Nutritional and Clinical Glycomics Research

Author: laurenmd (Page 3 of 9)

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

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Authors

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

Institutes
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.

Introduction
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.

Methods
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.

 

An Application of Mass Spectrometry for the Detection of Chemical Markers for Product Traceability

Evan Parker

Authors
Evan Parker; Carlito Lebrilla

Institutes
UC Davis, Davis, CA


Novel Aspect
First application of monodisperse polymers for encoding information in materials. Derivative methods could lead to commodity utility of mass spectrometry.

Introduction
Product safety of pharmaceuticals and food products being of paramount, industry expends great effort in the tracking and validation of product source and identity throughout the supply chain. Here, a method is described that uses a food safe, GRAS status, oligomer marker to encode binary information in bulk materials. Similar methods employ the use of dyes, fluorescent compounds, nanoparticles, microfabricated markers, and macroscopic markers. These methods tend to be either costly or easily replicated while often having the benefit of easy detection. In our method a host of monodisperse formulated polyethylene glycol oligomers can be added to a product to encode binary information detected and can be easily with MALDI MS.

Methods
We are using oligomers of polyethylene glycol (PEG) as single-unit characters in our encoding scheme. This category of molecule is useful because it is inert, nontoxic, and synthetic methods exist that would allow preparation at scale. As a proof of concept, eight oligomers of this compound ranging in length from 11 to 19 subunits were fractionated by reversed phase HPLC. The oligomers are then added to test mixtures and detected using MALDI mass spectrometry with DHB matrix. Both MALDI FT-ICR and MALDI-TOF were used to test the method. To find limits of detection we doped dilutions of a polydisperse mixture of PEG 600 into various matrices. Clear detection of the most abundant eight peaks constituted a positive hit.

Preliminary Results/Abstract
To prepare peg monomers, reversed phase chromatography was employed to separate mixtures of PEG 600 and PEG 1000 into their monodisperse components. Monitoring the absorption trace at 192 nanometers showed clear baseline separation even in overloading conditions necessary for preparation. Using this method, between 0.5 and 1 mg of each of the purified monodisperse components from 10 to 20 subunits long were collected. These collected markants were doped at approximately 100 ppm into both water and milk as a proof of concept for the actual encoding of information. We used mass spectrometry to collect the codes 11111111, 01001101, and 01010011 encoding to binary 255, and ASCII M and S respectively.
Using serial dilutions of polydisperse peg 600 we show that acceptable signal to noise can be achieved at concentration of 1 ppm by mass. To prove the utility of the markant in complex samples, various concentrations of the polydisperse solution were added to whole milk. We have shown that PEG can be readily detected at 50 ppm with a simple cleanup procedure entailing centrifugation and reversed phase SPE. To confirm the method’s utility as a surface marker, various amounts were sprayed on the surface of a cantaloupe and, after a week of storage on a shelf, PEGs were recovered via an extraction procedure entailing the peeling of the cantaloupe and vortexing of diced peel with water. We showed that the detection limit is below 0.1% spray concentration or 4 mg per 100 square centimeters or, roughly 50 mg for a 20 cm diameter cantaloupe that has been uniformly treated.

 

Peptidomics of human milk during lactation and mastitis

Andres - Copy

Authors
Andres GuerreroStephanie ContrerasDave Dallas; Lauren Wu; Jennifer Smilowitz; Daniela Barile; Bruce German; Carlito Lebrilla

Institutes
University of California, Davis, Davis, CA


Novel Aspect
Peptidomics analysis of milk yields bioactive peptides that vary during lactation and disease.

Introduction
Human milk, as other biofluids, contains proteolytic enzymes that produce endogenous peptides. A library of endogenous peptides found in human milk with post-translational modifications was created through the use of nano-LC and tandem mass spectrometry. This library was used to develop a method to identify peptides rapidly allowing monitoring with quantitation nearly 1000 peptides with nano-LC MS. The method was used to examine changes in the peptide abundances during lactation. A comprehensive understanding of the endogenous proteolytic activity in human milk can be achieved to compare different milk samples. In addition because many of the peptides have antimicrobial activity, we examined the effect of peptide production and abundances during diseases such as mastitis infection.

Methods
Peptide separation was performed using 25 ?L of human milk that was spiked with an internal standard. The samples were defatted and trichloroacetic acid was added to precipitate the peptides. The samples were cleaned up by solid phase extraction using a C-18 column. The samples were analyzed on a nano-LC Q-TOF with a C-18 chip to obtain MS and MS/MS data. The tandem mass spectrometry information from each sample was exported as an MGF file and uploaded to X! tandem to identify peptides present in each milk sample analyzed. When the peptides in milk were identified an in-house software was used to visualize the most abundant peptides present within a protein.

Preliminary Results/Abstract
The peptide milk library contains peptide fragments from different proteins along with their masses, retention times, and post-translational modifications that may occur on certain amino acids that include phosphorylation, oxidation, deamination, and water loss. The peptide milk library was used to identify peptides from different proteins of mothers with and without mastitis. Based on the analysis, we found that the vast majority of the peptides came from five proteins in milk: ?-casein, ?-casein, polymeric immunoglobulin, osteopontin, and butryophilin. By using an in-house software we were able to assign the peptide fragments within these proteins and identify locations where the abundances varied the most. This software also allowed us to locate the peptides within these proteins that had phosphorylation sites. For example, with ?-casein the peptides that had 1, 2, and 3 phosphorylation sites were all located at the N-terminus of the protein, whereas the peptides with no phosphorylation sites where found throughout the protein. Analysis of milk from mothers with mastitis were performed. The total peptide abundances from five proteins were summed over several time points to determine whether peptide abundances varied during mastitis. There was a general trend of a decrease in the peptide abundances around the time the mother had mastitis.

