Aberrant glycosylation is considered to be a hallmark of colorectal cancer (CRC), as demonstrated by various studies. While the N-glycosylation of cell lines and serum has been widely examined, the... Show moreAberrant glycosylation is considered to be a hallmark of colorectal cancer (CRC), as demonstrated by various studies. While the N-glycosylation of cell lines and serum has been widely examined, the analysis of cancer-associated N-glycans from tissues has been hampered by the heterogeneity of tumors and the complexity of N-glycan structures. To overcome these obstacles, we present a study using laser capture microdissection that makes it possible to largely deconvolute distinct N-glycomic signatures originating from different regions of heterogeneous tissues including cancerous, stromal, and healthy mucosa cells. N-glycan alditols were analyzed by means of porous graphitized carbon liquid chromatography-electrospray ionization tandem mass spectrometry, enabling the differentiation and structural characterization of isomeric species. In total, 116 N-glycans were identified that showed profound differences in expression among cancer, stroma, and normal mucosa. In comparison with healthy mucosa, the cancer cells showed an increase in α2-6 sialylation and monoantennary N-glycans, as well as a decrease in bisected N-glycans. Moreover, specific sialylated and (sialyl-)LewisA/X antigen-carrying N-glycans were exclusively expressed in cancers. In comparison with cancer, the stroma showed lower levels of oligomannosidic and monoantennary N-glycans, LewisA/X epitopes, and sulfation, as well as increased expression of (core-)fucosylation and α2-3 sialylation. Our study reveals the distinct N-glycomic profiles of different cell types in CRC and control tissues, proving the necessity of their separate analysis for the discovery of cancer-associated glycans. Show less
Gut microbiota of the gastrointestinal tract provide health benefits to the human host via bacterial metabolites. Bacterial butyrate has beneficial effects on intestinal homeostasis and is the... Show moreGut microbiota of the gastrointestinal tract provide health benefits to the human host via bacterial metabolites. Bacterial butyrate has beneficial effects on intestinal homeostasis and is the preferred energy source of intestinal epithelial cells, capable of inducing differentiation. It was previously observed that changes in the expression of specific proteins as well as protein glycosylation occur with differentiation. In this study, specific mucin O-glycans were identified that mark butyrate-induced epithelial differentiation of the intestinal cell line CaCo-2 (Cancer Coli-2), by applying porous graphitized carbon nano–liquid chromatography with electrospray ionization tandem mass spectrometry. Moreover, a quantitative proteomic approach was used to decipher changes in the cell proteome. It was found that the fully differentiated butyrate-stimulated cells are characterized by a higher expression of sialylated O-glycan structures, whereas fucosylation is downregulated with differentiation. By performing an integrative approach, we generated hypotheses about the origin of the observed O-glycome changes. These insights pave the way for future endeavors to study the dynamic O-glycosylation patterns in the gut, either produced via cellular biosynthesis or through the action of bacterial glycosidases as well as the functional role of these patterns in homeostasis and dysbiosis at the gut–microbiota interface. Show less
Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the second leading cause of cancer deaths worldwide. A well-known hallmark of cancer is altered glycosylation. Analyzing the... Show moreColorectal cancer (CRC) is the third most commonly diagnosed cancer and the second leading cause of cancer deaths worldwide. A well-known hallmark of cancer is altered glycosylation. Analyzing the N-glycosylation of CRC cell lines may provide potential therapeutic or diagnostic targets. In this study, an in-depth N-glycomic analysis of 25 CRC cell lines was conducted using porous graphitized carbon nano-liquid chromatography coupled to electrospray ionization mass spectrometry. This method allows for the separation of isomers and performs structural characterization, revealing profound N-glycomic diversity among the studied CRC cell lines with the elucidation of a number of 139 N-glycans. A high degree of similarity between the two N-glycan datasets measured on the two different platforms (porous graphitized carbon nano-liquid chromatography electrospray ionization tandem mass spectrometry (PGC-nano-LC-ESI-MS) and matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MALDI-TOF-MS)) was discovered. Furthermore, we studied the associations between glycosylation features, glycosyltransferases (GTs), and transcription factors (TFs). While no significant correlations between the glycosylation features and GTs were found, the association between TF CDX1 and (s)Le antigen expression and relevant GTs FUT3/6 suggests that CDX1 contributes to the expression of the (s)Le antigen through the regulation of FUT3/6. Our study provides a comprehensive characterization of the N-glycome of CRC cell lines, which may contribute to the future discovery of novel glyco-biomarkers of CRC. Show less
