Nutritional and Clinical Glycomics Research

Author: laurenmd (Page 2 of 9)

New Publications!!!

Mehra, R., et al. (2014). “Novel high-molecular weight fucosylated milk oligosaccharides identified in dairy streams.” PLoS One 9(5): e96040.
Oligosaccharides are the third largest component in human milk. This abundance is remarkable because oligosaccharides are not digestible by the newborn, and yet they have been conserved and amplified during evolution. In addition to encouraging the growth of a protective microbiota dominated by bifidobacteria, oligosaccharides have anti-infective activity, preventing pathogens from binding to intestinal cells. Although it would be advantageous adding these valuable molecules to infant milk formula, the technologies to reproduce the variety and complexity of human milk oligosaccharides by enzymatic/organic synthesis are not yet mature. Consequently, there is an enormous interest in alternative sources of these valuable oligosaccharides. Recent research has demonstrated that bovine milk and whey permeate also contain oligosaccharides. Thus, a thorough characterization of oligosaccharides in bovine dairy streams is an important step towards fully assessing their specific functionalities. In this study, bovine milk oligosaccharides (BMOs) were concentrated by membrane filtration from a readily available dairy stream called “mother liquor”, and analyzed by high accuracy MALDI FT-ICR mass spectrometry. The combination of HPLC and accurate mass spectrometry allowed the identification of ideal processing conditions leading to the production of Kg amount of BMO enriched powders. Among the BMOs identified, 18 have high-molecular weight and corresponded in size to the most abundant oligosaccharides present in human milk. Notably 6 oligosaccharides contained fucose, a sugar monomer that is highly abundant in human milk, but is rarely observed in bovine milk. This work shows that dairy streams represent a potential source of complex milk oligosaccharides for commercial development of unique dairy ingredients in functional foods that reproduce the benefits of human milk.

Ozcan, S., et al. (2014). “Glycosylated proteins preserved over millennia- N-glycan analysis of Tyrolean Iceman, Scythian Princess and Warrior.” Sci Rep 4: 4963.
An improved understanding of glycosylation will provide new insights into many biological processes. In the analysis of oligosaccharides from biological samples, a strict regime is typically followed to ensure sample integrity. However, the fate of glycans that have been exposed to environmental conditions over millennia has not yet been investigated. This is also true for understanding the evolution of the glycosylation machinery in humans as well as in any other biological systems. In this study, we examined the glycosylation of tissue samples derived from four mummies which have been naturally preserved: – the 5,300 year old “Iceman called Oetzi”, found in the Tyrolean Alps; the 2,400 year old “Scythian warrior” and “Scythian Princess”, found in the Altai Mountains; and a 4 year old apartment mummy, found in Vienna/Austria. The number of N-glycans that were identified varied both with the age and the preservation status of the mummies. More glycan structures were discovered in the contemporary sample, as expected, however it is significant that glycan still exists in the ancient tissue samples. This discovery clearly shows that glycans persist for thousands of years, and these samples provide a vital insight into ancient glycosylation, offering us a window into the distant past.

Smilowitz, J. T., et al. (2013). “Breast Milk Oligosaccharides- Structure-Function Relationships in the Neonate.” Annu Rev Nutr.
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. Expected final online publication date for the Annual Review of Nutrition Volume 34 is July 17, 2014. Please see for revised estimates.

Song, T., et al. (2014). “In-Depth Method for the Characterization of Glycosylation in Manufactured Recombinant Monoclonal Antibody Drugs.” Anal Chem.
The glycosylation in recombinant monoclonal antibody (rMab) drugs is a major concern in the biopharmaceutical industry as it impacts the drugs’ many attributes. Characterization is important but complicated by the intricate structures, microheterogeneity, and the limitations of current tools for structural analysis. In this study, we developed a liquid chromatography-mass spectrometry (LC-MS) N-glycan library based on eight commercial rMab drugs. A library of over 70 structures was developed for the rapid characterization of rMab. N-Glycans were separated on a porous graphitized carbon (PGC) column incorporated on a chip and then analyzed by an electrospray ionization hybrid quadrupole time-of-flight (ESI-Q-TOF) MS. The retention time and accurate mass for each N-glycan were recorded in the library. The complete structures were obtained through exoglycosidase sequencing. The results showed that most of the N-glycans between different antibodies are nearly the same with different abundances. The utility of this library enables one to identify structures in a rapid manner by matching LC retention times and accurate masses.


