-of-flight mass spectrometry (MS) is presented to simultaneously identify and quantify HMOs in the feces and urine of infants, of which 75 HMOs have previously been fully structurally elucidated. Matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance MS was employed for high-resolution and rapid compositional profiling. To demonstrate this novel method, samples from mother-infant dyads as well as samples from infants receiving infant formula fortified with dietary galacto-oligosaccharides or probiotic bifidobacteria were analyzed. Ingested oligosaccharides are demonstrated in high abundance in the infant feces and urine. While the method was developed to examine specimens from preterm infants, it is of general utility and can be used to monitor oligosaccharide consumption and utilization in term infants, children, and adults. This method may therefore provide diagnostic and therapeutic opportunities.

Many studies focused on the discovery of novel biomarkers for the diagnosis and treatment of disease states are facilitated by mass spectrometry-based technology. HPLC coupled to mass spectrometry is widely used; miniaturization of this technique using nano-liquid chromatography (LC)-mass spectrometry (MS) usually results in better sensitivity, but is associated with limited repeatability. The recent introduction of chip-based technology has significantly improved the stability of nano-LC-MS, but no substantial studies to verify this have been performed. To evaluate the temporal repeatability of chip-based nano-LC-MS analyses, N-glycans released from a serum sample were repeatedly analyzed using nLC-PGC-chip-TOF-MS on three non-consecutive days. With an average inter-day coefficient of variation of 4 %, determined on log10-transformed integrals, the repeatability of the system is very high. Overall, chip-based nano-LC-MS appears to be a highly stable technology, which is suitable for the profiling of large numbers of clinical samples for biomarker discovery.

Changes in glycosylation readily occur in cancer and other disease states. Thanks to recent advances in the development of analytical techniques and instrumentation, especially in mass spectrometry, it is now possible to identify blood-derived glycan-based biomarkers using glycomics strategies. This review is an overview of the developments made in the search for glycan-based cancer biomarkers and the technologies currently in use. It is anticipated that the progressing instrumental and bioinformatics developments will allow the identification of relevant glycan biomarkers for the diagnosis, early detection, and monitoring of cancer treatment with sufficient sensitivity and specificity for clinical use.

O-Glycans in saliva and tear isolated from patients suffering from ocular rosacea, a form of inflammatory ocular surface disease, were profiled, and their structures were elucidated using high resolution mass spectrometry. We have previously shown that certain structures, particularly sulfated oligosaccharides, increased in the tear and saliva of rosacea patients. In this study, the structures of these glycans were elucidated using primarily tandem mass spectrometry. There were important similarities in the glycan profiles of tears and saliva with the majority of the structures in common. The structures of the most abundant species common to both tear and saliva, which were also the most abundant species in both, were elucidated. For sulfated species, the positions of the sulfate groups were localized. The majority of the structures were new, with the sulfated glycans comprising mucin core 1- and core 2-type structures. As both saliva and tear are rich in mucins, it is suggested that the O-glycans are mainly components of mucins. The study further illustrates the strong correspondence between the glycans in the tear and saliva of ocular rosacea patients.