Zhang, J.; Zon, G. van der; Ma, J.; Mei, H.L.; Cabukusta, B.; Agaser, C.C.; ... ; Dijke, P. ten 2022
Epithelial-mesenchymal transition (EMT) is pivotal in the initiation and development of cancer cell metastasis. We observed that the abundance of glycosphingolipids (GSLs), especially ganglioside... Show moreEpithelial-mesenchymal transition (EMT) is pivotal in the initiation and development of cancer cell metastasis. We observed that the abundance of glycosphingolipids (GSLs), especially ganglioside subtypes, decreased significantly during TGF-beta-induced EMT in NMuMG mouse mammary epithelial cells and A549 human lung adenocarcinoma cells. Transcriptional profiling showed that TGF-beta/SMAD response genes and EMT signatures were strongly enriched in NMuMG cells, along with depletion of UDP-glucose ceramide glucosyltransferase (UGCG), the enzyme that catalyzes the initial step in GSL biosynthesis. Consistent with this finding, genetic or pharmacological inhibition of UGCG promoted TGF-beta signaling and TGF-beta-induced EMT. UGCG inhibition promoted A549 cell migration, extravasation in the zebrafish xenograft model, and metastasis in mice. Mechanistically, GSLs inhibited TGF-beta signaling by promoting lipid raft localization of the TGF-beta type I receptor (T beta RI) and by increasing T beta RI ubiquitination and degradation. Importantly, we identified ST3GAL5-synthesized a-series gangliosides as the main GSL subtype involved in inhibition of TGF-beta signaling and TGF-beta-induced EMT in A549 cells. Notably, ST3GAL5 is weakly expressed in lung cancer tissues compared to adjacent nonmalignant tissues, and its expression correlates with good prognosis. Show less
Aberrant expression of certain glycosphingolipids (GSLs) is associated withthe differentiation of acute myeloid leukemia (AML) cells. However, the expressionpatterns of GSLs in AML are still poorly... Show moreAberrant expression of certain glycosphingolipids (GSLs) is associated withthe differentiation of acute myeloid leukemia (AML) cells. However, the expressionpatterns of GSLs in AML are still poorly explored because of their complexity, the presenceof multiple isomeric structures, and tedious analytical procedures. In this study, weperformed an in-depth GSL glycan analysis of 19 AML cell lines using porous graphitizedcarbon liquid chromatography-mass spectrometry revealing strikingly different GSL glycanprofiles between the various AML cell lines. The cell lines of the M6 subtype showed a highexpression of gangliosides with alpha 2,3-sialylation and Neu5Gc, while the M2 and M5subtypes were characterized by high expression of (neo)lacto-series glycans and Lewis A/Xantigens. Integrated analysis of glycomics and available transcriptomics data revealed theassociation of GSL glycan abundances with the transcriptomics expression of certainglycosyltransferases (GTs) and transcription factors (TFs). In addition, correlations werefound between specific GTs and TFs. Our data reveal TFsGATA2,GATA1, andRUNX1as candidate inducers of the expression of gangliosides and sialylation via regulation of the GTsST3GAL2andST8SIA1.Inconclusion, we show that GSL glycan expression levels are associated with hematopoietic AML classifications and TF and GT geneexpression. Further research is needed to dissect the regulation of GSL expression and its role in hematopoiesis and associated malignancies. Show less
Zhang, J.; Zhang, Z.J.; Holst, S.; Bloechl, C.; Madunic, K.; Wuhrer, M.; ... ; Zhang, T. 2022
Pancreatic ductal adenocarcinoma (PDAC) is characterized by poor prognosis and high mortality. Transforming growth factor-beta (TGF-beta) plays a key role in PDAC tumor progression, which is often... Show morePancreatic ductal adenocarcinoma (PDAC) is characterized by poor prognosis and high mortality. Transforming growth factor-beta (TGF-beta) plays a key role in PDAC tumor progression, which is often associated with aberrant glycosylation. However, how PDAC cells respond to TGF-beta and the role of glycosylation therein is not well known. Here, we investigated the TGF-beta-mediated response and glycosylation changes in the PaTu-8955S (PaTu-S) cell line deficient in SMA-related and MAD-related protein 4 (SMAD4), a signal transducer of the TGF-beta signaling. PaTu-S cells responded to TGF-beta by upregulating SMAD2 phosphorylation and target gene expression. We found that TGF-beta induced expression of the mesenchymal marker N-cadherin but did not significantly affect epithelial marker E-cadherin expression. We also examined differences in N-glycans, O-glycans, and glycosphingolipid-linked