Nutritional and Clinical Glycomics Research

Category: Uncategorized (Page 2 of 6)

Determination of changes in cell surface glycosylation with cellular transformations


Dayoung Park; Narine Arabyan; Cynthia Williams; Ting Song; Bart Weimer; Carlito Lebrilla

University of California, Davis, Davis, CA

Novel Aspect
Methodology to analyze thousands of membrane glycan structures simultaneously with linkage and isomer differentiation and identify glycans involved in infection.

Cell membranes consist of proteins that contain short saccharide chains called glycans, which mediate nearly all interactions. In the gastrointestinal tract, a single layer of epithelial cells lines the inner layer as a protective interface between the embedded tissues and microorganisms that continually contact the internal organs. Regulation of these bacterial-host interactions is critical for host health. Using high resolution mass spectrometric techniques, a glycomic analysis was performed to characterize glycosylation changes on epithelial cell surfaces upon prolonged contact with foreign and resident bacteria of the gut. This new technique allows for rapid profiling and quantitation of membrane glycans based on retention time and accurate mass and was used to understand which glycans are involved in bacterial colonization and infection.

Epithelial colorectal adenocarcinoma Caco-2 cells were cultured in vitro. Selected bacterial genes that express glycosidases were knocked out. Upon infection with wildtype bacteria and the knockouts, cells were lysed using probe sonication and the membrane fraction was extracted from the resulting lysate by ultracentrifugation. Glycans were enzymatically released from isolated cell membrane glycoproteins and analyzed by microfluidic chip-based nano-LC quadrupole time-of-flight mass spectrometry. The total glycan profile of detected membrane glycans were constructed using a theoretical, retrosynthetic mass library, providing glycan compositions based on known synthetic pathways. Changes in relative abundances of individual N-glycans were characterized statistically. To elucidate exact structures, tandem MS was employed and reduced glycans were digested with exoglycosidases for linkage information and isomeric differentiation.

Preliminary Results/Abstract

The cell sample set was optimized using different cell amounts varying from one to four million cells. Samples with at least 2 million cells showed the highest glycan signal and reproducibility.
Caco-2 cell surface glycosylation is dominated by sialylated and fucosylated complex and hybrid glycans, comprising almost three fourths of the total number of glycans. When considering the relative intensities, however, high mannose glycans are among the most abundant. These results suggest that Caco-2 membranes have a large amount of terminal mannose residues, which may have functional significance in epithelial cells during infection.
Bacterial infection times were varied to observe changes in Caco-2 membrane glycosylation. From 0 min to 45 min, only slight variations in glycan profiles were observed. At the 1 hr time point, there was a significant change in glycan compositions and relative abundances. Notably, nondecorated and high mannose glycans increased in signal after 1 hr of infection but later decreased past 2 hrs. By the third hour of infection, the overall profile recovered and resembled that of the uninfected set.
During the course of infection, levels of bisecting and triantennary complex glycans were significantly altered. The most abundant glycan in the uninfected sample, a bisecting, monofucosylated, bisialylated complex glycan, decreased dramatically in signal post-infection, becoming suppressed by other high abundant glycans. An isomer of this glycan, which eluted at a later time, increased in abundance fifteen fold after infection. Terminal fucose and sialic acid residues on a glycan with more than two antennas may act as receptors for bacteria and utilized as a source of energy, carbon, and nitrogen. Deficient glycan degrading enzyme activity of the bacteria led to an accumulation of certain oligosaccharide substrates on the cell surface. On average, 176 glycan compositions were identified in the uninfected sample and 166 compositions for the infected sample.



Characterization of glycoengineered biopharmaceuticals

Andres - Copy

Andres Guerrero1; Yanhong Li1; Salem Alkanaimsh2; Lucas Arzola2; Bryce Hashimoto2; Minsook Hwang3; Aye Tu4; My Phu4; Abhaya Dandekar4; Bryce Falk3; Somen Nandi5; Raymond Rodriguez5; Karen McDonald2; Xi Chen1; Carlito Lebrilla1

1UC Davis, Chemistry Department, Davis, CA;2UC Davis, Chemical Engineering & Materials Science, Davis, CA; 3UC Davis, Plant Pathology, Davis, CA; 4UC Davis, Plant Science, Davis, CA; 5UC Davis, Molecular & Cellular Biology, Davis, CA

Novel Aspect:

A method for accurate glycan analysis was developed to monitor the production of a recombinant protein with multiple glycosylation sites.