Glycans and Glycoproteins in Mass Spectrometry

TWO-DAY COURSE, Saturday and Sunday
Glycans and Glycoproteins for Mass Spectrometry


Jon Amster
University of Georgia
Carlito Lebrilla
University of California, Davis
Ron Orlando
University of Georgia
Joe Zaia
Boston University

This course is designed for scientists who want to learn specific techniques for the MS and MS/MS characterization of glycans and glycoproteins. The course will address fundamental aspects of glycobiology, sample preparation and handling, mass spectrometry (hardware and software), and bioinformatic tools for interpretation of results.

Real-world examples of the application of these techniques will include characterization of intact glycoproteins, characterization of released glycans, analysis of complex mixtures of glycoproteins and glycans. The role of MS-based methods in interdisciplinary efforts to solve these complex problems will also be addressed.

Day 1
 Introduction to glycosylation
 Chemical manipulation/derivatization of oligosaccharides, GC-MS for linkage and composition analyses
 MALDI MS and ESI of oligosaccharides
 Tandem MS of oligosaccharides
 Glycoproteins — release and analyze, and glycomic approaches
 Glycopeptides, methods for site specific glycosylation analysis, glycoproteomic approaches

Day 2
 Glycoprotein applications and case studies
 Glycosaminoglycans: Background, fundamentals, Tandem MS
 Glycolipids
 Glycan Quantitation
 Bioinformatics

Pre-requisites: A basic knowledge of mass spectrometry and some rudimentary knowledge of biology
and chemistry is desirable.

Comprehensive site-specific characterization of glycoproteins using enzymes of varying cleavage specificities

Dr. Lebrilla

Carlito Lebrilla; Evan Parker; Michael Xin Sun; Jincui Huang; Andres Guerrero

UC Davis, Davis, CA

Novel Aspect
Software allows the use of any enzymatic system for the analysis of glycopeptides by LCMS thereby allowing target-oriented glycopeptide analysis.

Site-specific analysis of protein glycosylation is an active and evolving field of research. Successful application of this analysis with respect to bio-activity is hampered by inconsistent analysis, difficult to repeat results, and extraordinarily low throughput. We have developed a workflow that counteracts several of the difficulties often encountered when assigning glycosylation site-structure pairs. Difficulties often found include peptides too large for accurate mass determination, hard to confirm structures due to multiple sites, and enzymatic resistance to the proteolysis. Our method avoids these problems by using a host of site-specific and nonspecific proteases to generate glycopeptides depending on the specific need. Using our own software platform we are able to analyze all of the data in a directly comparable manner.

Both in-gel and in-solution digestions are used, although in-gel digestion provides sensitivity advantages. Shortly, 10 ?g of a target protein is reduced and alkylated prior to running on SDS-PAGE. Gel bands are cut, destained, and dried in a speed-vac prior to digestion with trypsin, pronase, or elastase in ammonium bicarbonate buffer. After digesting overnight the glycopeptides are extracted. Samples were dried and resuspended in 20 ?L prior to analysis on an Agilent HPLC-Chip/TOF MS. The resulting data can be exported to MGF and analyzed with in-house software utilizing accurate mass and fragment scoring to confirm identification.

Preliminary Results/Abstract
As a general example of the differences in digestion procedures, bovine fetuin and horseradish peroxidase serve as useful examples of potential pitfalls and benefits of each method. Horseradish perodidase is especially interesting because biochemical tools to cleave glycans in the presence of ?(1-3) linked fucose on the core glucoseamine are still new and difficult to obtain.

The N-glycosylation sites of bovine fetuin are an excellent example of why multiple proteases should be used to elucidate site-specific structures. Using the standard workflow of tryptic digestion, N-glycans on sites 99, 156, and 176 can be found in peptides of length 32, 15, and 28 respectively. When the potential for a single missed cleavage is taken into account peptides of length 49, 43, and 52 are possible. Using elastase as a proteolytic enzyme, peptides are much shorter, 4, 7, and 10 amino acids long at minimum and 11, 18, and 15 amino acids long with one missed cleavage. These small peptides are much easier to detect and have easier to interpret CID spectra. A further step we take is the use of nonspecific proteases; with nonspecific digestion peptides are observed from length 2 to 10 at all eight sites.As an additional example, horseradish peroxidase has eight glycosylation sites. Site 43, 216, and 228 are difficult to analyze by tryptic digestion due to the large peptide generated at site 43 and the fact that sites 216 and 218 share a tryptic peptide. While elastase could aid in the assignment of site 43, it actually complicates the assignment of site 216 and 228 further since the smallest peptide includes site 244 as well. Pronase performs excellently on this protein producing many peptides at each site from 2 to 10 amino acids long.