glycans in PaTu-S cells upon TGF-beta stimulation. TGF-beta treatment primarily induced N-glycome aberrations involving elevated levels of branching, core fucosylation, and sialylation in PaTu-S cells, in agreement with TGF-beta-induced changes in the expression of glycosylation-associated genes. In addition, we observed differences in O glycosylation and glycosphingolipid glycosylation profiles after TGF-beta treatment, including lower levels of sialylated Tn antigen and neoexpression of globosides. Furthermore, the expression of transcription factor sex-determining region Y-related high-mobility group box 4 was upregulated upon TGF-beta stimulation, and its depletion blocked TGF-beta-induced N-glycomic changes. Thus, TGF-beta-induced N-glycosylation changes can occur in a sex-determining region Y-related high-mobility group box 4-dependent and SMAD4-independent manner in the pancreatic PaTu-S cancer cell line. Our results open up avenues to study the relevance of glycosylation in TGF-beta signaling in SMAD4-inactivated PDAC. Show less
Rodriguez, E.; Boelaars, K.; Brown, K.; Madunic, K.; Ee, T. van; Dijk, F.; ... ; Kooyk, Y. van 2022
Pancreatic ductal adenocarcinoma (PDAC) remains one of the most aggressive malignancies with a 5-year survival rate of only 9%. Despite the fact that changes in glycosylation patterns during tumour... Show morePancreatic ductal adenocarcinoma (PDAC) remains one of the most aggressive malignancies with a 5-year survival rate of only 9%. Despite the fact that changes in glycosylation patterns during tumour progression have been reported, no systematic approach has been conducted to evaluate its potential for patient stratification. By analysing publicly available transcriptomic data of patient samples and cell lines, we identified here two specific glycan profiles in PDAC that correlated with progression, clinical outcome and epithelial to mesenchymal transition (EMT) status. These different glycan profiles, confirmed by glycomics, can be distinguished by the expression of O-glycan fucosylated structures, present only in epithelial cells and regulated by the expression of GALNT3. Moreover, these fucosylated glycans can serve as ligands for DC-SIGN positive tumour-associated macrophages, modulating their activation and inducing the production of IL-10. Our results show mechanisms by which the glyco-code contributes to the tolerogenic microenvironment in PDAC.Rodriguez et al. present a transcriptomic analysis of glycosylation associated gene profiles, including bulk patient sequencing, sc-RNA seq, cell lines and organoids, to examine glycosylation in PDAC. They find 2 specific glycan profiles correlating with progression, clinical outcome and EMT, and conclude that the glyco-code contributes to the tolerogenic microenvironment in PDAC. Show less
Cells are covered with a dense layer of carbohydrates, some of which are solely present on neoplastic cells. The so-called tumor-associated carbohydrate antigens (TACAs) are increasingly recognized... Show moreCells are covered with a dense layer of carbohydrates, some of which are solely present on neoplastic cells. The so-called tumor-associated carbohydrate antigens (TACAs) are increasingly recognized as promising targets for immunotherapy. These carbohydrates differ from those of the surrounding non-cancerous tissues and contribute to the malignant phenotype of the cancer cells by promoting proliferation, metastasis, and immunosuppression. However, due to tumor tissue heterogeneity and technological limitations, TACAs are insufficiently explored.Methods: A workflow was established to decode the colorectal cancer (CRC)-associated O-linked glycans from approximately 20,000 cell extracts. Extracts were obtained through laser capture microdissection of formalin fixed paraffin embedded tissues of both primary tumors and metastatic sites, and compared to healthy colon mucosa from the same patients. The released O-glycans were analyzed by porous graphitized carbon liquid chromatography-tandem mass spectrometry in negative ion mode.Results: Distinctive O-glycosylation features were found in cancerous, stromal and normal colon mucosal regions. Over 100 O-linked glycans were detected in cancerous regions with absence in normal mucosa. From those, six core 2 O-glycans were exclusively found in more than 33% of the cancers, carrying the terminal (sialyl-)Lewis(X/A) antigen. Moreover, two O-glycans were present in 72% of the analyzed cancers and 94% of the investigated cancers expressed at least one of these two O-glycans. In contrast, normal colon mucosa predominantly expressed core 3 O-glycans, carrying alpha 2-6-linked sialylation, (sulfo-)Lewis(X/A) and Sda antigens.Conclusion: In this study, we present a novel panel of highly specific TACAs, based upon differences in the glycomic profiles between CRC and healthy colon mucosa. These TACAs are promising new targets for development of innovative cancer immune target therapies and lay the foundation for the targeted treatment of CRC. Show less