Glycan analysis of recombinant proteins containing multiple glycosylation sites is analytically challenging especially during the initial phases of production when sample amount and purity are major limitations. In this work we propose a glycoprotein analysis method that addresses these potential limitations, provide site-specific glycan information and facilitate the quantitative comparison between samples.

The method was successfully applied during the production of a glycoengineered recombinant protein: human butyrylcholinesterase (BuChE). BuChE produced in Nicotiana benthamiana was sialytated in vitro to increase its therapeutic potential and mimic the human glycosylation pattern. We describe the glycopeptide characterization of the BuChE glycoforms expressed in different plant subcellular localizations and the glycosylation product, after every step of glycan remodeling, up to the final desired glycosylated product.


The analytical method employed glycopeptide analysis by in-gel digestion using a non-specific protease followed by mass spectrometry. Briefly, samples were desalted by C8 solid phase extraction, denatured and run on a SDS-PAGE gel. Excised protein bands were digested with pronase E and the resulting lysates were extracted and purified for glycopeptides using graphitized solid phase extraction. Tandem-MS analysis was performed on a nanoESI-LC-Q-TOF using a graphitized chip nanoLC column. Glycopeptides were identified using GP-Finder 3 (in-house software) and the corresponding signals quantified by ion-counting. The procedure was initially optimized using another human recombinant plant-made glycoprotein, lactotransferrin produced in Oryza sativa.

Preliminary Data:

The combination of gel electrophoresis and glycopeptide analysis facilitates the analysis of specific proteins in highly impure plant extracts. Additionally, the immobilization of the glycoprotein in the gel allows analysis on even very small amounts of sample. This analytical method was successfully applied to monitor glycosylation on intermediates and products through every step of the glycoprotein production.

Substrate selection

The glycoprotein analysis showed that subcellular localization affects the N-glycan composition of the glycoprotein. The BuChE recovered from the plant endoplasmic reticulum (ER) was almost exclusively glycosylated with high-mannose, while the apoplast targeted extract (APO) was predominantly composed by the basic N-glycan plant core. Based on these results, ER BuChE would require an additional enzymatic step (trim off the high-mannose structures) to reach the desired sialylated structures compared to APO BuChE. Hence, APO BuChE was chosen as a substrate.

Method validation

To obtain the desired glycoform from plant APO BuChE, sequential addition of N-acetylglucosamine, galactose and sialic acid is required using three enzymatic systems, namely N-acetylglucosaminyltransferase, β1,4-galactosyltransferase and α2,6-sialyltransferase. The activity of these enzymes was tested using plant recombinant lactotransferrin that, similarly to APO BuChE, exhibits basic plant core N-glycosylation. The analysis showed the systematic modification of the lactotransferrin N-glycosylation pattern after each redecoration step validating both, the in vitro glycan modification and our analytical approach.


As important as the glycan composition was to the product, the aim was product efficacy. A continuous feedback system was established between redecoration, glycoprotein analysis and protein bioactivity. APO BuChE N-glycan remodelation was performed under different conditions such as temperature and pH. At every step, product was analyzed using the analytical method to observe the progress. The final product contained at least 10%-30% of sialylation. Facilitated by our analytical method, further optimization of the redecoration process is expected to increase the degree of sialylation of the glycoprotein while keeping its activity.


Monitoring Responses of Antibody Glycosylation to HIV Infection

Cynthia Williams

Cynthia Williams; Anne Fenton; Lauren Nagy; Qiuting Hong; L. Renee Ruhaak; Satya Dandekar; Carlito Lebrilla

UC Davis, Davis, CA

Novel Aspect
Monitoring the degree of glycosylation with the absolute protein quantitation in plasma of HIV patients.

The immune system is highly dysregulated in patients with HIV infection. Current studies aim to understand this process to allow for better prevention, earlier diagnosis and a cure. Immunoglobulin concentrations are abnormal in HIV patients and they display altered glycosylation patterns. However, absolute protein quantitation, simultaneously, with site-specific glycosylation analysis of human plasma in HIV samples has previously not been achieved. We are developing methods for quantitation of glycosylated proteins on the protein-specific and site-specific level. In this study, we used multiple reaction monitoring (MRM) to quantitate immunoglobulins A, G, and M (IgA, IgG, and IgM) proteins and their site-specific glycans to examine whether immunoglobulin-specific glycosylation varies in HIV infection.