Aberrant glycosylation has been observed for decades in essentially all types of cancer, and is now well established as an indicator of carcinogenesis. Mining the glycome for biomarkers, however, requires analytical methods that can rapidly separate, identify, and quantify isomeric glycans. We have developed a rapid-throughput method for chromatographic glycan profiling using microfluidic chip-based nanoflow liquid chromatography (nano-LC)/mass spectrometry. To demonstrate the utility of this method, we analyzed and compared serum samples from epithelial ovarian cancer cases (n=46) and healthy control individuals (n=48). Over 250 N-linked glycan compound peaks with over 100 distinct N-linked glycan compositions were identified. Statistical testing identified 26 potential glycan biomarkers based on both compositional and structure-specific analyses. Using these results, an optimized model was created incorporating the combined abundances of seven potential glycan biomarkers. The receiver operating characteristic (ROC) curve of this optimized model had an area under the curve (AUC) of 0.96, indicating robust discrimination between cancer cases and healthy controls. Rapid-throughput chromatographic glycan profiling was found to be an effective platform for structure-specific biomarker discovery., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Determining protein-specific glycosylation in protein mixtures remains a difficult task. A common approach is to use gel electrophoresis to isolate the protein followed by glycan release from the identified band. However, gel bands are often composed of several proteins. Hence, release of glycans from specific bands often yields products not from a single protein but a composite. As an alternative, we present an approach whereby glycans are released with peptide tags allowing verification of glycans bound to specific proteins. We term the process in-gel nonspecific proteolysis for elucidating glycoproteins (INPEG). INPEG combines rapid gel separation of a protein mixture with in-gel nonspecific proteolysis of protein bands followed by tandem mass spectrometry (MS) analysis of the resulting N- and O-glycopeptides. Here, in-gel digestion is shown for the first time with nonspecific and broad specific proteases such as Pronase, proteinase K, pepsin, papain, and subtilisin. Tandem MS analysis of the resulting glycopeptides separated on a porous graphitized carbon (PGC) chip was achieved via nanoflow liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (nano-LC/Q-TOF MS). In this study, rapid and automated glycopeptide assignment was achieved via an in-house software (Glycopeptide Finder) based on a combination of accurate mass measurement, tandem MS data, and predetermined protein identification (obtained via routine shotgun analysis). INPEG is here initially validated for O-glycosylation (κ casein) and N-glycosylation (ribonuclease B). Applications of INPEG were further demonstrated for the rapid determination of detailed site-specific glycosylation of lactoferrin and transferrin following gel separation and INPEG analysis on crude bovine milk and human serum, respectively.

Bifidobacterium longum subsp. infantis is a common member of the intestinal microbiota in breast-fed infants and capable of metabolizing human milk oligosaccharides (HMO). To investigate the bacterial response to different prebiotics, we analyzed both cell wall associated and whole cell proteins in B. infantis. Proteins were identified by LC-MS/MS followed by comparative proteomics to deduce the protein localization within the cell. Enzymes involved in the metabolism of lactose, glucose, galactooligosaccharides, fructooligosaccharides and HMO were constitutively expressed exhibiting less than two-fold change regardless of the sugar used. In contrast, enzymes in N-Acetylglucosamine and sucrose catabolism were induced by HMO and fructans, respectively. Galactose-metabolizing enzymes phosphoglucomutase, UDP-glucose 4-epimerase and UTP glucose-1-P uridylytransferase were expressed constitutively, while galactokinase and galactose-1-phosphate uridylyltransferase, increased their expression three fold when HMO and lactose were used as substrates for cell growth. Cell wall-associated proteomics also revealed ATP-dependent sugar transport systems associated with consumption of different prebiotics. In addition, the expression of 16 glycosyl hydrolases revealed the complete metabolic route for each substrate. Mucin, which possesses O-glycans that are structurally similar to HMO did not induced the expression of transport proteins, hydrolysis or sugar metabolic pathway indicating B. infantis do not utilize these glycoconjugates.