Blochl, C.; Wang, D.; Madunic, K.; Lageveen-Kammeijer, G.S.M.; Huber, C.G.; Wuhrer, M.; Zhang, T. 2021
Acute myeloid leukemia (AML) is characterized by a dysregulated expansion of poorly differentiated myeloid cells. Although patients are usually treated effectively by chemotherapy, a high rate of... Show moreAcute myeloid leukemia (AML) is characterized by a dysregulated expansion of poorly differentiated myeloid cells. Although patients are usually treated effectively by chemotherapy, a high rate of relapsed or refractory disease poses a major hurdle in its treatment. Recently, several studies have proposed implications of protein glycosylation in the pathobiology of AML including chemoresistance. Accordingly, associations have been found between specific glycan epitopes and the outcome of the disease. To advance this poorly studied field, we performed an exploratory glycomics study characterizing 21 widely used AML cell lines. Exploiting the benefits of porous graphitized carbon chromatography coupled to tandem mass spectrometry (PGC nano-LC-MS2), we qualitatively and quantitatively profiled N- and O-linked glycans. AML cell lines exhibited distinct glycan fingerprints differing in relevant glycan traits correlating with their cellular phenotype as classified by the FAB system. By implementing transcriptomics data, specific glycosyltransferases and hematopoietic transcription factors were identified, which are candidate drivers of the glycan phenotype of these cells. In conclusion, we report the varying expression of glycan structures across a high number of AML cell lines, including those associated with poor prognosis, identified underlying glycosyltransferases and transcription factors, and provide insights into the regulation of the AML glycan repertoire. Show less
Kotsias, M.; Madunic, K.; Nicolardi, S.; Kozak, R.P.; Gardner, R.A.; Jansen, B.C.; ... ; Wuhrer, M. 2021
The study of protein O-glycosylation is important in biological research as O-glycans have been reported to regulate a multitude of molecular and cell biology processes occurring in cancer. It is... Show moreThe study of protein O-glycosylation is important in biological research as O-glycans have been reported to regulate a multitude of molecular and cell biology processes occurring in cancer. It is known that alterations in O-glycosylation are involved in the development and progression of cancer. Their easy accessibility makes in vitro established cell lines suitable and useful models for studying biological mechanisms in disease. However, the O-glycosylation analysis of large numbers of samples, as required in systems biology and biomarker discovery studies, is often challenging. In the present study, O-glycans from three human colorectal cancer cell lines and two human pancreatic cancer cell lines were released by semi-automated, high throughput reductive beta-elimination and analysed using ultrahigh resolution MALDI-FT-ICR MS. Automated data integration and processing was performed using MassyTools, where the analyte was automatically included for relative quantitation based on a range of selection criteria including signal-to-noise ratio, mass error and isotopic pattern quality scores. A total of 126 O-glycan compositions, ranging from a single monosaccharide to large oligosaccharides exhibiting complex glycan motifs, were detected. The use of ultrahigh resolution MALDI-FTICR MS enabled glycan identification and quantitation in the matrix region of the spectrum. This approach has the potential to be used for O-glycosylation analysis of large numbers of samples, such as patient sample cohorts. Show less
The desolvation and ionization process of analytes can significantly be improved by enriching the nebulizing gas with a dopant (dopant enriched nitrogen (DEN) gas) in the electrospray source.... Show moreThe desolvation and ionization process of analytes can significantly be improved by enriching the nebulizing gas with a dopant (dopant enriched nitrogen (DEN) gas) in the electrospray source. However, for the analysis of released glycans in negative ion mode, the usage of DEN gas remains largely unexplored. For this purpose, we investigated the effect of different polar protic solvents (methanol, ethanol, and isopropanol) as well as using solely the nebulizing gas or ambient air on the ionization and charge state distribution of released N- and O-glycans. Compared to the standard acetonitrile enriched nitrogen gas, isopropanol showed the highest increase in regards to peak areas. Moreover, it showed large benefits for the identification of glycan structures at high sensitivity as the increased precursor intensities subsequently resulted in higher intensities in tandem MS mode. While similar effects are noted for both neutral and sialylated species, the most significant effect was observed for early eluting glycans where very low acetonitrile concentrations were present in the eluent. The best results in terms of S/N ratios were obtained with methanol, with less effect on the MS/MS signal enhancement. Therefore, the use of this dopant would be particularly beneficial for high sensitivity MS-mode applications. In conclusion, isopropanol enriched DEN gas greatly improves the detection of both N-and O-glycan species and their tandem mass spectra, particularly for the early eluting species whose ionization in the absence of DEN gas is hindered by low organic concentrations. Show less
