Stephanie Holst

Linkage-Specific in Situ Sialic Acid Derivatization for N-Glycan Mass Spectrometry Imaging of Formalin-Fixed Paraffin-Embedded Tissues.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging is a rapidly evolving field in which mass spectrometry techniques are applied directly on tissues to characterize the spatial distribution of various molecules such as lipids, protein/peptides, and recently also N-glycans. Glycans are involved in many biological processes and several glycan changes have been associated with different kinds of cancer, making them an interesting target group to study. An important analytical challenge for the study of glycans by MALDI mass spectrometry is the labile character of sialic acid groups which are prone to in-source/postsource decay, thereby biasing the recorded glycan profile. We therefore developed a linkage-specific sialic acid derivatization by dimethylamidation and subsequent amidation and transferred this onto formalin-fixed paraffin-embedded (FFPE) tissues for MALDI imaging of N-glycans. Our results show (i) the successful stabilization of sialic acids in a linkage specific manner, thereby not only increasing the detection range, but also adding biological meaning, (ii) that no noticeable lateral diffusion is induced during to sample preparation, (iii) the potential of mass spectrometry imaging to spatially characterize the N-glycan expression within heterogeneous tissues.

Automation of High-Throughput Mass Spectrometry-Based Plasma N-Glycome Analysis with Linkage-Specific Sialic Acid Esterification

Glycosylation is a post-translational modification of key importance with heterogeneous structural characteristics. Previously, we have developed a robust, high-throughput MALDI-TOF-MS method for the comprehensive profiling of human plasma N-glycans. In this approach, sialic acid residues are derivatized with linkage-specificity, namely the ethylation of α2,6-linked sialic acid residues with parallel lactone formation of α2,3-linked sialic acids. In the current study, this procedure was used as a starting point for the automation of all steps on a liquid-handling robot system. This resulted in a time-efficient and fully standardized procedure with throughput times of 2.5 h for a first set of 96 samples and approximately 1 h extra for each additional sample plate. The mass analysis of the thus-obtained glycans was highly reproducible in terms of relative quantification, with improved interday repeatability as compared to that of manual processing.

Investigations on Aberrant Glycosylation of Glycosphingolipids in Colorectal Cancer Tissues Using Liquid Chromatography and Matrix-Assisted Laser Desorption Time-of-Flight Mass Spectrometry (MALDI-TOF-MS)

Cancer is a leading cause of death and alterations of glycosylation are characteristic features of malignant cells. Colorectal cancer is one of the most common cancers and its exact causes and biology are not yet well understood. Here, we compared glycosylation profiles of colorectal tumor tissues and corresponding control tissues of 13 colorectal cancer patients to contribute to the understanding of this cancer. Using MALDI-TOF(/TOF)-MS and 2-dimensional LC-MS/MS we characterized enzymatically released and 2-aminobenzoic acid labeled glycans from glycosphingolipids. Multivariate data analysis revealed significant differences between tumor and corresponding control tissues. Main discriminators were obtained, which represent the overall alteration in glycosylation of glycosphingolipids during colorectal cancer progression, and these were found to be characterized by (1) increased fucosylation, (2) decreased acetylation, (3) decreased sulfation, (4) reduced expression of globo-type glycans, as well as (5) disialyl gangliosides. The findings of our current research confirm former reports, and in addition expand the knowledge of glycosphingolipid glycosylation in colorectal cancer by revealing new glycans with discriminative power and characteristic, cancer-associated glycosylation alterations. The obtained discriminating glycans can contribute to progress the discovery of biomarkers to improve diagnostics and patient treatment.

Two-Dimensional N-Glycan Distribution Mapping of Hepatocellular Carcinoma Tissues by MALDI-Imaging Mass Spectrometry.