Plasma samples from 26 male patients and 11 controls were collected and HIV infected patients were broken into 3 groups: No Therapy (n=11), Therapy (n=11), and Long Term Non Progressors (n=4). Two uL of patient sera and IgA, IgG, and IgM standards were digested using trypsin after reduction and alkylation. Protein and glycopeptide quantitation was performed using a QqQ MS in dynamic MRM mode coupled with a UPLC system. Unglycosylated peptides were used for absolute protein quantitation. Site-specific glycosylation was normalized to absolute protein abundances to determine degree of glycosylation.

Preliminary Results/Abstract
Presented here is a high-throughput MRM method for absolute quantitation of IgG, IgA and IgM and their glycosylation analysis directly from 2 uL of serum. A 10 &11-minute UPLC gradient was used with a C18 stationary phase and were employed in the development of the method to quantify protein and site-specific glycan abundances for IgG and IgA & IgM. IgG, A, and M concentrations, as well as subclasses and glycosylation patterns were monitored through 14 MRM transitions for peptides and 53 for glycopeptides.
Our rapid throughput platform was applied to HIV samples (26 patients and 11 controls). For IgG, the peptide common from each subclass was used to quantify the total IgG content. Because this peptide is common to all four subclasses, it correlates linearly to the total IgG protein abundances. Average IgG concentrations were 10.1, 12.3, 13.7, and 19.2 mg/mL in HIV Negative (Neg), Therapy (Tx), Long Term Non-Progessors (LTNP), and No Therapy (No Tx) cases, respectively. Differences with statistical significance were observed in the protein concentrations and in specific IgG glycoforms as well. Galactose deficient glycopeptides were observed to be significantly higher in the HIV positive cases. In IgA, there were no differences observed on the protein level, but on the glycosylation level, Hex4HexNAc4NeuAc1 was shown to be significantly lower in the Tx groups. In comparison to the IgG analysis, the IgM protein concentration levels were only elevated in the No Tx cases. Interestingly, although there were only 4 patient samples within the LTNP group, a positive association was observed between levels of high mannose type glycans and the LTNP response. These results show that the immune responses of HIV patients are clearly altered and that glycosylation is a very important factor.


New Publications! 3/17/2014

Khaldi, N., et al. (2014). “Predicting the important enzyme players in human breast milk digestion.” J Agric Food Chem.
Human milk is known to contain several proteases, but little is known about whether these enzymes are active, which proteins they cleave and their relative contribution to milk protein digestion in vivo. We analyzed the mass spectrometry-identified protein fragments found in pooled human milk by comparing their cleavage sites with the enzyme specificity patterns of an array of enzymes. The results indicate that several enzymes are actively taking part in the digestion of human milk proteins within the mammary gland, including plasmin and/or trypsin, elastase, cathepsin D, pepsin, chymotrypsin, a glutamyl endopeptidase-like enzyme and proline endopeptidase. Two proteins were most affected by enzyme hydrolysis: beta-casein and polymeric immunoglobulin receptor. In contrast, other highly abundant milk proteins such as alpha-lactalbumin and lactoferrin appear to have undergone no proteolytic cleavage. We also show that a peptide sequence containing a known anti-microbial peptide is released in breast milk by elastase and cathepsin D.

Kim, K., et al. (2014). “Evaluation of Glycomic Profiling as a Diagnostic Biomarker for Epithelial Ovarian Cancer.” Cancer Epidemiol Biomarkers Prev.
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. Cancer Epidemiol Biomarkers Prev; 1-11. (c)2014 AACR.

2014 Publications

Dallas, D.C., A. Guerrero, N. Khaldi, R. Borghese, A. Bhandari, M.A. Underwood, C.B. Lebrilla, J.B. German, and D. Barile, A Peptidomic Analysis of Human Milk Digestion in the Infant Stomach Reveals Protein-Specific Degradation Patterns. J Nutr, 2014.

Dallas, D.C., A. Guerrero, E.A. Parker, R.C. Robinson, J. Gan, J.B. German, D. Barile, and C.B. Lebrilla, Current peptidomics- Applications, purification, identification, quantification, and functional analysis. Proteomics, 2014.

