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AbstractGastric cancer (GC) is one of the leading causes of cancer-related death worldwide, largely because of difficulties in early diagnosis. Despite accumulating evidence indicating that aberrant glycosylation is associated with GC, site-specific localization of the glycosylation to increase specificity and sensitivity for clinical use is still an analytical challenge. Here, we created an analytical platform with a targeted glycoproteomic approach for GC biomarker discovery. Unlike the conventional glycomic approach with untargeted mass spectrometric profiling of released glycan, our platform is characterized by three key features: it is a target-protein-specific, glycosylation-site-specific, and structure-specific platform with a one-shot enzyme reaction. Serum haptoglobin enriched by immunoaffinity chromatography was subjected to multispecific proteolysis to generate site-specific glycopeptides and to investigate the macroheterogeneity and microheterogeneity. Glycopeptides were identified and quantified by nano liquid chromatography–mass spectrometry and nano liquid chromatography–tandem mass spectrometry. Ninety-six glycopeptides, each corresponding to a unique glycan/glycosite pairing, were tracked across all cancer and control samples. Differences in abundance between the two groups were marked by particularly high magnitudes. Three glycopeptides exhibited exceptionally high control-to-cancer fold changes along with receiver operating characteristic curve areas of 1.0, indicating perfect discrimination between the two groups. From the results taken together, our platform, which provides biological information as well as high sensitivity and reproducibility, may be useful for GC biomarker discovery.Graphical abstractᅟ

Glycomic analysis is the comprehensive determination of glycan (oligosaccharide) structures with quantitative information in a biological sample. Rapid-throughput glycomics is complicated due to the lack of a template, which has greatly facilitated analysis in the field of proteomics. Furthermore, the large similarities in structures make fragmentation spectra (as obtained in electron impact ionization and tandem mass spectrometry) less definitive for identification as it has been in metabolomics. In this study, we develop a concept of rapid-throughput glycomics on human milk oligosaccharides, which have proven to be an important bioactive component of breast milk, providing the infant with protection against pathogenic infection and supporting the establishment of a healthy microbiota. To better understand the relationship between diverse oligosaccharides structures and their biological function as anti-pathogenic and prebiotic compounds, large human studies are needed, which necessitate rapid- to high-throughput analytical platforms. Herein, a complete glycomics methodology is presented, evaluating the most effective human milk oligosaccharide (HMO) extraction protocols, the linearity and reproducibility of the nano-liquid chromatography chip time-of-flight mass spectrometry (nano-LC chip-TOF MS) method, and the efficacy of newly developed, in-house software for chromatographic peak alignment that allows for rapid data analysis. High instrument stability and retention time reproducibility, together with the successful automated alignment of hundreds of features in hundreds of milk samples, allow for the use of an HMO library for rapid assignment of fully annotated structures. Graphical Abstract ᅟ

This study examines the genes coding for enzymes involved in bovine milk oligosaccharide metabolism by comparing the oligosaccharide profiles with the expressions of glycosylation-related genes. Fresh milk samples (n = 32) were collected from four Holstein and Jersey cows at days 1, 15, 90 and 250 of lactation and free milk oligosaccharide profiles were analyzed. RNA was extracted from milk somatic cells at days 15 and 250 of lactation (n = 12) and gene expression analysis was conducted by RNA-Sequencing. A list was created of 121 glycosylation-related genes involved in oligosaccharide metabolism pathways in bovine by analyzing the oligosaccharide profiles and performing an extensive literature search. No significant differences were observed in either oligosaccharide profiles or expressions of glycosylation-related genes between Holstein and Jersey cows. The highest concentrations of free oligosaccharides were observed in the colostrum samples and a sharp decrease was observed in the concentration of free oligosaccharides on day 15, followed by progressive decrease on days 90 and 250. Ninety-two glycosylation-related genes were expressed in milk somatic cells. Most of these genes exhibited higher expression in day 250 samples indicating increases in net glycosylation-related metabolism in spite of decreases in free milk oligosaccharides in late lactation milk. Even though fucosylated free oligosaccharides were not identified, gene expression indicated the likely presence of fucosylated oligosaccharides in bovine milk. Fucosidase genes were expressed in milk and a possible explanation for not detecting fucosylated free oligosaccharides is the degradation of large fucosylated free oligosaccharides by the fucosidases. Detailed characterization of enzymes encoded by the 92 glycosylation-related genes identified in this study will provide the basic knowledge for metabolic network analysis of oligosaccharides in mammalian milk. These candidate genes will guide the design of a targeted breeding strategy to optimize the content of beneficial oligosaccharides in bovine milk.