Developments in mass spectrometry (MS)-based analyses of glycoproteins have been important to study changes in glycosylation related to disease. Recently, the characteristic pattern of oxonium ions... Show moreDevelopments in mass spectrometry (MS)-based analyses of glycoproteins have been important to study changes in glycosylation related to disease. Recently, the characteristic pattern of oxonium ions in glycopeptide fragmentation spectra had been used to assign different sets of glycopeptides. In particular, this was helpful to discriminate between O-GalNAc and O-GlcNAc. Here, we thought to investigate how such information can be used to examine quantitative proteomics data. For this purpose, we used tandem mass tag (TMT)-labeled samples from total cell lysates and secreted proteins from three different colorectal cancer cell lines. Following automated glycopeptide assignment (Byonic) and evaluation of the presence and relative intensity of oxonium ions, we observed that, in particular, the ratio of the ions at m/z 144.066 and 138.055, respectively, could be used to discriminate between O-GlcNAcylated and O-GalNAcylated peptides, with concomitant relative quantification between the different cell lines. Among the O-GalNAcylated proteins, we also observed anterior gradient protein 2 (AGR2), a protein which glycosylation site and status was hitherto not well documented. Using a combination of multiple fragmentation methods, we then not only assigned the site of modification, but also showed different glycosylation between intracellular (ER-resident) and secreted AGR2. Overall, our study shows the potential of broad application of the use of the relative intensities of oxonium ions for the confident assignment of glycopeptides, even in complex proteomics datasets. Show less
The thick mucus layer of the gut provides a barrier to infiltration of the underlying epithelia by both the normal microbiota and enteric pathogens. Some members of the microbiota utilise mucin... Show moreThe thick mucus layer of the gut provides a barrier to infiltration of the underlying epithelia by both the normal microbiota and enteric pathogens. Some members of the microbiota utilise mucin glycoproteins as a nutrient source, but a detailed understanding of the mechanisms used to breakdown these complex macromolecules is lacking. Here we describe the discovery and characterisation of endo-acting enzymes from prominent mucin-degrading bacteria that target the polyLacNAc structures within oligosaccharide side chains of both animal and human mucins. These O-glycanases are part of the large and diverse glycoside hydrolase 16 (GH16) family and are often lipoproteins, indicating that they are surface located and thus likely involved in the initial step in mucin breakdown. These data provide a significant advance in our knowledge of the mechanism of mucin breakdown by the normal microbiota. Furthermore, we also demonstrate the potential use of these enzymes as tools to explore changes in O-glycan structure in a number of intestinal disease states. Epithelial cells that line the gut secrete complex glycoproteins that form a mucus layer to protect the gut wall from enteric pathogens. Here, the authors provide a comprehensive characterisation of endo-acting glycoside hydrolases expressed by mucin-degrading members of the microbiome that are able to cleave the O-glycan chains of a range of different animal and human mucins. Show less
Zhang, T.; Madunic, K.; Holst, S.; Zhang, J.; Jin, C.S.; Dijke, P. ten; ... ; Wuhrer, M. 2020
Changes in glycosylation signatures of cells have been associated with pathological processes in cancer as well as infectious and autoimmune diseases. The current protocols for comprehensive... Show moreChanges in glycosylation signatures of cells have been associated with pathological processes in cancer as well as infectious and autoimmune diseases. The current protocols for comprehensive analysis ofN-glycomics andO-glycomics derived from cells and tissues often require a large amount of biological material. They also only allow the processing of very limited numbers of samples at a time. Here we established a workflow for sequential release ofN-glycans andO-glycans based on PVDF membrane immobilization in 96-well format from 5 x 10(5)cells. Released glycans are reduced, desalted, purified, and reconstituted, all in 96-well format plates, without