Mass spectrometric analysis of peptides contained in enzymatically digested hydrolysates of proteins is increasingly being used to characterize potentially bioactive or otherwise interesting hydrolysates. However, when preparations containing mixtures of enzymes are used, from either biological or experimental sources, it is unclear which of these enzymes have been most important in hydrolyzing the sample. We have developed a tool to rapidly evaluate the evidence for which enzymes are most likely to have cleaved the sample. EnzymePredictor, a web-based software, has been developed to (i) identify the protein sources of fragments found in the hydrolysates and map them back on it, (ii) identify enzymes that could yield such cleavages, and (iii) generate a colored visualization of the hydrolysate, the source proteins, the fragments, and the predicted enzymes. It tabulates the enzymes ranked according to their cleavage counts. The provision of odds ratio and standard error in the table permits users to evaluate how distinctively particular enzymes may be favored over other enzymes as the most likely cleavers of the samples. Finally, the method displays the cleavage not only according to peptides, but also according to proteins, permitting evaluation of whether the cleavage pattern is general across all proteins, or specific to a subset. We illustrate the application of this method using milk hydrolysates, and show how it can rapidly identify the enzymes or enzyme combinations used in generating the peptides. The approach developed here will accelerate the identification of enzymes most likely to have been used in hydrolyzing a set of mass spectrometrically identified peptides derived from proteins. This has utility not only in understanding the results of mass spectrometry experiments, but also in choosing enzymes likely to yield similar cleavage patterns. EnzymePredictor can be found at http://bioware.ucd.ie/∼enzpred/Enzpred.php.

Human milk oligosaccharides (HMOs), as an abundant and bioactive component of breast milk, work in many ways to promote the health of breast fed infants. The expression of HMOs has been shown to vary in accordance with Lewis blood type and secretor status, as women of different blood types differ in the expression of α1,2 fucosyltransferase (FUT2) and α1,3/4 fucosyltransferase (FUT3). In this study, HMOs were extracted from the milk of 60 women from The Gambia, Africa with various Lewis and secretor blood types. The HMOs were profiled using high resolution HPLC-Chip/TOF mass spectrometry. Notably, the amounts of fucosylation varied significantly between Le(a+b-) nonsecretors, Le(a-b+) and Le(a-b-) secretors, and Le(a-b-) nonsecretors. With higher frequency of expression of the recessive Lewis negative and nonsecretor phenotypes in West African populations, the HMO profiles of several milks from women of these phenotypes were examined, demonstrating decreased amounts of total oligosaccharide abundance and lower relative amounts of fucosylation. Also in this study, four specific fucosylated structures (2'FL, LNFP I, LDFT, and LNDFH I) were determined to be specific and sensitive glycan markers for rapidly determining secretor status without the need for serological testing.

Structure-specific characterization and quantitation is often required for effective functional studies of oligosaccharides. Inside the gut, HMOs are preferentially bound and catabolized by the beneficial bacteria. HMO utility by these bacteria employs structure-specific catabolism based on a number of glycosidases. Determining the activity of these enzymes requires accurate quantitation of a large number of structures. In this study, we describe a method for the quantitation of human milk oligosaccharide (HMO) structures employing LC/MS and isotopically labeled internal standards. Data analysis was accomplished with a newly developed software tool, LC/MS Searcher, that employs a reference structure library to process LC/MS data yielding structural identification with accurate quantitation. The method was used to obtain a meta-enzyme analysis of bacteria, the simultaneous characterization of all glycosidases employed by bacteria for the catabolism of milk oligosaccharides. Analysis of consumed HMO structures confirmed the utility of a β-1,3-galactosidase in Bifidobacterium longum subsp. infantis ATCC 15697 (B. infantis). In comparison, Bifidobacterium breve ATCC 15700 showed significantly less HMO catabolic activity compared to B. infantis.

Breast milk is the ideal nutrition for term infants but must be supplemented to provide adequate growth for most premature infants. Human milk oligosaccharides (HMOs) are remarkably abundant and diverse in breast milk and yet provide no nutritive value to the infant. HMOs appear to have at least two major functions: prebiotic activity (stimulation of the growth of commensal bacteria in the gut) and protection against pathogens. Investigations of HMOs in milk from women delivering preterm have been limited. We present the first detailed mass spectrometric analysis of the fucosylation and sialylation in HMOs in serial specimens of milk from 15 women delivering preterm and 7 women delivering at term using nanohigh performance liquid chromatography chip/time-of-flight mass spectrometry. A mixed-effects model with Levene's test was used for the statistical analyses. We find that lacto-N-tetraose, a core HMO, is both more abundant and more highly variable in the milk of women delivering preterm. Furthermore, fucosylation in preterm milk is not as well regulated as in term milk, resulting in higher within and between mother variation in women delivering preterm vs term. Of particular clinical interest, the α1,2-linked fucosylated oligosaccharide 2'-fucosyllactose, an indicator of secretor status, is not consistently present across lactation of several mothers that delivered preterm. The immaturity of HMO production does not appear to resolve over the time of lactation and may have relevance to the susceptibility of premature infants to necrotizing enterocolitis, late onset sepsis, and related neurodevelopmental impairments.

