(Sialyl)Lewis Antigen Expression on Glycosphingolipids, N-, and O-Glycans in Colorectal Cancer Cell Lines is Linked to a Colon-Like Differentiation Program.

Alterations in the glycomic profile are a hallmark of cancer, including colorectal cancer (CRC). While, the glycosylation of glycoproteins and glycolipids has been widely studied for CRC cell lines and tissues, a comprehensive overview of CRC glycomics is still lacking due to the usage of different samples and analytical methods. In this study, we compared glycosylation features of N-, O-glycans, and glycosphingolipid glycans for a set of 22 CRC cell lines, all measured by porous graphitized carbon nano-liquid chromatography-tandem mass spectrometry. An overall, high abundance of (sialyl)Lewis antigens for colon-like cell lines was found, while undifferentiated cell lines showed high expression of H blood group antigens and α2-3/6 sialylation. Moreover, significant associations of glycosylation features were found between the three classes of glycans, such as (sialyl)Lewis and H blood group antigens. Integration of the datasets with transcriptomics data revealed positive correlations between (sialyl)Lewis antigens, the corresponding glycosyltransferase FUT3 and transcription factors CDX1, ETS, HNF1/4A, MECOM, and MYB. This indicates a possible role of these transcription factors in the upregulation of (sialyl)Lewis antigens, particularly on glycosphingolipid glycans, via FUT3/4 expression in colon-like cell lines. In conclusion, our study provides insights into the possible regulation of glycans in CRC and can serve as a guide for the development of diagnostic and therapeutic biomarkers.

In-Depth Glycoproteomic Assay of Urinary Prostatic Acid Phosphatase.

Prostate-specific antigen (PSA) is a well-known clinical biomarker in prostate cancer (PCa) diagnosis, but a better test is still needed, as the serum-level-based PSA quantification exhibits limited specificity and comes with poor predictive value. Prior to PSA, prostatic acid phosphatase (PAP) was used, but it was replaced by PSA because PSA improved the early detection of PCa. Upon revisiting PAP and its glycosylation specifically, it appears to be a promising new biomarker candidate. Namely, previous studies have indicated that PAP glycoforms differ between PCa and non-PCa individuals. However, an in-depth characterization of PAP glycosylation is still lacking. In this study, we established an in-depth glycoproteomic assay for urinary PAP by characterizing both the micro- and macroheterogeneity of the PAP glycoprofile. For this purpose, PAP samples were analyzed by capillary electrophoresis coupled to mass spectrometry after affinity purification from urine and proteolytic digestion. The developed urinary PAP assay was applied on a pooled DRE (digital rectal examination) urine sample from nine individuals. Three glycosylation sites were characterized, namely N94, N220, and N333, via N-glycopeptide analysis. Taking sialic acid linkage isomers into account, a total of 63, 27, and 4 N-glycan structures were identified, respectively. The presented PAP glycoproteomic assay will enable the determination of potential glycomic biomarkers for the early detection and prognosis of PCa in cohort studies.

An Integrated Glycosylation Signature of Rheumatoid Arthritis.

Rheumatoid arthritis (RA) Is a highly prevalent autoimmune disease that affects the joints but also various other organs. The disease is characterized by autoantibodies that are often already observed pre-disease. Since the 1980s, it has been known that antibody glycosylation is different in RA as compared to control individuals. While the literature on glycosylation changes in RA is dominated by reports on serum or plasma immunoglobulin G (IgG), our recent studies have indicated that the glycosylation changes observed for immunoglobulin A (IgA) and total serum N-glycome (TSNG) may be similarly prominent, and useful in differentiating between the RA patients and controls, or as a proxy of the disease activity. In this study, we integrated and compared the RA glycosylation signatures of IgG, IgA and TSNG, all determined in the pregnancy-induced amelioration of rheumatoid arthritis (PARA) cohort. We assessed the association of the altered glycosylation patterns with the disease, autoantibody positivity and disease activity. Our analyses indicated a common, composite glycosylation signature of RA that was independent of the autoantibody status.

Serum N-Glycosylation RPLC-FD-MS Assay to Assess Colorectal Cancer Surgical Interventions.

A newly developed analytical strategy was applied to profile the total serum N-glycome of 64 colorectal cancer (CRC) patients before and after surgical intervention. In this cohort, it was previously found that serum N-glycome alterations in CRC were associated with patient survival. Here, fluorescent labeling of serum N-glycans was applied using procainamide and followed by sialic acid derivatization specific for α2,6- and α2,3-linkage types via ethyl esterification and amidation, respectively. This strategy allowed efficient separation of specific positional isomers on reversed-phase liquid chromatography-fluorescence detection-mass spectrometry (RPLC-FD-MS) and complemented the previous glycomics data based on matrix-assisted laser desorption/ionization (MALDI)-MS that did not include such separations. The results from comparing pre-operative CRC to post-operative samples were in agreement with studies that identified a decrease in di-antennary structures with core fucosylation and an increase in sialylated tri- and tetra-antennary N-glycans in CRC patient sera. Pre-operative abundances of N-glycans showed good performance for the classification of adenocarcinoma and led to the revisit of the previous MALDI-MS dataset with regard to histological and clinical data. This strategy has the potential to monitor patient profiles before, during, and after clinical events such as treatment, therapy, or surgery and should also be further explored.

Software-Assisted Data Processing Workflow for Intact Glycoprotein Mass Spectrometry.