Stability Analysis of Oligosaccharides


Lauren D. Wu; Angela Zivkovic; Sarah Totten; L. Renee Ruhaak; Carlito B. Lebrilla

University of California, Davis, CA

Novel Aspect
The stability of oligosaccharides in human breast milk during sample handling and storage is assessed for the first time.

Human milk oligosaccharides (HMOs) are one of the more abundant bioactives in human milk. They are now known to influence the development of healthy bacteria in the infant’s gut. Recently, our group has developed a TOF-MS based analytical method, including an extensive library, for the in-depth characterization of a mother’s HMO profile. Using this method, we have shown variations in HMO composition between different mothers, but limited effort has been made to assess sample stability during storage and sample handling. Therefore, we here assess the effects of long-term storage (>1 year) and multiple freeze-thaw cycles on the HMO profile of a donor milk sample.

Fresh breast milk (BM) was obtained from a single donor mother and was aliquoted and stored at -80°C. The HMOs from one aliquot were extracted immediately (BM Fresh), while another was stored for 1 year (BM Storage). BM Fresh was processed under consecutive freeze-thaw cycles in the same day it was received, with HMO separation and purification following each thaw cycle. HMOs were analyzed using nano-LC/MS and were stored for one year to observe the stability of purified compounds. BM Storage was processed under the same conditions and analyzed alongside the HMOs purified one year earlier. Upon data processing, absolute abundances were compared between samples analyzed within the same instrumental run, and relative abundances were assessed between runs.

Preliminary Results/Abstract

Using the obtained three data sets (BM fresh, BM Fresh at year 1, and BM Storage), we were able to observe oligosaccharide stability after freeze-thaw cycles, purified HMO stability, and long-term storage including its effect on freeze-thaw cycles. We first focused on the effects of unstable temperature conditions, where we compared a fresh milk sample to multiple freeze-thaw cycles using absolute abundance in ion counts and percent relative abundances. There was a significant difference in absolute abundance of total sialylated compounds in two of the cycles, but no distinct pattern. The purified HMOs were saved to test the stability of purified compounds after one year. There were no significant differences between each freeze-thaw cycle.

We then tested storage stability by comparing the HMOs from a fresh milk sample to the same milk that was stored frozen. These samples were analyzed one year apart using the same instrument parameters. Because of the interday instrument variation, we only assessed relative abundances. Fucosylation and sialylation in purified HMOs and extracted HMOs from frozen samples were more abundant than HMOs extracted from fresh milk, while non-fucosylated neutrals were lower in abundance.

To observe how storage affects the compound stability during freeze-thaw cycles, we extracted HMOs from frozen milk samples and processed them in the same conditions. There was no distinct trend of HMOs increasing or decreasing in relative or absolute abundances between the first freeze-thaw cycle to each subsequent set. The compounds in this batch seemed to remain relatively stable even after one year of storage.

Although there is some variation in sialylation during freeze-thaw cycles, HMOs do not exhibit a distinct pattern where oligosaccharides can be assumed to be degrading. While other biomolecules are known to change after unstable sample handling and storage conditions, these factors are insignificant to HMO analysis.



A systems approach to protein-specific glycosylation analyses of serum glycoproteins for cancer diagnosis.


Renee Ruhaak1; Carol Stroble1, 2; Qiuting Hong1; Suzanne Miyamoto2; Kyoungmi Kim1; Gary Leiserowitz2; Carlito Lebrilla1

1UC Davis, Davis, CA; 2UC Davis Medical Center, Sacramento, CA

Novel Aspect
A QQQ-MS approach is presented for the discovery and validation of serum protein and glycopeptide levels as discriminators of disease.

Protein glycosylation has been proposed as a new source of potential biomarkers for diseases as diverse as cancer and infection. We have previously reported on the use of serum glycans for the diagnosis of ovarian cancer (OC). However, these studies have focused on released glycans thereby eliminating protein-specific information. Shotgun glycoproteomics analyses, which would provide protein-specific information, have thus far not been widely successful due to the large heterogeneity in protein glycosylation. Instead, we have focused on protein-specific glycosylation using a targeted approach. Multiple reaction monitoring on QQQ-MS is an excellent tool for protein- and site- specific quantification of protein glycosylation. We developed transitions for many abundant serum proteins, and apply the method towards glycopeptide biomarker discovery for ovarian cancer.