additional staining or derivatization. Glycans are then analyzed with porous graphitized carbon nano-liquid chromatography coupled to tandem mass spectrometry using negative-mode electrospray ionization, enabling the chromatographic resolution and structural elucidation of glycan species including many compositional isomers. The approach was demonstrated using glycoprotein standards and further applied to analyze the glycosylation of the murine mammary gland NMuMG cell line. The developed protocol allows the analysis ofN- andO-glycans from relatively large numbers of samples in a less time consuming way with high repeatability. Inter- and intraday repeatability of the fetuinN-glycan analysis showed two median intraday coefficients of variations (CVs) of 7.6% and 8.0%, and a median interday CV of 9.8%. Median CVs of 7.9% and 8.7% for the main peaks ofN- andO-glycans released from the NMuMG cell line indicate a very good repeatability. The method is applicable to purified glycoproteins as well as to biofluids and cell- or tissue-based samples. Show less
Alterations in protein glycosylation in colorectal cancer (CRC) have been extensively studied using cell lines as models. However, little is known about their O-glycome and the differences in... Show moreAlterations in protein glycosylation in colorectal cancer (CRC) have been extensively studied using cell lines as models. However, little is known about their O-glycome and the differences in glycan biosynthesis in different cell types. To provide a better understanding of the variation in O-glycosylation phenotypes and their association with other molecular features, an in-depth O-glycosylation analysis of 26 different CRC cell lines was performed. The released O-glycans were analysed on porous graphitized carbon nano-liquid chromatography system coupled to a mass spectrometer via electrospray ionization (PGC-nano-LC-ESI-MS/MS) allowing isomeric separation as well as in-depth structural characterization. Associations between the observed glycan phenotypes with previously reported cell line transcriptome signatures were examined by canonical correlation analysis. Striking differences are observed between the O-glycomes of 26 CRC cell lines. Unsupervized principal component analysis reveals a separation between well-differentiated colon-like and undifferentiated cell lines. Colon-like cell lines are characterized by a prevalence of I-branched and sialyl Lewis x/a epitope carrying glycans, while most undifferentiated cell lines show absence of Lewis epitope expression resulting in dominance of truncated alpha 2,6-core sialylated glycans. Moreover, the expression of glycan signatures associates with the expression of glycosyltransferases that are involved in their biosynthesis, providing a deeper insight into the regulation of glycan biosynthesis in different cell types. This untargeted in-depth screening of cell line O-glycomes paves the way for future studies exploring the role of glycosylation in CRC development and drug response leading to discovery of novel targets for the development of anti-cancer antibodies. Show less
Vreeker, G.C.M.; Nicolardi, S.; Madunic, K.; Kotsias, M.; Burgt, Y.E.M. van der; Wuhrer, M. 2020
Glycosylation analysis from biological samples is often challenging due to the high complexity of the glycan structures found in these samples. In the present study N- and O- glycans from human... Show moreGlycosylation analysis from biological samples is often challenging due to the high complexity of the glycan structures found in these samples. In the present study N- and O- glycans from human colorectal cancer cell lines and human plasma were analyzed using ultrahigh resolution MALDI-FTICR-MS. N-glycans were enzymatically released from cell lines and plasma proteins, whereas beta-elimination was used for the release of O-glycans from the cells. The purified samples were mass analyzed using a 15T MALDI-FTICR-MS system, with additional MS/MS (collision-induced dissociation) experiments for O-glycan identifications. A total of 104 O-glycan and 62 N-glycan compositions were observed in the spectra obtained from colorectal cancer cell line samples. In the cell line N-glycan spectra, the highest intensity signals originated from high-mannose glycans, next to the presence of various complex type glycans. Notably, in the O-glycan spectra mono- and disaccharide signals were observed, which are difficult to detect using alternative glycomic platforms such as porous graphitized carbon LC-MS. In the N-glycan spectra from plasma, isobaric species were resolved in MALDI-FTICR-MS spectra using absorption mode whereas these overlapped in magnitude mode. The use of ultrahigh resolution MALDI-FTICR-MS for the analysis of glycans in complex mixtures enables us to confidently analyze glycans in the matrix region of the spectrum and to differentiate isobaric glycan species. (C) 2019 The Authors. Published by Elsevier B.V. Show less