A method is described for the rapid identification of oligosaccharides employing a library of tandem MS spectra. Identification is aided by software that compares the sample tandem MS to those in the library. The method incorporates quadrupole time-of-flight mass spectrometry along with an annotated oligosaccharide (OS) structure library and the MassHunter Personal Compound Database and Library (PCDL) software. With an automated spectra search, OS structures in different samples are readily identified. This method is shown to be useful in the study of milk oligosaccharides but can be readily applied to oligosaccharide pools in other biological tissues.

Breastfeeding is one of the main factors guiding the composition of the infant gut microbiota in the first months of life. This process is shaped in part by the high amounts of human milk oligosaccharides that serve as a carbon source for saccharolytic bacteria such as Bifidobacterium species. Infant-borne bifidobacteria have developed various molecular strategies for utilizing these oligosaccharides as a carbon source. We hypothesized that these species also interact with N-glycans found in host glycoproteins that are structurally similar to free oligosaccharides in human milk. Endo-β-N-acetylglucosaminidases were identified in certain isolates of Bifidobacterium longum subsp. longum, B. longum subsp. infantis, and Bifidobacterium breve, and their presence correlated with the ability of these strains to deglycosylate glycoproteins. An endoglycosidase from B. infantis ATCC 15697, EndoBI-1, was active toward all major types of N-linked glycans found in glycosylated proteins. Its activity was not affected by core fucosylation or extensive fucosylation, antenna number, or sialylation, releasing several N-glycans from human lactoferrin and immunoglobulins A and G. Extensive N-deglycosylation of whole breast milk was also observed after coincubation with this enzyme. Mutation of the active site of EndoBI-1 did not abolish binding to N-glycosylated proteins, and this mutant specifically recognized Man(3)GlcNAc(2)(α1-6Fuc), the core structure of human N-glycans. EndoBI-1 is constitutively expressed in B. infantis, and incubation of the bacterium with human or bovine lactoferrin led to the induction of genes associated to import and consumption of human milk oligosaccharides, suggesting linked regulatory mechanisms among these glycans. This work reveals an unprecedented interaction of bifidobacteria with host N-glycans and describes a novel endoglycosidase with broad specificity on diverse N-glycan types, potentially a useful tool for glycoproteomics studies.

Human serum glycomics is a promising method for finding cancer biomarkers but often lacks the tools for streamlined data analysis. The Glycolyzer software incorporates a suite of analytic tools capable of identifying informative glycan peaks out of raw mass spectrometry data. As a demonstration of its utility, the program was used to identify putative biomarkers for epithelial ovarian cancer from a human serum sample set. A randomized, blocked, and blinded experimental design was used on a discovery set consisting of 46 cases and 48 controls. Retrosynthetic glycan libraries were used for data analysis and several significant candidate glycan biomarkers were discovered via hypothesis testing. The significant glycans were attributed to a glycan family based on glycan composition relationships and incorporated into a linear classifier motif test. The motif test was then applied to the discovery set to evaluate the disease state discrimination performance. The test provided strongly predictive results based on receiver operator characteristic curve analysis. The area under the receiver operator characteristic curve was 0.93. Using the Glycolyzer software, we were able to identify a set of glycan biomarkers that highly discriminate between cases and controls, and are ready to be formally validated in subsequent studies., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Milk is an important fluid in glycobiology because it contains a number of short carbohydrate chains either free or as glycoconjugates. These compounds as a class are the most abundant component and benefit the infant by developing and maintaining the infant's gut flora. New and emerging methods for oligosaccharide analysis have been developed to study milk. These methods allow for the rapid profiling of oligosaccharide mixtures with quantitation. With these tools, the role of oligosaccharide in milk is being understood. They further point to how oligosaccharide analysis can be performed, which until now has been very difficult and have lagged significantly those of other biopolymers.