Protein standards were used to develop MRM transitions. Standards of the 9 proteins immunogobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), haptoglobin (HP), transferrin (TF), alpha-1-acid glycoprotein (AGP), alpha-1- antitrypsin (A1AT), alpha-2-macroglobulin (A2MG) and complement C3 (C3) were treated with DTT and IAA prior to digestion using trypsin as well as trypsin/chymotrypsin. First, their site-specific glycosylation patterns were determined using nLC-chip-Q-TOF mass spectrometry. Subsequently, quantitative MRM transitions were developed for peptides and glycopeptides using UPLC-QQQ-MS. The absolute protein concentration was determined using the peptide signals. Glycopeptide signals were normalized to the absolute protein amount to determine the protein- and site- specific glycosylation pattern. The method was applied to serum samples of ovarian cancer patients and their controls.

Preliminary Results/Abstract
For each of the proteins, site-specific glycosylation patterns were determined using literature search and Q-TOF fragmentation data. Based on these data, transitions were developed corresponding to glycopeptides as well as nonglycosylated peptides, where the latter allows for protein quantification. The proteins IgG, IgA, IgM, HP, TF, AGP, A1AT, A2MG and C3 were targeted thus far. For each of the glycopeptides, transitions were developed to fragments with m/z 204 (HexNAc) or m/z 366 (Hex-HexNAc), except for high-mannose glycopeptides where transitions were developed to the peptide+HexNAc fragment. The method is highly accurate with RSD’s reported for the glycopeptides of less than 10%.
The method was applied to a sample set of 40 ovarian cancer cases and their age-matched controls for biomarker discovery. Levels of 18 glycopeptides of IgG, 13 glycopeptides of IgA and 10 glycopeptides of IgM were shown to alter significantly (either over- or under-expressed) with OC at a false discovery rate of <0.05. Multiplex classifiers combining multiple glycopeptides together were developed for each of the proteins with the highest accuracy for determining ovarian cancer of 91.1% when combining five glycopeptides of IgG. For IgA, multiplexing resulted in a highest accuracy of 88%, while the highest accuracy was 80% for IgM.
The method was then applied to an independent test set for validation. When testing the multiplex classifiers developed using the training set in samples of the independent test set, classification accuracies of cancer state of 90%, 80% and 74% were achieved for IgG, IgA and IgM, respectively. We are currently developing a model combining the glycopeptides from the different proteins that could allow the definition of a targeted QQQ-based OC diagnostic test with greater accuracy. This is the first attempt at quantitation of the glycosylation of several proteins simultaneously and shows the potential of targeted glycopeptide analysis as a diagnostic tool.


Absolute Quantitation of Human Milk Proteins and their Glycoforms using Multiple Reaction Monitoring (MRM)

Jincui Huang

Jincui Huang; Qiuting Hong; Rocchina Sabia; Carlito Lebrilla

UC Davis, Davis, CA

Novel Aspect
Rapid-throughput method of quantitation of human milk proteins and their glycoforms was developed.

Human milk has long been recognized as not only a source of nutrition but for a number of functional components to the newborn. Milk proteins are highly glycosylated, yet the roles of glycosylation in the function of milk is limited by the lack of tools to quantitate various protein glycoforms. We have developed a new MRM approach to quantitate human milk proteins and their glycosylation, giving the capability to monitor glycosylation at site-specific level. The process involves identifying the best peptides for protein quantitation, mapping the site-specific glycan heterogeneity of the glycoproteins, and characterizing the fragmentation patterns of specific glycopeptides to determine the best transitions for MRM. The method advances our understanding of the role of glycan-conjugates in human health.

Quantitation of human milk proteins and their peptide-conjugated glycans directly facilitates the differential analysis of distinct glycoforms associated with the state of lactation. To be useful, the method must be rapid throughput. In a 96-well plate, standard protein mixture (200 µg of human lactoferrin, ?-lactalbumin, 100 µg of IgA, and 20 µg of IgG, IgM, antitrysin and lysozyme) and 25 µL of human milk samples were reduced and alkylated prior to trypsin digestion at 37?C for 18 hr. The resulting peptides and glycopeptides were cleaned and enriched via solid phase extraction (SPE) with C-18 cartridges. The mixture was profiled using nano-LC-chip-QTOF mass spectrometer and quantified using UPLC-ESI-QqQ mass spectrometer.