Dallas, D.C., V. Weinborn, J.M. de Moura Bell, M. Wang, E.A. Parker, A. Guerrero, K.A. Hettinga, C.B. Lebrilla, J.B. German, and D. Barile, Comprehensive peptidomic and glycomic evaluation reveals that sweet whey permeate from colostrum is a source of milk protein-derived peptides and oligosaccharides Food Res Int, 2014. 63(Pt B): p. 203-209.

Dallas, D.C., C.J. Smink, R.C. Robinson, T. Tian, A. Guerrero, E.A. Parker, J.T. Smilowitz, K.A. Hettinga, M.A. Underwood, C.B. Lebrilla, J.B. German, and D. Barile, Endogenous Human Milk Peptide Release Is Greater after Preterm Birth than Term Birth.. J Nutr, 2014.

De Leoz, M.L., K.M. Kalanetra, N.A. Bokulich, J.S. Strum, M.A. Underwood, J.B. German, D.A. Mills, and C.B. Lebrilla, Human Milk Glycomics and Gut Microbial Genomics in Infant Feces Show a Correlation between Human Milk Oligosaccharides and Gut Microbiota- A Proof-of-Concept StudyJ Proteome Res, 2014.

Guerrero, A., D.C. Dallas, S. Contreras, S. Chee, E.A. Parker, X. Sun, L. Dimapasoc, D. Barile, J.B. German, and C.B. Lebrilla, Mechanistic peptidomics- factors that dictate specificity in the formation of endogenous peptides in human milk. Mol Cell Proteomics, 2014. 13(12): p. 3343-51.

Guerrero, A., L. Lerno, D. Barile, and C.B. Lebrilla, Top-Down Analysis of Highly Post-Translationally Modified Peptides by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry J Am Soc Mass Spectrom, 2014.

Holton, T.A., V. Vijayakumar, D.C. Dallas, A. Guerrero, R.A. Borghese, C.B. Lebrilla, J.B. German, D. Barile, M.A. Underwood, D.C. Shields, and N. Khaldi, Following the digestion of milk proteins from mother to baby J Proteome Res, 2014. 13(12): p. 5777-83.

Kailemia, M.K., L.R. Ruhaak, C.B. Lebrilla, and I.J. Amster,Oligosaccharide Analysis by Mass Spectrometry- A Review of Recent Developments.: A Review Of Recent Developments. Anal Chem, 2013.

Khaldi, N., V. Vijayakumar, D.C. Dallas, A. Guerrero, S. Wickramasinghe, J.T. Smilowitz, J.F. Medrano, C.B. Lebrilla, D.C. Shields, and J.B. German, Predicting the important enzyme players in human breast milk digestion. J Agric Food Chem, 2014.

Kim, K., L.R. Ruhaak, U.T. Nguyen, S.L. Taylor, L. Dimapasoc, C. Williams, C. Stroble, S. Ozcan, S. Miyamoto, C.B. Lebrilla, and G.S. Leiserowitz, Evaluation of Glycomic Profiling as a Diagnostic Biomarker for Epithelial Ovarian Cancer. Cancer Epidemiol Biomarkers Prev, 2014.

Hong, Q., L.R. Ruhaak, S.M. Totten, J.T. Smilowitz, J.B. German, and C.B. Lebrilla, Label Free Absolute Quantitation of Oligosaccharides using Multiple Reaction Monitoring., 2014.

Hua, S., M. Saunders, L.M. Dimapasoc, S.H. Jeong, B.J. Kim, S. Kim, M. So, K.S. Lee, J.H. Kim, K.S. Lam, C.B. Lebrilla, and H.J. An, Differentiation of Cancer Cell Origin and Molecular Subtype by Plasma Membrane N-Glycan Profiling.. J Proteome Res, 2013.

Huang, J., H. Lee, A.M. Zivkovic, J.T. Smilowitz, N. Rivera, J.B. German, and C.B. Lebrilla, Glycomic Analysis of High Density Lipoprotein Shows a Highly Sialylated Particle J Proteome Res, 2014.

Mehra, R., D. Barile, M. Marotta, C.B. Lebrilla, C. Chu, and J.B. German, Novel high-molecular weight fucosylated milk oligosaccharides identified in dairy streams. PLoS One, 2014. 9(5): p. e96040.