Precise profiling of polar lipids including gangliosides and sulfatides is a necessary step in understanding the diverse physiological role of these lipids. We have established an efficient method for the profiling of polar lipids using reversed-phase nano high-performance liquid chromatography microfluidic chip quadrupole time-of-flight mass spectrometry (nano-HPLC-chip Q-TOF/MS). A microfluidic chip design provides improved chromatographic performance, efficient separation, and stable nanospray while the advanced high-resolution mass spectrometer allowed for the identification of complex isobaric polar lipids such as NeuAc- and NeuGc-containing gangliosides. Lipid classes were identified based on the characteristic fragmentation product ions generated during data-dependent tandem mass spectrometry (MS/MS) experiments. Each class was monitored by a postprocessing precursor ion scan. Relatively simple quantitation and identification of intact ions was possible due to the reproducible retention times provided by the nano-HPLC chip. The method described in this paper was used to profile polar lipids from mouse brain, which was found to contain 17 gangliosides and 13 sulfatides. Types and linkages of the monosaccharides and their acetyl modifications were identified by low-energy collision-induced dissociation (CID) (40 V), and the type of sphingosine base was identified by higher energy CID (80 V). Accurate mass measurements and chromatography unveiled the degree of unsaturation and hydroxylation in the ceramide lipid tails.

The purpose of this study was to study changes in glycosylation in tear and saliva obtained from control and ocular rosacea patients in order to identify potential biomarkers for rosacea. Tear fluid was collected from 51 subjects (28 healthy controls and 23 patients with ocular rosacea). Saliva was collected from 42 of the same subjects (25 controls and 17 patients). Pooled and individual samples were examined to determine overall glycan profiles and individual variations in glycosylation. O-and N- glycans were released from both patients and control subjects. Released glycans were purified and enriched by solid-phase extraction (SPE) with graphitized carbon. Glycans were eluted based on glycan size and polarity. SPE fractions were then analyzed by high-resolution mass spectrometry. Glycan compositions were assigned by accurate masses. Their structures were further elucidated by tandem mass spectrometric using collision-induced dissociation (CID), and specific linkage information was obtained by exoglycosidase digestion. N- and O-glycans were released from 20-μL samples without protein identification, separation, and purification. Approximately 50 N-glycans and 70 O-glycans were globally profiled by mass spectrometry. Most N-glycans were highly fucosylated, while O-glycans were sulfated. Normal tear fluid and saliva contain highly fucosylated glycans. The numbers of sulfated glycans were dramatically increased in tear and saliva of rosacea patients compared to controls. Glycans found in tear and saliva from roseatic patients present highly quantitative similarity. The abundance of highly fucosylated N-glycans in the control samples and sulfated O-glycans in ocular rosacea patient samples may lead to the discovery of an objective diagnostic marker for the disease., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Free oligosaccharides are key components of human milk and play multiple roles in the health of the neonate, by stimulating growth of selected beneficial bacteria in the gut, participating in development of the brain, and exerting antipathogenic activity. However, the concentration of oligosaccharides is low in mature bovine milk, normally used for infant formula, compared with both human colostrum and mature human milk. Characterization of bovine milk oligosaccharides in different breeds is crucial for the identification of viable sources for oligosaccharide purification. An improved source of oligosaccharides can lead to infant formula with improved oligosaccharide functionality. In the present study we have analyzed milk oligosaccharides by high-performance liquid chromatography chip quadrupole time-of-flight mass spectrometry and performed a detailed data analysis using both univariate and multivariate methods. Both statistical tools revealed several differences in oligosaccharide profiles between milk samples from the two Danish breeds, Jersey and Holstein-Friesians. Jersey milk contained higher relative amounts of both sialylated and the more complex neutral fucosylated oligosaccharides, while the Holstein-Friesian milk had higher abundance of smaller and simpler neutral oligosaccharides. The statistical analyses revealed that Jersey milk contains levels of fucosylated oligosaccharides significantly higher than that of Holstein-Friesian milk. Jersey milk also possesses oligosaccharides with a higher degree of complexity and functional residues (fucose and sialic acid), suggesting it may therefore offer advantages in term of a wider array of bioactivities.