Preliminary Results/Abstract
Our strategy allows both the determination of protein concentrations as well as the absolute and relative abundances of the different glycoforms in a given sample. The proteins, human lactoferrin (hLF), ?-lactalbumin, IgA, IgG, IgM, lysozyme and antitrypsin, are the most abundant in milk and represent nearly 80% of the whey protein abundances. For each protein two or three unique peptides were identified as being robust for quantitation. Nano?ow LC?MS analysis with the Q-TOF instrument allowed identi?cation of the glycopeptides (from hLF, IgA, IgM, IgG and antitrypsin) based on the accurate mass and the tandem MS. Glycopeptides were characterized by their fragmentation pattern to obtain the best transitions. For example, hLF has three potential N-glycosylation sites, but only two glycosites were occupied having mainly complex type N-glycans. In total, more than 10 glycoforms were observed at Asn156 and 5 glycoforms were present at Asn497.
UPLC separation allowed rapid-throughput quantitation using a 16-min gradient for monitoring hundreds of peptide and glycopeptide transitions. Over 100 glycopeptides were monitored from the five glycoproteins.
This rapid-throughput platform was tested for its reproducibility and accuracy, and applied to a clinical study with 171 human milk samples from 46 mothers at four time points during lactation. Within a 2-day run, protein concentration and protein glycosylation level were monitored and quantitated. hLF concentration decreased from 2.3 mg/ to 1.5 mg/mL, while ?-lactalbumin remained around 3.8 mg/mL over lactation. Lysozyme was the only protein found increasing concentration from 0.08 mg/mL to 0.16 mg/mL. By normalizing the glycopeptide signals to the protein abundances, hLF glycosylation at both glycosites showed a four-fold decrease during lactation.


Glycan Site Mapping of Glycoproteins in Serum


Qiuting Hong; Evan Parker; Ting Song; Carlito Lebrilla

Chemsitry, UC, Davis, Davis, CA

Novel Aspect
A comprehensive site-specific glycan map for plasma glycoproteins

Glycosylation analysis on the site-specific level provides both protein and glycosylation information to facilitate biomarker discovery. However, site-specific analysis remains a difficult task, and there is no comprehensive glycan map even for the most abundant plasma glycoproteins. We are in the process of mapping the glycosylation and the site-specific heterogeneity in plasma proteins. Trypsin produces consistent peptides for quantitation but may provide limited glycosite information. Nonspecific protease yields better glycosite coverage but data analysis is complicated. To obtain comprehensive glycan maps several protease methods are used. A software tool for examining tandem MS of glycopeptides is also proven to be highly useful. The method can be applied to any glycoprotein and will further facilitate our efforts on disease biomarker discovery.

Glycosylation analysis was performed on commercially available plasma proteins including IgG, IgM, IgA, transferrin, haptoglobin, alpha-2-macroglobulin, alpha-1-antitrypsin, C3 complement, and alpha-1-acid glycoprotein. Glycopeptides were profiled using an Agilent 6520 nano-LC-Chip/QTOF MS, and identified using in-house software, GPFinder. Both trypsin and pronase (nonspecific protease), using both in-gel and in-solution digestion techniques, were applied to obtain a more comprehensive glycan map. Proteins were treated with DTT and IAA before enzyme digestion in a 37?C water bath. Trypsin digestions were performed overnight, and the resulting mixtures were used directly for LC-MS analysis. Pronase digestions were performed 1 hour for the in-gel and 3 hours for the in-solution approaches. The resulting sample was purified using C18 or graphitized zip-tip before MS analysis.