Ozcan, S., B.J. Kim, G. Ro, J.H. Kim, T.L. Bereuter, C. Reiter, L. Dimapasoc, D. Garrido, D.A. Mills, R. Grimm, C.B. Lebrilla, and H.J. An, Glycosylated proteins preserved over millennia- N-glycan analysis of Tyrolean Iceman, Scythian Princess and Warrior.. Sci Rep, 2014. 4: p. 4963.

Ozcan, S., D.A. Barkauskas, L. Renee Ruhaak, J. Torres, C.L. Cooke, H.J. An, S. Hua, C.C. Williams, L.M. Dimapasoc, J. Han Kim, M. Camorlinga-Ponce, D. Rocke, C.B. Lebrilla, and J.V. Solnick, Serum glycan signatures of gastric cancer.. Cancer Prev Res (Phila), 2014. 7(2): p. 226-35.

Ruhaak, L.R., C. Stroble, M.A. Underwood, and C.B. Lebrilla, Detection of milk oligosaccharides in plasma of infants.. Anal Bioanal Chem, 2014.

Smilowitz, J.T., C.B. Lebrilla, D.A. Mills, J.B. German, and S.L. Freeman, Breast Milk Oligosaccharides- Structure-Function Relationships in the Neonate. Annu Rev Nutr, 2013.

Song, T., S. Ozcan, A. Becker, and C.B. Lebrilla, In-Depth Method for the Characterization of Glycosylation in Manufactured Recombinant Monoclonal Antibody Drugs. Anal Chem, 2014.

Totten, S.M., L.D. Wu, E.A. Parker, J.C. Davis, S. Hua, C. Stroble, L.R. Ruhaak, J.T. Smilowitz, J.B. German, and C.B. Lebrilla, Rapid-throughput glycomics applied to human milk oligosaccharide profiling for large human studies. Anal Bioanal Chem, 2014. 406(30): p. 7925-35.

Underwood, M.A., K.M. Kalanetra, N.A. Bokulich, M. Mirmiran, D. Barile, D.J. Tancredi, J.B. German, C.B. Lebrilla, and D.A. Mills, Prebiotic Oligosaccharides In Premature Infants, 2014. 58(3): p. 352-60.

New Publications!

Dallas, D. C., A. Guerrero, N. Khaldi, P. A. Castillo, W. F. Martin, J. T. Smilowitz, C. L. Bevins, D. Barile, J. B. German and C. B. Lebrilla (2013). “Extensive in vivo Human Milk Peptidomics Reveals Specific Proteolysis Yielding Protective Antimicrobial Peptides.” J Proteome Res 12(5): 2295-2304.
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 beta-casein, however no peptide fragments from the major milk proteins lactoferrin, alpha-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.

Ruhaak, L. R., U. T. Nguyen, C. Stroble, S. L. Taylor, A. Taguchi, S. M. Hanash, C. B. Lebrilla, K. Kim and S. Miyamoto (2013). “Enrichment strategies in glycomics based lung cancer biomarker development.” Proteomics Clin Appl.
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. 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 (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. This article is protected by copyright. All rights reserved.

Strum, J. S., C. C. Nwosu, S. Hua, S. R. Kronewitter, R. R. Seipert, R. J. Bachelor, H. J. An and C. B. Lebrilla (2013). “Automated Assignments of N- and O-Site Specific Glycosylation with Extensive Glycan Heterogeneity of Glycoprotein Mixtures.” Anal Chem.
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.


Determination of Extensive Glycosylation on Glycoproteins and Glycolipids in High-density Lipoprotein