Human milk lactoferrin (hmLF) is the most abundant glycoprotein present in human milk and displays a broad range of protective functions in the gut of newborn infants. hmLF is N-glycosylated, but little is known about the lactation stage-related development of the glycosylation phenotype. hmLF glycosylation from milk samples from five donors during the first 10 weeks of lactation was assessed and observed to be more diverse than previously reported. During this period dynamic changes in glycosylation were observed corresponding to a decrease in glycosylation in the second week followed by an increase in total glycosylation as well as higher order fucosylation thereafter. Gene expression analysis was performed in milk somatic cells from a sixth subject. It was found that fucosyltransferase expression increased during entire period, whereas expression of genes for the oligosaccharyl transferase complex decreased in the second week. The effect of hmLF glycosylation was examined for the protein's ability to affect bacterial binding to epithelial cells. hmLF significantly inhibited pathogen adhesion and purified hmLF glycans significantly reduced Salmonella invasion of colonic epithelial cells to levels associated with non-invasive deletion mutants. This study indicates that hmLF glycosylation is tightly regulated by gene expression and that glyco-variation is involved in modulating pathogen association.

Attenuated total reflection Fourier transform infrared spectroscopy was used to monitor the formation of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), DMPC: lactosylceramide, and DMPC: GD3 lipid bilayers onto a zinc selenide surface. Infrared absorption peak position, bandwidth, and intensity were all used to monitor the formation, acyl chain ordering, and chemical environment within each bilayer. The results from this study indicate that the addition of glycosphingolipids into a DMPC lipid bilayer introduces decreases in both, acyl chain ordering, and homogeneity within the bilayer. GD3:DMPC lipid bilayers possess lipid chain characteristics that are indiscernible from those present in the lactosylceramide:DMPC bilayer, while possessing different structural head groups, indicating that the head group has little influence on the underlying lipid structure. Differences in the phosphate hydration are, however, evident between the three types of bilayer, with phosphate hydration decreasing in the order LacCer:DMPC (1223.4 cm(-1))>DMPC only (1226 cm(-1))>GD3:DMPC (1229.6 cm(-1))., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Mass spectrometry has been coupled with flash liquid chromatography to yield new capabilities for isolating nonchromophoric material from complicated biological mixtures. A flash liquid chromatography/tandem mass spectrometry (LC/MS/MS) method enabled fraction collection of milk oligosaccharides from biological mixtures based on composition and structure. The method is compatible with traditional gas pressure-driven flow flash chromatography widely employed in organic chemistry laboratories. The online mass detector enabled real-time optimization of chromatographic parameters to favor separation of oligosaccharides that would otherwise be indistinguishable from coeluting components with a nonspecific detector. Unlike previously described preparative LC/MS techniques, we have employed a dynamic flow connection that permits any flow rate from the flash system to be delivered from 1 to 200 ml/min without affecting the ionization conditions of the mass spectrometer. A new way of packing large amounts of graphitized carbon allowed the enrichment and separation of milligram quantities of structurally heterogeneous mixtures of human milk oligosaccharides (HMOs) and bovine milk oligosaccharides (BMOs). Abundant saccharide components in milk, such as lactose and lacto-N-tetraose, were separated from the rarer and less abundant oligosaccharides that have greater structural diversity and biological functionality. Neutral and acidic HMOs and BMOs were largely separated and enriched with a dual binary solvent system., (Published by Elsevier Inc.)