The glycome, i.e. the glycan components of a biological source, has been widely reported to change with disease states. However, mining the glycome for biomarkers is complicated by glycan structural heterogeneity. Nanoflow liquid chromatography, or nano-LC, addresses the problem by providing a sensitive and quantitative method of separating and profiling glycans and glycopeptides. The first chapter of this thesis provides an overview of glycomic analysis by nanoflow liquid chromatography. Recent advances in analytical technology and methodology are presented that enhance and augment the advantages offered by nano-LC, especially when combined with mass spectrometry. Particular emphasis is placed on methods and technologies that allow structure-specific glycan profiling. The second chapter describes the development of a method for isomer-specific profiling of native N-glycans from human serum based on porous graphitized carbon nano-LC combined with mass spectrometry. The inclusion of isomer-specific characterization is expected to uncover more robust glycan biomarkers with higher specificity than compositional (MS-only) profiling. The N-glycan separation capabilities of chip-based porous graphitized carbon nano-LC are characterized in detail. The utility of this method for structure-specific biomarker discovery is demonstrated with a small pilot study on prostate cancer patients (n = 8) with good and bad prognoses. The third chapter describes the streamlining and optimization of the isomer-specific N-glycan profiling method to increase throughput and reproducibility, thereby enabling the discovery of sensitive and specific biomarkers for cancer. To demonstrate the utility of this method, serum samples from epithelial ovarian cancer cases (n = 46) and healthy control individuals (n = 48) were analyzed and compared. The N-glycan profiling results were used to create an optimized model for robust discrimination between epithelial ovarian cancer cases and controls, demonstrating the effectiveness of this platform for structure-specific biomarker discovery. The fourth chapter moves beyond profiling of released N-glycans to examine site-specific glycosylation analysis. This goal was accomplished via nano-LC separation and MS characterization of glycopeptides. Isomer-specific glycan profiles were thus obtained in the context of their location and relative abundance on a glycoprotein. The method was validated using well-characterized glycoprotein standards and then applied to the isomer-specific, site-specific characterization and quantitation of both N- and O-glycosylation on selected glycoproteins.

As a major component of bovine milk, oligosaccharides are an important source of potentially bioactive or functional food ingredients. Due to their similarity to human milk, recent studies have shown that bovine milk oligosaccharides (bMOs) may serve as important modulators of microbiotic activity, either as inhibitors of pathogen binding, or as bacteria-specific carbon sources for certain classes of beneficial bacteria. This thesis closely examines bMO structure and biosynthesis by correlating glycomic and transcriptomic data in order to obtain a more complete picture of the bovine milk glycome. The first chapter of this thesis explores the genetic mechanisms underlying bovine milk oligosaccharide synthesis. The bovine somatic (milk) cell transcriptome was profiled via RNA sequencing in order to establish gene expression patterns during different stages of lactation. These results were correlated with the oligosaccharide profile of the bovine milk produced at these stages of lactation. The second chapter of this thesis closely examines the structural characteristics of bovine milk oligosaccharides (bMOs). Porous graphitized carbon, an isomer-sensitive stationary phase, was used to separate bMOs isolated from bovine colostrum. Structural elucidation was then accomplished by a combination of linkage-specific exoglycosidase digestions and structure specific nano-LC/MS/MS fragmentation.