Jincui Huang
Jincui Huang; Hyeyoung Lee; Angela Zivkovic; Jennifer Smilowitz; Bruce German; Carlito Lebrilla
UC Davis, Davis, CA
NOVEL ASPECT: Extensive site-specific glycosylation of HDL proteins and glycolipid analysis indicate that HDL is a highly glycosylated and sialylated particle.
High-density lipoprotein (HDL) particles are composed of lipids and lipoproteins and are considered as a strong inverse predictor of risk of cardiovascular disease. The function of HDL in health and diseases is related to its role in reverse cholesterol transport and in its interactions with cell membrane receptors, enzymes, and lipid-transport proteins. The proteins and lipids of HDL are potentially glycosylated. Because of the role of glycosylation in recognition, protein folding, and protein half-life in blood, it may contribute significantly to the overall function of HDL However, very little is known concerning the glycosylation of  HDL. Using glycomics, glycolipidomics and glycoproteomics approaches, we have determined for the first time the site-specific glycosylation in proteins and lipid glycosylation of HDL.
Isolation of HDL was performed by ultracentrifugation. N-Linked glycans were released from the HDL solution using PNGase F. Pronase E, covalently coupled to CNBr activated sepharose beads, was reacted to the HDL by incubating at 37 ºC overnight. The digestion mixtures were desalted and enriched via solid phase extraction (SPE) prior to the analyses. Extraction of gangliosides was performed by mixing water, methanol and chloroform with a HDL solution after centrifugation. The supernatant was collected and cleaned by SPE. MS and MS/MS analyses of the glycopeptides and gangliosides were achieved via nano-LC/Q-TOF MS with a nano-LC column integrated in a micro-chip packed with porous graphitized carbon for glycan/glycopeptides and C18 for gangliosides.
Proteomics analysis of HDL particles revealed a list of apolipoproteins as well as the HDL associated plasma proteins, approximately 60% of which are glycosylated.Glycans from HDL were separated with a PGC stationary phase and corresponded to over 60 N-linked glycan compounds with over 20 distinct compositions. Each composition produced two or more chromatographic peaks corresponding to structural isomers. Sialylated glycans including complex type with bi- or tri- antenna were found to be the dominant glycan types. Interestingly, a biantennary glycan with two sialic acids was the most abundant glycan species as is found in blood. The structures of N-glycans from HDL associated glycoproteins were further elucidated by tandem MS.Comprehensive glycan-microheterogeneity of HDL associated glycoproteins was determined using non-specific proteolysis and analysis with nano-LC/Q-TOF MS.  An in-house software, Glycopeptide Finder, was used to interpret the tandem MS. This method lead to the characterization of both N- or O-glycans whose site was determined based on the associated peptide tags. In all, over 50 glycopeptides corresponding to 20 glycosites from more than 10 glycoproteins were observed and determined in the HDL protein mixture. These glycopeptides were a mixture of N-linked glycopeptides (mainly biantennary complex type sialylated glycans) and O-linked glycopeptides (mainly sialylated). The results yielded comprehensive glycoprotein analysis with detailed glycan microheterogeneity.Ganglioside analysis showed that GM3 (monosialoganglioside, NeuAc2–3Gal1–4Glc–Cer) and GD3 (disialoganglioside, NeuAc2–8NeuAc2–3Gal1–4Glc–Cer) are the most abundant glycolipids in human HDL. A proportion of 75% GM3 and 25% GD3 were obtained. Both GM3 and GD3 are composed of heterogeneous ceramide lipids, including d34:1, d39:1, d40:1 and d41:1.

Cell Membrane Glycan Profiling Differentiates Cancer Cell Origin and Molecular Subtype