The isolation of whey proteins from human and bovine milks followed by profiling of their entire N-glycan repertoire is described. Whey proteins resulting from centrifugation and ethanol precipitation of milk were treated with PNGase F to release protein-bound N-glycans. Once released, N-glycans were analyzed via nanoflow liquid chromatography coupled with quadrupole time-of-flight mass spectrometry following chromatographic separation on a porous graphitized carbon chip. In all, 38 N-glycan compositions were observed in the human milk sample while the bovine milk sample revealed 51 N-glycan compositions. These numbers translate to over a hundred compounds when isomers are considered and point to the complexity of the mixture. High mannose, neutral, and sialylated complex/hybrid glycans were observed in both milk sources. Although NeuAc sialylation was observed in both milk samples, the NeuGc residue was only observed in bovine milk and marks a major difference between human and bovine milks. To the best of our knowledge, this study is the first MS based confirmation of NeuGc in milk protein bound glycans as well as the first comprehensive N-glycan profile of bovine milk proteins. Tandem MS was necessary for resolving complications presented by the fact that (NeuGc:Fuc) corresponds to the exact mass of (NeuAc:Hex). Comparison of the relative distribution of the different glycan types in both milk sources was possible via their abundances. While the human milk analysis revealed a 6% high mannose, 57% sialylation, and 75% fucosylation distribution, a 10% high mannose, 68% sialylation, and 31% fucosylation distribution was observed in the bovine milk analysis. Comparison with the free milk oligosaccharides yielded low sialylation and high fucosylation in human, while high sialylation and low fucosylation are found in bovine. The results suggest that high fucosylation is a general trait in human, while high sialylation and low fucosylation are general features of glycosylation in bovine milk.

Glycosylation is one of the most common yet diverse post-translational modifications. Information on glycan heterogeneity and glycosite occupancy is increasingly recognized as crucial to understanding glycoprotein structure and function. Yet, no approach currently exists with which to holistically consider both the proteomic and glycomic aspects of a system. Here, we developed a novel method of comprehensive glycosite profiling using nanoflow liquid chromatography/mass spectrometry (nano-LC/MS) that shows glycan isomer-specific differentiation on specific sites. Glycoproteins were digested by controlled non-specific proteolysis in order to produce informative glycopeptides. High-resolution, isomer-sensitive chromatographic separation of the glycopeptides was achieved using microfluidic chip-based capillaries packed with graphitized carbon. Integrated LC/MS/MS not only confirmed glycopeptide composition but also differentiated glycan and peptide isomers and yielded structural information on both the glycan and peptide moieties. Our analysis identified at least 13 distinct glycans (including isomers) corresponding to five compositions at the single N-glycosylation site on bovine ribonuclease B, 59 distinct glycans at five N-glycosylation sites on bovine lactoferrin, 13 distinct glycans at one N-glycosylation site on four subclasses of human immunoglobulin G, and 20 distinct glycans at five O-glycosylation sites on bovine κ-casein. Porous graphitized carbon provided effective separation of glycopeptide isomers. The integration of nano-LC with MS and MS/MS of non-specifically cleaved glycopeptides allows quantitative, isomer-sensitive, and site-specific glycoprotein analysis.