 

New 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.

In vitro digestion of isolated milk proteins results in milk peptides with a variety of actions. However, it remains unclear to what degree protein degradation occurs in vivo in the infant stomach and whether peptides previously annotated for bioactivity are released. This study combined nanospray liquid chromatography separation with time-of-flight mass spectrometry, comprehensive structural libraries, and informatics to analyze milk from 3 human mothers and the gastric aspirates from their 4- to 12-d-old postpartum infants. Milk from the mothers contained almost 200 distinct peptides, demonstrating enzymatic degradation of milk proteins beginning either during lactation or between milk collection and feeding. In the gastric samples, 649 milk peptides were identified, demonstrating that digestion continues in the infant stomach. Most peptides in both the intact milk and gastric samples were derived from beta-casein. The numbers of peptides from beta-casein, lactoferrin, alpha-lactalbumin, lactadherin, kappa-casein, serum albumin, bile salt-associated lipase, and xanthine dehydrogenase/oxidase were significantly higher in the gastric samples than in the milk samples (P < 0.05). A total of 603 peptides differed significantly in abundance between milk and gastric samples (P < 0.05). Most of the identified peptides have previously identified biologic activity. Gastric proteolysis occurs in the term infant in the first 2 wk of life, releasing biologically active milk peptides with immunomodulatory and antibacterial properties of clinical relevance to the proximal intestinal tract. Data are available via ProteomeXchange (identifier PXD000688).

 

Rapid Assays of Bacteria Binding to Oligosaccharides

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Authors
Qian Wang1; Zachery Lewis2, 3; Andres Guerrero1; David Mills2, 3; Carlito Lebrilla1

Institutes
1Department of Chemistry, UC Davis, Davis, CA; 2Department of Viticulture & Enology, UC Davis, Davis, CA; 3Department of Food Science & Technology, UC Davis, Davis, CA


Novel Aspect
A fast and simple method employing direct MALDI MS to compare the affinities between oligosaccharides and bacteria.

Introduction
A necessary step of bacterial colonization and infection is the adherence of the bacteria to host cell surfaces. The adherence factors of bacteria are called adhesins, which consist of two major types: protein adhesins and carbohydrate adhesins. The adherence factors of host cells which adhesins recognize are called receptors, which are usually carbohydrate or peptide residues on the surface of host cells. The specific adherence of adhesins to receptors determines the tissue specificity of binding. In this study, the goal is to develop a rapid mass spectrometry method with the use of MALDI TOF/TOF mass spectrometer to analyze the specific affinities between different bacteria and oligosaccharides which are a major type of receptors.

Methods
First, oligosaccharides solutions are applied on the MALDI spots and the MALDI plate (with immobilized phenylboronic acid on the surface which is able to bind to oligosaccharides covalently) is incubated in humidified atmosphere. After that, the unbound oligosaccharides are washed away. Then bacteria suspension is applied on the MALDI spots and the MALDI plate is incubated at 37 oC. The unbound bacteria are washed away after incubation (For the control spots, unbound bacteria are not washed away). Lastly the matrix is applied and mass spectra are obtained. If the mass spectrum matches that of the control spot, then the affinity is approved. If no (or weak) mass spectrum is observed then the affinity is disproved (or the affinity is weak).

Preliminary Data
We studied the amount of oligosaccharides to saturate the MALDI spots. If the spots are not saturated by oligosaccharides, then bacteria may bind to the plate directly which will produce false signal. Different amounts of oligosaccharides were applied on each spot and mass spectra were obtained. Starting from the spot with 10-10 mole oligosaccharides, the peak of sodiated molecular ion of the oligosaccharide was observed suggesting that the spot had been saturated. So 10-10 mole of oligosaccharides is enough to saturate a spot.

The mass spectra of the control spots were obtained. Bifidobacterium longum subsp. infantis ATCC 15697, a commensal bacteria abundant in breast fed infants, Lacto-N-tetraose, disialyllacto-N-tetraose, 3-sialyllactose, 3’-sialyllactose, 6’-sialyllactose and Lewis B tetrasaccharide were used to probe the binding interactions. Bacteria that were bound to the specific oligosaccharide-activated surfaces were analyzed by direct MALDI. The mass spectra from these compounds were the same suggesting that mass spectra were not dependent on the oligosaccharides. Escherichia Coli DH5-alpha was also applied to the surface. These bacteria yielded spectra that were distinct from B. infantis. The mass spectra of B. infantis and E. Coli with the same oligosaccharide are unique and reproducible suggesting that this method is able to differentiate different types of bacteria. An effective washing method to remove unbound bacteria was developed to probe the various bacterial surface and determine the specific affinity of the bacteria to the oligosaccharides.

 

 

Determination of changes in cell surface glycosylation with cellular transformations

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

Institutes
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.

Introduction
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.

Methods
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

Authors
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

Institutes
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.

Introduction:

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.

Methods:

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.

Optimization

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

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

Institutes
UC Davis, Davis, CA


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

Introduction
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.

Methods
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.

New Publications! 2/2014

Hong, Q., et al. (2014). “Label Free Absolute Quantitation of Oligosaccharides using Multiple Reaction Monitoring.Anal Chem.

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 post source 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 tri-saccharide 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 five 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.

 

Ozcan, S., et al. (2014). “Serum glycan signatures of gastric cancer.” Cancer Prev Res (Phila) 7(2): 226-235.

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. Cancer Prev Res; 7(2); 226-35. (c)2013 AACR.

 

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