Serenus Hua1; Lauren Dimapasoc2; Mary Saunders²; Bum Jin Kim1; Seung Hyup Jeong1; Kit S. Lam², Carlito Lebrilla2Hyun Joo An1
1GRAST, Chungnam National University, Daejeon, SOUTH KOREA; 2University of California, Davis, Davis, CA
NOVEL ASPECT: Stark differences in cancer cell membrane glycosylation can be exploited to create an MS-based biopsy
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, biopsy results are inconclusive. Moreover, even when primary cancer origin can be identified, phenotypic subtypes are rarely differentiated, often leading to inefficient or ineffective treatment. Glycomic profiling of the cell membrane offers an alternate route towards cancer diagnosis. In this study, isomer-sensitive nano-LC/MS and -LCMS/MS were used to obtain a detailed, structure-specific profile of the different N-glycan structures present on cancer cell membranes. Application of this method to biopsy samples may provide complementary or supplementary information that can be used to aid cancer diagnosis and guide treatment.
Cells were harvested from cell lines representing various subtypes of breast, lung, cervical, ovarian, and lymph node cancer. After gentle lysing, cell membranes were isolated by ultracentrifugation. N-glycans were released enzymatically, and then enriched by graphitized carbon solid phase extraction.
Native glycans were reproducibly profiled and characterized by chip-based nano-LC/MS and -LC/MS/MS. Using established human glycan structure/composition libraries, glycan signals were rapidly identified and sorted into biologically relevant classes and categories, such as high-mannose glycans, hybrid glycans, truncated complex glycans, fucosylated or sialylated complex glycans, etc. Statistical methods including Pearson correlation, hierarchical clustering, and t-tests were used to correlate or differentiate the various cell lines.
Chip-based nano-LC/MS analysis of the cell membrane N-glycomes provided high retention time reproducibility and quantitative precision. Structure-sensitive N-glycan profiling identified hundreds of glycan peaks per cell line, including multiple isomers for most compositions.
Most cancer cell lines exhibited high  levels (~30 to 60% relative abundance) of high-mannose glycosylation, an established hallmark of cancer. However, significant differences between the individual cell lines were easily observable. Hierarchical clustering based on Pearson correlation coefficients was used to quickly compare and separate each cell line according to originating organ and disease subtype. For example, a comparison of four B-cell lymphoma cell lines easily clustered together two cell lines originating from endemic and sporadic Burkitt’s lymphoma patients (from Nigeria and America, respectively) with a correlation coefficient R of 0.9744 while simultaneously differentiating both of them from the other two B-cell lymphoma cell lines.
Similar comparisons were able to differentiate several breast cancer cell lines from a (non-cancerous) fibrocystic breast cell line; and HPV-infected cervical carcinoma cells from non-HPV-infected cervical carcinoma cells.
To demonstrate the diagnostic  possibilities of this method, simple dichotomous keys were created. 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 and identified. More closely-related cell lines were differentiated based on several-fold differences in the abundances of individual glycans. For example, lung carcinoma cell lines NCI-H358 and A549 were differentiated by parallel six-fold differences between the abundances of biosynthetically-related N-glycans Hex3HexNAc2Fuc, Hex3HexNAc3Fuc, and Hex3HexNAc4Fuc (which each differ from the next by only one HexNAc). In clinical settings, similar keys might allow a diagnostician to quickly and rapidly identify different cancer cell types based on a glycomic profile.

Digestomics of human milk proteins in term and premature infants

David Dallas1; Andres Guerrero1; Nora Khaldi2; Bruce German1; Daniela Barile1; Mark Underwood1; Carlito Lebrilla1
1University of California, Davis, Davis, CA; 2UC Dublin, Dublin, Ireland
NOVEL ASPECT: This study represents a novel application of high-throughput peptidomics to investigate milk protein digestion in term and premature infants.
Decades of research demonstrate that human milk protein digestion releases peptide fragments with beneficial antimicrobial and immunomodulatory functions for the infant. However, as previous studies were based on in vitro digestion of milk, the relevance of these findings is unknown. For the first time, we analyze peptides released from human milk in the infant stomach in vivo. Released peptide sequences are determined via our novel, high-throughput peptidomics technique and potential functions are examined. Because premature infants have lower acid and enzyme production in the stomach than term infants, we hypothesize that premature infants do not produce the same functional milk peptides as term infants. This study compares digestion between these infant groups to identify missing functional peptides in premature infants.
In the UC Davis Medical School’s neonatal intensive care unit, gastric samples were obtained from term and premature infants after 2 hours of digestion. Infants already intubated for medical conditions preventing normal feeding, but without digestive disorders were selected. Peptides were extracted from all samples in 96-well plate format by centrifugal delipidation, acid precipitation of proteins, and C18 solid phase extraction clean-up.Peptides were measured by nano-LC-Q-TOF with automated peak selection for tandem fragmentation. Tandem spectra were searched in X!Tandem with no enzyme selected.
A library of identified peptides with exact mass and retention time was applied back to all samples for peak volume extraction. Univariate statistical testing was employed for peptide abundance comparison between term and premature infants.

Analysis of peptides with the LC-MS/MS technique revealed hundreds of unique peptides in all mother’s milk and infant gastric samples. The number of unique peptides increased 3-fold from intact milk to gastric samples, demonstrating that digestion does occur in the infant stomach. Specific proteins that were not digested in the mammary gland, like lactoferrin and alpha-lactalbumin, are degraded within the infant stomach of both term and premature infants. Evidence for digestion in the stomach is surprising because conditions in the infant stomach are far from optimal for the only known infant gastric protease:
pepsin. Enzyme analysis of the cleavage sites revealed that pepsin is active at low levels in the infant stomach as early as the first week of life. Moreover, this analysis revealed that milk proteases that were initially blocked by antiproteases in the mammary gland become functional in the infant stomach and are responsible for the majority of cleavages. This demonstrates that milk continues to guide its own digestion within the infant gut. The factor causing activation of these milk enzymes in the infant stomach remains unknown.Statistical analysis of the differences in gastric digestion between term and premature infants is underway. Identification of milk enzymes as the main contributors to gastric digestion demonstrate that differences in proteases and antiproteases between term and premature mother’s milk may contribute to differences in peptide release in the stomach.