Oligosaccharides in human milk strongly influence the composition of the gut microflora of neonates. Because it is now clear that the microflora play important roles in the development of the infant immune system, human milk oligosaccharides (HMO) are studied frequently. Milk samples contain complex mixtures of HMO, usually comprising several isomeric structures that can be either linear or branched. Traditionally, HMO profiling was performed using HPLC with fluorescence or UV detection. By using porous graphitic carbon liquid chromatography MS, it is now possible to separate and identify most of the isomers, facilitating linkage-specific analysis. Matrix-assisted laser desorption ionization time-of-flight analysis allows fast profiling, but does not allow isomer separation. Novel MS fragmentation techniques have facilitated structural characterization of HMO that are present at lower concentrations. These techniques now facilitate more accurate studies of HMO consumption as well as Lewis blood group determinations.

Over forty-five complex free oligosaccharides (of which several are novel) have been isolated and chemically characterized by gas chromatography and high resolution and high mass accuracy matrix-assisted laser desorption/ionization mass spectrometry (MALDI-FTICR MS) in red and white wines, Grignolino and Chardonnay, respectively. Oligosaccharides with a degree of polymerization between 3 and 14 were separated from simple monosaccharides and disaccharides by solid-phase extraction. The concentrations of free oligosaccharides were over 100 mg/L in both red and white wines. The free oligosaccharides-characterized for the first time in the present study-include hexose-oligosaccharides, xyloglucans, and arabinogalactans and may be the natural byproduct of the degradation of cell wall polysaccharides. The coupled gas chromatography and accurate mass spectrometry approach revealed an effective method to characterize and quantify complex functional oligosaccharides in both red and white wine.

Most cell membrane proteins are known or predicted to be glycosylated in eukaryotic organisms, where surface glycans are essential in many biological processes including cell development and differentiation. Nonetheless, the glycosylation on cell membranes remains not well characterized because of the lack of sensitive analytical methods. This study introduces a technique for the rapid profiling and quantitation of N- and O-glycans on cell membranes using membrane enrichment and nanoflow liquid chromatography/mass spectrometry of native structures. Using this new method, the glycome analysis of cell membranes isolated from human embryonic stem cells and somatic cell lines was performed. Human embryonic stem cells were found to have high levels of high mannose glycans, which contrasts with IMR-90 fibroblasts and a human normal breast cell line, where complex glycans are by far the most abundant and high mannose glycans are minor components. O-Glycosylation affects relatively minor components of cell surfaces. To verify the quantitation and localization of glycans on the human embryonic stem cell membranes, flow cytometry and immunocytochemistry were performed. Proteomics analyses were also performed and confirmed enrichment of plasma membrane proteins with some contamination from endoplasmic reticulum and other membranes. These findings suggest that high mannose glycans are the major component of cell surface glycosylation with even terminal glucoses. High mannose glycans are not commonly presented on the surfaces of mammalian cells or in serum yet may play important roles in stem cell biology. The results also mean that distinguishing stem cells from other mammalian cells may be facilitated by the major difference in the glycosylation of the cell membrane. The deep structural analysis enabled by this new method will enable future mechanistic studies on the biological significance of high mannose glycans on stem cell membranes and provide a general tool to examine cell surface glycosylation.

Glycosylation is one of the most common post-translational modifications of proteins and has been shown to change with various pathological states including cancer. Global glycan profiling of human serum based on mass spectrometry has already led to several promising markers for diseases. The changes in glycan structure can result in altered monosaccharide composition as well as in the linkages between the monosaccharides. High-throughput glycan structural elucidation is not possible because of the lack of a glycan template to expedite identification. In an effort toward rapid profiling and identification of glycans, we have constructed a library of structures for the serum glycome to aid in the rapid identification of serum glycans. N-Glycans from human serum glycoproteins are used as a standard and compiled into a library with exact structure (composition and linkage), liquid chromatography retention time, and accurate mass. Development of the library relies on highly reproducible nanoLC-MS retention times. Tandem MS and exoglycosidase digestions were used for structural elucidation. The library currently contains over 300 entries with 50 structures completely elucidated and over 60 partially elucidated structures. This database is steadily growing and will be used to rapidly identify glycans in unknown biological samples.