Yoon-Woo Kim

Mass spectrometry-based N-linked glycomic profiling as a means for tracking pancreatic cancer metastasis

The aberrant glycosylation profile on the surface of cancer cells has been recognized for its potential diagnostic value towards assessing tumor progression. In this study, we initially investigate N-glycan profiles on the surface of normal (HPDE) and cancerous (Capan-1, Panc-1, and MIA PaCa-2) pancreatic cell lines, which are from different sites of pancreatic tumor. The enzymatically deglycosylated total N-glycans are permethylated via a quantitative solid-phase method and then analyzed by using MALDI-TOF MS and MALDI-QIT-TOF MS. We demonstrate that the level of high-mannose type glycans is higher among Capan-1 cells-pancreatic cancer cells that have metastasized to the liver-than that observed among Panc-1 and MIA PaCa-2 cells-pancreatic cancer cells from the pancreas duct head and tail regions, respectively. Furthermore, the relative abundance of highly-branched sialyted N-glycans is significantly up-regulated on Panc-1 and MIA PaCa-2 pancreatic cancer cells compared to that of normal HPDE pancreas cells. Taken together, these results indicate that specific N-glycosylation profile changes in pancreatic cancer cells can be used to not only distinguish between normal and cancerous cells but also provide more information on their location and metastatic potential.

Stable isotopic labeling-based quantitative targeted glycomics (i-QTaG).

Mass spectrometry (MS) analysis combined with stable isotopic labeling is a promising method for the relative quantification of aberrant glycosylation in diseases and disorders. We developed a stable isotopic labeling‐based quantitative targeted glycomics (i‐QTaG) technique for the comparative and quantitative analysis of total N‐glycans using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). We established the analytical procedure with the chemical derivatizations (i.e., sialic acid neutralization and stable isotopic labeling) of N‐glycans using a model glycoprotein (bovine fetuin). Moreover, the i‐QTaG using MALDI‐TOF MS was evaluated with various molar ratios (1:1, 1:2, 1:5) of 13C6/12C6‐2‐aminobenzoic acid‐labeled glycans from normal human serum. Finally, this method was applied to direct comparison of the total N‐glycan profiles between normal human sera (n = 8) and prostate cancer patient sera (n = 17). The intensities of the N‐glycan peaks from i‐QTaG method showed a good linearity (R2 > 0.99) with the amount of the bovine fetuin glycoproteins. The ratios of relative intensity between the isotopically 2‐AA labeled N‐glycans were close to the theoretical molar ratios (1:1, 1:2, 1:5). We also demonstrated that the up‐regulation of the Lewis antigen (∼82%) in sera from prostate cancer patients. In this proof‐of‐concept study, we demonstrated that the i‐QTaG method, which enables to achieve a reliable comparative quantitation of total N‐glycans via MALDI‐TOF MS analysis, has the potential to diagnose and monitor alterations in glycosylation associated with disease states or biotherapeutics.

Comparative N-Linked Glycan Analysis of Wild-Type and α1,3-Galactosyltransferase Gene Knock-Out Pig Fibroblasts Using Mass Spectrometry Approaches

Carbohydrate antigens expressed on pig cells are considered to be major barriers in pig-to-human xenotransplantation. Even after α1,3-galactosyltransferase gene knock-out (GalT-KO) pigs are generated, potential non-Gal antigens are still existed. However, to the best of our knowledge there is no extensive study analyzing N-glycans expressed on the GalT-KO pig tissues or cells. Here, we identified and quantified totally 47 N-glycans from wild-type (WT) and GalT-KO pig fibroblasts using mass spectrometry. First, our results confirmed the absence of galactose-alpha-1,3-galactose (α-Gal) residue in the GalT-KO pig cells. Interestingly, we showed that the level of overall fucosylated N-glycans from GalT-KO pig fibroblasts is much higher than from WT pig fibroblasts. Moreover, the relative quantity of the N-glycolylneuraminic acid (NeuGc) antigen is slightly higher in the GalT-KO pigs. Thus, this study will contribute to a better understanding of cellular glycan alterations on GalT-KO pigs for successful xenotransplantation.

Highly sensitive glycosylation analysis of membrane glycoproteins avoiding polymeric contaminants

We established a ‘seize-and-release’ purification method to eliminate polyhexose contaminants for a highly sensitive glycan profiling. Pig liver membrane lysates were pretreated with sodium dodecyl sulfate (SDS) surfactant and subsequently dialyzed to remove polyhexose contaminants. From the purified membrane glycoproteins, glycans were released and identified by mass spectrometry. As a result, we clearly obtained N- and O-glycan profiles of a pig liver, which were not achieved without any pre-treatments. This technique demonstrates a powerful approach for enhancing the sensitivity of MS-based glycan profiling.

The Xeno-glycomics database (XDB): a relational database of qualitative and quantitative pig glycome repertoire

SUMMARY In recent years, the improvement of mass spectrometry-based glycomics techniques (i.e. highly sensitive, quantitative and high-throughput analytical tools) has enabled us to obtain a large dataset of glycans. Here we present a database named Xeno-glycomics database (XDB) that contains cell- or tissue-specific pig glycomes analyzed with mass spectrometry-based techniques, including a comprehensive pig glycan information on chemical structures, mass values, types and relative quantities. It was designed as a user-friendly web-based interface that allows users to query the database according to pig tissue/cell types or glycan masses. This database will contribute in providing qualitative and quantitative information on glycomes characterized from various pig cells/organs in xenotransplantation and might eventually provide new targets in the α1,3-galactosyltransferase gene-knock out pigs era. AVAILABILITY The database can be accessed on the web at http://bioinformatics.snu.ac.kr/xdb.

A solid-phase screening method for identification of glycan-binding cells

Glycan immobilization and epitope recognition on a solid support is useful for functional glycoproteomics and analysis of protein-protein interactions. However, the lack of high-throughput experimental methods for the examination of cell-glycan interactions means that identifying glycan-binding cells remains a challenge. Herein, we describe a tool which enables the screening of glycanbinding cells that specifically recognize the glycan epitopes released from the membrane glycoproteins of cells and tissues. Biotinylated pig kidney N-glycans were immobilized on streptavidin-conjugated solid supports and then incubated with human immune cell lines. U937 cells discriminated between the α-galactose (α-Gal) and non-Gal pig N-glycans on the solid support. This screening strategy could aid exploration of cell-protein or cell-cell interactions.