Approximately 10% of the identified peptides were homologous with known antimicrobial or immunomodulatory peptides. Differences in these functional peptides may contribute to the variance in health outcomes between term and premature infants.

Mass Spectrometry Based Glycan Arrays for Determining Specificity of Glycosidases in Bacteria

Sarah Totten; Santiago Ruiz-Moyano; David Mills; Carlito Lebrilla
University of California Davis, Davis, CA
NOVEL ASPECT: The application of mass spectrometry based glycan arrays and high-throughput HMO analysis to characterizing the specificity of glycosidases in bacteria
Human milk oligosaccharides function as prebiotics for beneficial bacteria that occupy the gut of breast fed infants. Certain species of Bifidobacteria are equipped with the glycosidases necessary for oligosaccharide digestion and have demonstrated the ability to metabolize HMOs.  The mechanism and specificity of HMO consumption of each subspecies has not been fully characterized. In this study, the digestion of a glycan array (over 100 human milk oligosaccharide species extracted from a pooled milk sample) is rapidly profiled using sensitive, high-throughput nano-LC Chip/TOF mass spectrometry-based methods for a series of glycosidases.  The resulting digestion glycoprofile provides insight to the specificity of the enzymes used by gut bacteria to selectively metabolize milk oligosaccharides.
Pooled milk was first defatted via centrifugation and depleted of protein by ethanol precipitation.
The isolated HMOs were then reduced to alditol form using NaBH4.  Solid phase extraction on graphitized carbon cartridges (GCC) was used for desalting and enrichment. HMOs were eluted with 40% acetonitrile in 0.05% trifluoroacetic acid (v/v).  SPE fractions were dried and reconstituted to approximately 1.5 mg/mL. The reduced HMO pool was then digested by a β-galactosidase isolated from B. longum subsp. infantis ATCC15697 (Blon_2016) for 1 hour at 37°C, pH 4.5. The digestion mixture was then filtered using Millipore C18 ZipTip pipette tips.  The HMO pool was profiled using nano-LC Chip/TOF mass spectrometry on a PGC chip both before (undigested control) and after digestion.
Over 200 oligosaccharides were detected in the pooled milk sample before digestion, to which monosaccharide composition was assigned based on accurate mass (typically within ±5 ppm).  An annotated in-house HMO library containing 75 neutral and anionic species was used to rapidly assign specific structure using the reproducible LC retention time and CID fragmentation patterns. Using chip-TOF mass spectrometry, the digestion of individual HMO structures was quantitated with respect to the undigested control by normalzing the absolute peak intensity of the digested sample to that of the control.  The total oligosaccharide abundance decreased in the digested sample by 32%, almost all of which can be attributed to the digestion of neutral, non-fucosylated species, whose abundance decreased by 80%. Isomers of m/z 710.23 Lacto-N-tetraose and lacto-N-neotetraose, and isomers of m/z 1075.41 lacto-N-hexaose, lacto-N-neohexaose and para lacto-N-hexaose were consumed to the greatest extents (the abundance decreased by more than 90% in the digested sample).  The formation of peaks with m/z 548.23 and m/z 751.30 in the digested sample indicate the loss of one or two galactose residues from the structures listed above, respectively, demonstrating that the β-galactosidase cleaves both β1-3 and β1-4 galactoses in the terminal positions.  When the above core structures are fucosylated, they are digested more selectively. Monofucosylated isomers lacto-n-fucopentaose I, II, and III were not cleaved by the enzyme, however monofucosylated structures of increasing degrees of polymerization with un-decorated terminal galactoses were almost completely digested, suggesting that the presence of a fucose residue, either linked to the same GlcNAc as the terminal galactose or linked to the terminal galactose itself, blocks the cleavage of that terminal galactose on both type 1 and type 2 chains.The mass  spectrometry-based characterization of the enzymatic digestion of the glycan array described above by additional galactosidases, fucosidases, and sialidases is currently underway.
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