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Fatty liver disease progresses through stages of fat accumulation and inflammation to nonalcoholic steatohepatitis (NASH), fibrosis and cirrhosis, and eventually hepatocellular carcinoma (HCC). Currently available diagnostic tools for HCC lack sensitivity and specificity. In this study, we investigated the use of circulating serum glycoproteins to identify a panel of potential prognostic markers that may be indicative of progression from the healthy state to NASH and further to HCC. Serum samples were processed and analyzed using a novel high-throughput glycoproteomics platform. Our initial dataset contained healthy, NASH, and HCC serum samples. We analyzed 413 glycopeptides, representing 57 abundant serum proteins, and compared among the three phenotypes. We studied the normalized abundance of common glycoforms and found 40 glycopeptides with statistically significant differences in abundances in NASH and HCC compared to controls. Summary level relative abundances of core-fucosylated, sialylated, and branched glycans containing glycopeptides were higher in NASH and HCC as compared to controls. We replicated some of our findings in an independent set of samples of individuals with benign liver conditions and HCC. Our results may be of value in the management of liver diseases. Data generated in this work can be downloaded from MassIVE (https://massive.ucsd.edu) with identifier MSV000088809.

Proteins on cell membrane are modified by N- and O-glycans. N-Glycans have been extensively characterized using advanced separation and mass spectrometry techniques. However, O-glycans remain a challenge, because of the lack of universal enzymes to release them and the large background abundances of N-glycans. Here, we report a method for in-depth structural analysis and quantitation of O-glycans derived from human cell membrane. O-Glycans were chemically released from isolated cell membrane glycoproteins following N-glycan and lipid/glycolipid removal by PNGase F digestion and Folch extraction, respectively. Released O-glycans were purified by an optimized protocol to eliminate interference from small molecules and degraded proteins. Cell surface O-glycans were then analyzed using a nanoLC-chip-QTOF mass spectrometer with a porous graphitized carbon (PGC) column, while the N-glycans and glycolipids isolated from the same cell membrane fractions were analyzed in parallel using previously reported methods. The monosaccharide compositions and linkages of the detected O-glycans were identified by exoglycosidase digestion facilitated with tandem mass spectrometry (MS/MS). Using this method, we identified 44 cell membrane O-glycan isomers with MS/MS, and, among them, we unambiguously characterized 25 O-glycan structures with exoglycosidase digestion to create a library with their complete structures, accurate masses, and retention times. In this process, we identified and characterized unexpected mannose oligomers that are α(1-2/3) linked. This library enabled the identification and quantification of unique cell surface O-glycans from different cell lines and the study of specific O-glycan changes during cell differentiation.

Analysis of serum protein glycovariants has the potential to identify new biomarkers of human disease. However, the inability to rapidly quantify glycans in a site-specific fashion remains the major barrier to applying such biomarkers clinically. Advancements in sample preparation and glycopeptide quantification are thus needed to better bridge glycoscience with biomarker discovery research. We present here the successful utilization of several sample preparation techniques, including multienzyme digestion and glycopeptide enrichment, to increase the repertoire of glycopeptides that can be generated from serum glycoproteins. These techniques combined with glycopeptide retention time prediction and UHPLC-QqQ conditions optimization were then used to develop a dynamic multiple-reaction monitoring (dMRM)-based strategy to simultaneously monitor over 100 glycosylation sites across 50 serum glycoproteins. In total, the abundances of over 600 glycopeptides were simultaneously monitored, some of which were identified by utilizing theoretically predicted ion products and presumed m/ z values. The dMRM method was found to have good sensitivity. In the targeted dMRM mode, the limit of quantitation (LOQ) of nine standard glycoproteins reached femtomole levels with dynamic ranges spanning 3-4 orders of magnitude. The dMRM-based strategy also showed high reproducibility with regards to both instrument and sample preparation performance. The high coverage of the serum glycoproteins that can be quantitated to the glycopeptide level makes this method especially suitable for the biomarker discovery from large sample sets. We predict that, in the near future, biomarkers, such as these, will be deployed clinically, especially in the fields of cancer and autoimmunity.