Preliminary Results/Abstract
We have determined the site-specific glycosylation of the top 10 plasma glycoproteins using both specific and nonspecific proteolysis. Many of these proteins have only partial assignments or no site-specific assignments for protein glycosylation, despite being the most abundant proteins. For proteins, like alpha-2-marcoglobulin that have fewer lysine and arginine, trypsin produces large glycopeptides and multiple glycosites on single peptides. For alpha-2-marcoglobulin, only one glycosite (N1424) can be mapped out using trypsin. Pronase produces smaller peptides and yields site-specific glycoform information for all eight glycosites of alpha-2-marcoglobulin. It was found that glycosites N247, N410, N869 have both high mannose type and sialylated complex type glycans, whereas the other five glycosites contain sialylated and neutral complex type glycans.
For some proteins, the enzymes provide complimentary information. Take IgA as an example, trypsin digestion provides glycosylation information for three out of seven glycosites, N144(IgA1), N131(IgA2), and N205(IgA2). Pronase digestion was found to yield glycan information the same sites N144(IgA1) and N131(IgA2), but also other sites N340(IgA1). Glycoforms on glycosite N340(IgA1) and N205(IgA2) are all fucosylated, while glycoforms on N144(IgA1) and N131(IgA2) are all nonfucosylated. No glycans were detected on the remaining sites and are believed to unoccupied in our protein standard.
In summary, a comprehensive glycan map for 43 glycosites from the 10 most abundant plasma glycoproteins has been obtained using different proteolysis approaches and the in-house software, GPFinder. We showed that glycoforms are very specific on different glycosites. This study is part of a larger one where we plan to map protein glycosylation in plasma proteins beginning with the most abundant proteins. The site-specific glycosylation for low abundant plasma proteins will be investigated using protein enrichment methods to facilitate their analyses.


Method for analysis of glycan degradation products in the feces of breast-fed newborns



Jasmine C. C. Davis; Sarah M. Totten; Carlito B. Lebrilla

UC Davis, Davis, CA

Novel Aspect
This research aimed to create a library of intact and degraded milk glycans found in infant feces from microbial catabolism.

Oligosaccharides continue to be one of the most difficult biomolecules to analyze. Analysis is, however, aided by the use of biological rules followed by glycosyl transferases and glycosidases in their modification of the nascent glycan structure. Human milk oligosaccharides (HMOs) will interact with bacteria in the infant’s digestive tract, but the nature of these interactions is often unknown. Furthermore, the interactions often lead to the production of new types of oligosaccharides that are degradation products from the bacterial consumption. In this this research, we developed a method for determining glycan products due to the interactions of bacteria with glycoconjugates in milk. We are able to rapidly identify glycan structures and correlate the enzymes responsible for those oligosaccharide products.

Fecal samples were diluted and shaken overnight. Proteins in the supernatant were precipitated with ethanol, and the resulting glycans were reduced to their alditol form with NaBH4. The samples were cleaned up with solid phase extraction, the eluents evaporated, and before analysis the samples were reconstituted in water.

Extracted glycans were analyzed on a nano-HPLC-Chip/TOF MS system followed by identification and quantitation with Agilent Mass Hunter Qualitative Analysis. Structures were confirmed using collision induced dissociation (CID) on a nano-HPLC-Chip/Q-TOF MS.

To monitor bacterial consumption, an HMO pool was digested with a ß-galactosidase, incubated, and C18 zip-tip cleaned up. Spectra of the consumed HMOs were compared to that of the undigested HMO pool, and CID was used for structural confirmation.

Preliminary Results/Abstract
Consumption studies were performed to determine the degradation products of a ß-galactosidase strain, as well as the specificity of that strain. Our intact HMO library contains specific linkages of isomers, so we are able to determine the specificity of the enzyme by comparing the spectra and abundances of isomers from an undigested HMO pool with the consumed pool. The masses and compositions of the digested products were added to a previous HMO library in order to create a fecal library.

Not only are there exoglycosidases that cleave glycans, but there are also enzymes that can cleave N-glycans from glycoproteins. There are no free N-glycans in breast milk, but they were discovered in the fecal samples analyzed, so those structures, along with their degraded products, were also taken into account. Compositions of intact N-glycans from a theoretical library were added to the fecal library.

Structures of the extracted glycans, both intact and degraded, were confirmed using CID. The CID spectra of the N-glycans were not consistent with the N-glycans containing their intact chitobiose core, and it appeared that the N-acetylglucosamines (GlcNAcs) on their reducing ends were missing from their structures, suggesting the glycan was cleaved by an endoglycosidase. The possible digested N-glycans were also added to the fecal library to incorporate all possible glycan compositions.

Our library was created with specific isomer information, with identification based on composition, exact mass, and retention time. We can monitor the identified degradation products in fecal samples based on the data from the consumption studies. It is possible to tell the difference between most HMOs and N-glycans based on their differences in composition and retention times. HMOs and N-glycans both have specific biological rules in how glycosyl transferases add monosaccharides to their core structures, so there are certain compositions that either glycan can have.


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