A new mass spectrometry imaging approach to simultaneously map the two-dimensional distribution of N-glycans in tissues has been recently developed. The method uses Matrix Assisted Laser Desorption Ionization Imaging Mass Spectrometry (MALDI-IMS) to spatially profile the location and distribution of multiple N-linked glycan species released by peptide N-glycosidase F in frozen or formalin-fixed tissues. Multiple formalin-fixed human hepatocellular carcinoma tissues were evaluated with this method, resulting in a panel of over 30 N-glycans detected. An ethylation reaction of extracted N-glycans released from adjacent slides was done to stabilize sialic acid containing glycans, and these structures were compared to N-glycans detected directly from tissue profiling. In addition, the distribution of singly fucosylated N-glycans detected in tumor tissue microarray cores were compared to the histochemistry staining pattern of a core fucose binding lectin. As this MALDI-IMS workflow has the potential to be applied to any formalin-fixed tissue block or tissue microarray, the advantages and limitations of the technique in context with other glycomic methods are also summarized.

N-glycosylation Profiling of Colorectal Cancer Cell Lines Reveals Association of Fucosylation with Differentiation and Caudal Type Homebox 1 (CDX1)/Villin mRNA Expression

Various cancers such as colorectal cancer (CRC) are associated with alterations in protein glycosylation. CRC cell lines are frequently used to study these (glyco)biological changes and their mechanisms. However, differences between CRC cell lines with regard to their glycosylation have hitherto been largely neglected. Here, we comprehensively characterized the N-glycan profiles of 25 different CRC cell lines, derived from primary tumors and metastatic sites, in order to investigate their potential as glycobiological tumor model systems and to reveal glycans associated with cell line phenotypes. We applied an optimized, high-throughput membrane-based enzymatic glycan release for small sample amounts. Released glycans were derivatized to stabilize and differentiate between α2,3- and α2,6-linked N-acetylneuraminic acids, followed by N-glycosylation analysis by MALDI-TOF(/TOF)-MS. Our results showed pronounced differences between the N-glycosylation patterns of CRC cell lines. CRC cell line profiles differed from tissue-derived N-glycan profiles with regard to their high-mannose N-glycan content but showed a large overlap for complex type N-glycans, supporting their use as a glycobiological cancer model system. Importantly, we could show that the high-mannose N-glycans did not only occur as intracellular precursors but were also present at the cell surface. The obtained CRC cell line N-glycan features were not clearly correlated with mRNA expression levels of glycosyltransferases, demonstrating the usefulness of performing the structural analysis of glycans. Finally, correlation of CRC cell line glycosylation features with cancer cell markers and phenotypes revealed an association between highly fucosylated glycans and CDX1 and/or villin mRNA expression that both correlate with cell differentiation. Together, our findings provide new insights into CRC-associated glycan changes and setting the basis for more in-depth experiments on glycan function and regulation.

Multimodal Mass Spectrometry Imaging of N-Glycans and Proteins from the Same Tissue Section.

On-tissue digestion matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) can be used to record spatially correlated molecular information from formalin-fixed, paraffin-embedded (FFPE) tissue sections. In this work, we present the in situ multimodal analysis of N-linked glycans and proteins from the same FFPE tissue section. The robustness and applicability of the method are demonstrated for several tumors, including epithelial and mesenchymal tumor types. Major analytical aspects, such as lateral diffusion of the analyte molecules and differences in measurement sensitivity due to the additional sample preparation methods, have been investigated for both N-glycans and proteolytic peptides. By combining the MSI approach with extract analysis, we were also able to assess which mass spectral peaks generated by MALDI-MSI could be assigned to unique N-glycan and peptide identities.

