Maria Lorna A. De Leoz

Glycomics and disease markers

Glycomics is the comprehensive study of all glycans expressed in biological systems. The biosynthesis of glycan relies on a number of highly competitive processes involving glycosyl transferases. Glycosylation is therefore highly sensitive to the biochemical environment and has been implicated in many diseases including cancer. Recently, interest in profiling the glycome has increased owing to the potential of glycans for disease markers. In this regard, mass spectrometry is emerging as a powerful technique for profiling the glycome. Global glycan profiling of human serum based on mass spectrometry has already led to several potentially promising markers for several types of cancer and diseases.

High-mannose glycans are elevated during breast cancer progression

Alteration in glycosylation has been observed in cancer. However, monitoring glycosylation changes during breast cancer progression is difficult in humans. In this study, we used a well-characterized transplantable breast tumor mouse model, the mouse mammary tumor virus-polyoma middle T antigen, to observe early changes in glycosylation. We have previously used the said mouse model to look at O-linked glycosylation changes with breast cancer. In this glycan biomarker discovery study, we examined N-linked glycan variations during breast cancer progression of the mouse model but this time doubling the number of mice and blood draw points. N-glycans from total mouse serum glycoproteins were profiled using matrix-assisted laser desorption/ionization Fourier transform-ion cyclotron resonance mass spectrometry at the onset, progression, and removal of mammary tumors. We observed four N-linked glycans, m/z 1339.480 (Hex3HexNAc), 1485.530 (Hex3HexNAc4Fuc), 1809.639 (Hex5HexNAc4Fuc), and 1905.630 (Man9), change in intensity in the cancer group but not in the control group. In a separate study, N-glycans from total human serum glycoproteins of breast cancer patients and controls were also profiled. Analysis of human sera using an internal standard showed the alteration of the low-abundant high-mannose glycans, m/z 1419.475, 1581.528, 1743.581, 1905.634 (Man6–9), in breast cancer patients. A key observation was the elevation of a high-mannose type glycan containing nine mannoses, Man9, m/z 1905.630 in both mouse and human sera in the presence of breast cancer, suggesting an incompletion of the glycosylation process that normally trims back Man9 to produce complex and hybrid type oligosaccharides.

The development of retrosynthetic glycan libraries to profile and classify the human serum N-linked glycome.

Annotation of the human serum N‐linked glycome is a formidable challenge but is necessary for disease marker discovery. A new theoretical glycan library was constructed and proposed to provide all possible glycan compositions in serum. It was developed based on established glycobiology and retrosynthetic state‐transition networks. We find that at least 331 compositions are possible in the serum N‐linked glycome. By pairing the theoretical glycan mass library with a high mass accuracy and high‐resolution MS, human serum glycans were effectively profiled. Correct isotopic envelope deconvolution to monoisotopic masses and the high mass accuracy instruments drastically reduced the amount of false composition assignments. The high throughput capacity enabled by this library permitted the rapid glycan profiling of large control populations. With the use of the library, a human serum glycan mass profile was developed from 46 healthy individuals. This paper presents a theoretical N‐linked glycan mass library that was used for accurate high‐throughput human serum glycan profiling. Rapid methods for evaluating a patients glycome are instrumental for studying glycan‐based markers.

Human milk glycomics and gut microbial genomics in infant feces show a correlation between human milk oligosaccharides and gut microbiota: A proof-of-concept study

Human milk oligosaccharides (HMOs) play a key role in shaping and maintaining a healthy infant gut microbiota. This article demonstrates the potential of combining recent advances in glycomics and genomics to correlate abundances of fecal microbes and fecal HMOs. Serial fecal specimens from two healthy breast-fed infants were analyzed by bacterial DNA sequencing to characterize the microbiota and by mass spectrometry to determine abundances of specific HMOs that passed through the intestinal tract without being consumed by the luminal bacteria. In both infants, the fecal bacterial population shifted from non-HMO-consuming microbes to HMO-consuming bacteria during the first few weeks of life. An initial rise in fecal HMOs corresponded with bacterial populations composed primarily of non-HMO-consuming Enterobacteriaceae and Staphylococcaeae. This was followed by decreases in fecal HMOs as the proportion of HMO-consuming Bacteroidaceae and Bifidobacteriaceae increased. Analysis of HMO structures with isomer differentiation revealed that HMO consumption is highly structure-specific, with unique isomers being consumed and others passing through the gut unaltered. These results represent a proof-of-concept and are consistent with the highly selective, prebiotic effect of HMOs in shaping the gut microbiota in the first weeks of life. The analysis of selective fecal bacterial substrates as a measure of alterations in the gut microbiota may be a potential marker of dysbiosis.