The review focuses on recent aspects (last three years) of glycosylation analyses that provide relevant information about cancer. It includes recent development in glycan and protein enrichment methods for discovery of cancer markers. It will however focus on the recent technological developments in mass spectrometry (MS), bioinformatics and separation methods as they apply toward identifying cancer markers. More specifically, it will cover advances in matrix-assisted laser desorption/ionization (MALDI), electrospray ionization (ESI), capillary electrophoresis (CE), and liquid chromatography (LC) coupled to mass spectrometry. The discussions will include glycans, recently identified as potential markers for cancer that have been discovered using the highlighted technologies. We will also discuss emerging glycoproteomic techniques and site-specific methods, and how these methods are being utilized for cancer biomarker discovery. The large amount of data and the complexity of glycoproteomic analysis have been the impetus for developing bioinformatic methods for assigning glycosylation sites and characterizing the potentially very large site- or microheterogeneity. This review will cover the most recent advancements in biomarker discovery of N- and O-glycosylation of proteins as well as the glycolipids. This group collectively constitutes glycosylation on the cell membrane or the glycocalyx. The review will also highlight methods that are highly reproducible, with low coefficient of variation (CV), and scalable for large sample sets. The reader is also referred to other notable earlier reviews on glycomic biomarkers for cancer. Mereiter et al. describe the recent glycomic effort in gastrointestinal cancer.1 A review focused on N-glycomic analysis of colorectal cancer has been published by Sethi and Fanayan.2 N-Glycan, O-glycan, and glycolipid characteristics of colorectal cancer were reviewed by Holst et al.3 Muchena et al. have provided a more general review of glycan biomarkers covering up to the current review period.4 The field of glycoscience also covers a broad area of structures and may include highly anionic (glycosaminoglycans) and monosaccharide (e.g. O-GlcNAc) modifications that require their specific and unique sets of analytical tools. The latter topics are not covered in this review. 1.1. Background of Glycosylation and Cancer There is nearly 50 years of research illustrating that changes in glycosylation accompany cancer.5 Glycosylation is a dynamic process intimately involved in key processes in cells, including cell-cell and cell-extracellular communication as well as cell-cell adhesion, and cellular metabolism. Glycans expressed in several types of glycoconjugates are known to change during cancer genesis and progression.6 These changes increase the structural heterogeneity and alter the functions of cells.7 Glycosylation has been found to enable tumor-induced immunomodulation and metastasis.8–10 The cell-surface structures allow the immune cells to differentiate self/normal cells from non-self/abnormal cells.11 For example, terminal residues on N-glycans, such as sialic acids, are involved in immunity and cell-cell communication.12 Changes in glycosylation of adhesion proteins can largely influence their binding properties, leading to altered cell-cell or cell-matrix contacts.13 Other types of glycans are also involved in cancer. Gangliosides and sphingolipids are involved in transmembrane communication vital in tumor cell growth and invasion.14 Glycosaminoglycans are involved in tumor cell migration15 and motility.16–18 The search for effective markers is aided by the understanding of how glycans are synthesized. The glycan biosynthetic process is a non-template process involving multiple enzymes, some performing competing activities. It is estimated that more than 300 metabolic enzymes, composed of glycosyltransferases and glycosidases, are involved in the biosynthesis and processing of glycans.19–20 The best-known series of pathways belongs to the production of N-glycans (Figure 1). They illustrate the large degree of structural heterogeneity in glycosylation. N-Glycans are produced in a step-wise process beginning with the production of high mannose structures on a lipid, which are transferred to the nascent polypeptide chain to guide protein folding. Once folded, the glycans are then trimmed back and extended to form complex and hybrid structures. The folded protein can be secreted with glycans that range from early in the process to yield high mannose structures to later in the process corresponding to complex or hybrid structures. The number of structures for one glycosylation site can vary by a large degree, from a handful for transferrin21 to over 70 structures for IgG, the most abundant serum glycoprotein.22–23 Open in a separate window Figure 1. Representation of the glycosylation pathway of proteins. The pathway illustrates the complexity and heterogeneity of structures. The proteins may exit the pathway with various levels of glycosylation.

The online coupling of capillary electrophoresis with ambient direct analysis in real time mass spectrometry (DART-MS) was realized by a coaxial tip interface. The analytes eluted from capillary electrophoresis (CE) were directly ionized by the metastable helium flux produced by DART and transferred into MS for the detection, with which the online separation and simultaneous detection were achieved. The CE-DART-MS can tolerate higher concentrations of detergents and salts than traditional CE-electrospray ionization (ESI)-MS and avoided the difficulties of collecting CE effluent and cleaning the interface, which simplified the experimental procedures and shortened the analysis time. The performance of the technique was successfully verified by capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) using a mixture of 4-aminoantipyrine, zolmitriptan, and quinine. This online technique showed good repeatability with the relative standard deviations (RSDs; n = 5) of 0.56-1.23% for the retention times and 2.01-7.41% for the peak areas. The quantitative analysis of 4-aminoantipyrine was accomplished in the range of 0.01-0.50 mg/mL with the linear correlation coefficient of 0.9995 and limit of detection of 14.7 fmol. Compared with CE-ESI-MS, the ion suppression effects of nonvolatile salts and detergents were efficiently minimized. The signal intensity remained constant when the concentrations reached 100 mM for sodium borate and 30 mM for SDS (in 30 mM sodium borate buffer). In addition, the proposed method was successfully applied to the detection of the endogenous caffeine in Chinese white tea.