Glycosylation Changes Triggered by the Differentiation of Monocytic THP-1 Cell Line into Macrophages

The human acute monocytic leukemia cell line THP-1 is widely used as an in vitro phagocytic cell model because it exhibits several immune properties similar to native monocyte-derived macrophages. In this study, we investigated the alteration of N- and O-linked glycans as well as glycosphingolipids, during THP-1 differentiation, combining mass spectrometry, flow cytometry, and quantitative real-time PCR. Mass spectrometry revealed that macrophage differentiation led to a marked upregulation of expression of GM3 ganglioside as well as an increase in complex-type structures, particularly triantennary glycans, occurring at the expense of high-mannose N-glycans. Moreover, we observed a slight decrease in the proportion of multifucosylated N-glycans and α2,6-sialylation. The uncovered changes in glycosylation correlated with variations of gene expression of relevant glycosyltransferases and glycosidases including sialyltransferases, β-N-acetylglucosaminyltransferases, fucosyltransferases, and neuraminidase. Furthermore, using flow cytometry and antibodies directed against glycan structures, we confirmed that the alteration of glycosylation occurs at the cell surface of THP-1 macrophage-like cells. Altogether, we established that macrophagic maturation of THP-1 induces dramatic modifications of the surface glycosylation pattern that may result in differential interaction of monocytic and macrophagic THP-1 with immune or bacterial lectins.

Profiling of different pancreatic cancer cells used as models for metastatic behaviour shows large variation in their N -glycosylation

To characterise pancreatic cancer cells from different sources which are used as model systems to study the metastatic behaviour in pancreatic ductal adenocarcinoma (PDAC), we compared the N-glycan imprint of four PDAC cells which were previously shown to differ in their galectin-4 expression and metastatic potential in vivo. Next to the sister cell lines Pa-Tu-8988S and Pa-Tu-8988T, which were isolated from the same liver metastasis of a PDAC, this included two primary PDAC cell cultures, PDAC1 and PDAC2. Additionally, we extended the N-glycan profiling to a normal, immortalized pancreatic duct cell line. Our results revealed major differences in the N-glycosylation of the different PDAC cells as well as compared to the control cell line, suggesting changes of the N-glycosylation in PDAC. The N-glycan profiles of the PDAC cells, however, differed vastly as well and demonstrate the diversity of PDAC model systems, which ultimately affects the interpretation of functional studies. The results from this study form the basis for further biological evaluation of the role of protein glycosylation in PDAC and highlight that conclusions from one cell line cannot be generalised, but should be regarded in the context of the corresponding phenotype.

High-Throughput and High-Sensitivity Mass Spectrometry-Based N -Glycomics of Mammalian Cells

The current protocols for glycomic analysis of cells often require a large quantity of material (5-20 million cells). In order to analyze the N-glycosylation from small amounts of cells (≤1 million) as obtained from, for example, primary cell lines or cell sorting, and in a higher throughput approach, we set up a robust 96-well format PVDF-membrane based N-glycan release protocol followed by linkage-specific sialic acid stabilization, cleanup, and MALDI-TOF-MS analysis. We further evaluated the influence of PNGase F incubation time on the N-glycan profile.

Serum sialylation changes in cancer

Cancer is a major cause of death in both developing and developed countries. Early detection and efficient therapy can greatly enhance survival. Aberrant glycosylation has been recognized to be one of the hallmarks of cancer as glycans participate in many cancer-associated events. Cancer-associated glycosylation changes often involve sialic acids which play important roles in cell-cell interaction, recognition and immunological response. This review aims at giving a comprehensive overview of the literature on changes of sialylation in serum of cancer patients. Furthermore, the methods available to measure serum and plasma sialic acids as well as possible underlying biochemical mechanisms involved in the serum sialylation changes are surveyed. In general, total serum sialylation levels appear to be increased with various malignancies and show a potential for clinical applications, especially for disease monitoring and prognosis. In addition to overall sialic acid levels and the amount of sialic acid per total protein, glycoprofiling of specific cancer-associated glycoproteins, acute phase proteins and immunoglobulins in serum as well as the measurements of sialylation-related enzymes such as sialidases and sialyltransferases have been reported for early detection of cancer, assessing cancer progression and improving prognosis of cancer patients. Moreover, sialic-acid containing glycan antigens such as CA19–9, sialyl Lewis X and sialyl Tn on serum proteins have also displayed their value in cancer diagnosis and management whereby increased levels of these factors positively correlated with metastasis or poor prognosis.