Glycomic Approach for Potential Biomarkers on Prostate Cancer: Profiling of N-Linked Glycans in Human Sera and pRNS Cell Lines

Prostate cancer is a leading cause of cancer death among men. Currently available screening test measures prostate-specific antigen (PSA) to detect prostate cancer. However, this test produces false positive values that often lead to negative biopsies. Therefore, a more reliable diagnostic tool is needed. Glycans in serum are of particular interest as around half of all proteins are glycosylated. In this study, N-linked glycans were enzymatically released by PNGase F from prostate epithelial cell lines (pRNS) expressing wild type or mutant androgen receptors and a small set of human serum samples. Released glycans were purified and partitioned into neutral and acidic components by solid phase extraction (SPE) using graphitized carbon cartridges. The SPE fractions were analyzed by matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI FT-ICR MS). Significant changes in some high-mannose and fucosylated biantennary complex N-linked glycans were observed in the serum of prostate cancer patients.

A quantitative and comprehensive method to analyze human milk oligosaccharide structures in the urine and feces of infants

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

Human Serum Processing and Analysis Methods for Rapid and Reproducible N-Glycan Mass Profiling

Glycans constitute a new class of compounds for biomarker discovery. Glycosylation is a common post-translational modification and is often associated with transformation to malignancy. To analyze glycans, they are released from proteins, enriched, and measured with mass spectrometry. For biomarker discovery, repeatability at every step of the process is important. Locating and minimizing the process variability is key to establishing a robust platform stable enough for biomarker discovery. Understanding the variability of the measurement devices helps understand the variability associated with the chemical processing. This report explores the potential use of methods expediting the enzymatic release of glycans such as a microwave reactor and automation of the solid-phase extraction with a robotic liquid handler. The study employs matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance mass spectrometry but would be suitable with any mass spectrometry method. Methods for system-wide data analysis are examined because proper metrics for evaluating the performance of glycan sample preparation procedures are not well established.

Human milk oligosaccharides in premature infants: absorption, excretion, and influence on the intestinal microbiota

Background:Human milk oligosaccharides (HMOs) shape the intestinal microbiota in term infants. In premature infants, alterations in the intestinal microbiota (dysbiosis) are associated with risk of necrotizing enterocolitis (NEC) and sepsis, and the influence of HMOs on the microbiota is unclear.Methods:Milk, urine, and stool specimens from 14 mother–premature infant dyads were investigated by mass spectrometry for HMO composition. The stools were analyzed by next-generation sequencing to complement a previous analysis.Results:Percentages of fucosylated and sialylated HMOs were highly variable between individuals but similar in urine, feces, and milk within dyads. Differences in urine and fecal HMO composition suggest variability in absorption. Secretor status of the mother correlated with the urine and fecal content of specific HMO structures. Trends toward higher levels of Proteobacteria and lower levels of Firmicutes were noted in premature infants of nonsecretor mothers. Specific HMO structures in the milk, urine, and feces were associated with alterations in fecal Proteobacteria and Firmicutes.Conclusion:HMOs may influence the intestinal microbiota in premature infants. Specific HMOs, for example those associated with secretor mothers, may have a protective effect by decreasing pathogens associated with sepsis and NEC, while other HMOs may increase dysbiosis in this population.

The Glycolyzer: Automated Glycan Annotation Software for High Performance Mass Spectrometry and Its Application to Ovarian Cancer Glycan Biomarker Discovery

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

Detecting glycan cancer biomarkers in serum samples using MALDI FT-ICR mass spectrometry data

MOTIVATION The development of better tests to detect cancer in its earliest stages is one of the most sought-after goals in medicine. Especially important are minimally invasive tests that require only blood or urine samples. By profiling oligosaccharides cleaved from glycosylated proteins shed by tumor cells into the blood stream, we hope to determine glycan profiles that will help identify cancer patients using a simple blood test. The data in this article were generated using matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI FT-ICR MS). We have developed novel methods for analyzing this type of mass spectrometry data and applied it to eight datasets from three different types of cancer (breast, ovarian and prostate). RESULTS The techniques we have developed appear to be effective in the analysis of MALDI FT-ICR MS data. We found significant differences between control and cancer groups in all eight datasets, including two structurally related compounds that were found to be significantly different between control and cancer groups in all three types of cancer studied.