Intact protein analysis by mass spectrometry is important for several applications such as assessing post-translational modifications and biotransformation. In particular, intact protein analysis allows the detection of proteoforms that are commonly missed by other approaches such as proteolytic digestion followed by bottom-up analysis. Two quantification methods are mainly used for intact protein data quantification, namely the extracted ion and deconvolution approaches. However, a consensus with regard to a single best practice for intact protein data processing is lacking. Furthermore, many data processing tools are not fit-for-purpose and, as a result, the analysis of intact proteins is laborious and lacks the throughput required to be implemented for the analysis of clinical cohorts. Therefore, in this study, we investigated the application of a software-assisted data analysis and processing workflow in order to streamline intact protein integration, annotation, and quantification via deconvolution. In addition, the assessment of orthogonal data sets generated via middle-up and bottom-up analysis enabled the cross-validation of cleavage proteoform assignments present in seminal prostate-specific antigen (PSA). Furthermore, deconvolution quantification of PSA from patients' urine revealed results that were comparable with manually performed quantification based on extracted ion electropherograms. Overall, the presented workflow allows fast and efficient processing of intact protein data. The raw data is available on MassIVE using the identifier MSV000086699.

In-Depth Analysis of the N-Glycome of Colorectal Cancer Cell Lines.

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 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.

Neutron-encoded diubiquitins to profile linkage selectivity of deubiquitinating enzymes.

Deubiquitinating enzymes are key regulators in the ubiquitin system and an emerging class of drug targets. These proteases disassemble polyubiquitin chains and many deubiquitinases show selectivity for specific polyubiquitin linkages. However, most biochemical insights originate from studies of single diubiquitin linkages in isolation, whereas in cells all linkages coexist. To better mimick this diubiquitin substrate competition, we develop a multiplexed mass spectrometry-based deubiquitinase assay that can probe all ubiquitin linkage types simultaneously to quantify deubiquitinase activity in the presence of all potential diubiquitin substrates. For this, all eight native diubiquitins are generated and each linkage type is designed with a distinct molecular weight by incorporating neutron-encoded amino acids. Overall, 22 deubiquitinases are profiled, providing a three-dimensional overview of deubiquitinase linkage selectivity over time and enzyme concentration.

Transcriptionally imprinted glycomic signatures of acute myeloid leukemia.

BACKGROUND: Acute myeloid leukemia (AML) is a genetically and phenotypically heterogeneous disease that has been suffering from stagnant survival curves for decades. In the endeavor toward improved diagnosis and treatment, cellular glycosylation has emerged as an interesting focus area in AML. While mechanistic insights are still limited, aberrant glycosylation may affect intracellular signaling pathways of AML blasts, their interactions within the microenvironment, and even promote chemoresistance. Here, we performed a meta-omics study to portray the glycomic landscape of AML, thereby screening for potential subtypes and responsible glyco-regulatory networks. RESULTS: Initially, by integrating comprehensive N-, O-, and glycosphingolipid (GSL)-glycomics of AML cell lines with transcriptomics from public databases, we were able to pinpoint specific glycosyltransferases (GSTs) and upstream transcription factors (TFs) associated with glycan phenotypes. Intriguingly, subtypes M5 and M6, as classified by the French-American-British (FAB) system, emerged with distinct glycomic features such as high (sialyl) Lewisx/a ((s)Lex/a) and high sialylation, respectively. Exploration of transcriptomics datasets of primary AML cells further substantiated and expanded our findings from cell lines as we observed similar gene expression patterns and regulatory networks that were identified to be involved in shaping AML glycan signatures. CONCLUSIONS: Taken together, our data suggest transcriptionally imprinted glycomic signatures of AML, reflecting their differentiation status and FAB classification. This study expands our insights into the emerging field of AML glycosylation and paves the way for studies of FAB class-associated glycan repertoires of AML blasts and their functional implications.

Analysis of Immunogenic Galactose-α-1,3-galactose-Containing N-Glycans in Beef, Mutton, and Pork Tenderloin by Combining Matrix-Assisted Laser Desorption/Ionization-Mass Spectroscopy and Capillary Electrophoresis Hyphenated with Mass Spectrometry via Electrospray Ionization.

Severe allergic reactions to certain types of meat following tick bites have been reported in geographic regions which are endemic with ticks. This immune response is directed to a carbohydrate antigen (galactose-α-1,3-galactose or α-Gal), which is present in glycoproteins of mammalian meats. At the moment, asparagine-linked complex carbohydrates (N-glycans) with α-Gal motifs in meat glycoproteins and in which cell types or tissue morphologies these α-Gal moieties are present in mammalian meats are still unclear. In this study, we analyzed α-Gal-containing N-glycans in beef, mutton, and pork tenderloin and provided for the first time the spatial distribution of these types of N-glycans in various meat samples. Terminal α-Gal-modified N-glycans were found to be highly abundant in all analyzed samples (55, 45, and 36% of N-glycome in beef, mutton, and pork, respectively). Visualizations of the N-glycans with α-Gal modification revealed that this motif was mainly present in the fibroconnective tissue. To conclude, this study contributes to a better understanding of the glycosylation biology of meat samples and provides guidance for processed meat products, in which only meat fibers are required as an ingredient (i.e., sausages or canned meat).

N-Glycan Isomer Differentiation by Zero Flow Capillary Electrophoresis Coupled to Mass Spectrometry.

Isomeric N-glycans often vastly differ in their biological activities, hence the need for methods that allow resolving and structurally characterizing them in biological material. Here, we established a zero flow approach using capillary electrophoresis in combination with (tandem) mass spectrometry to allow structural characterization of isomeric N-glycans at high sensitivity. Additionally, diagnostic fragment ion ratios were identified, indicative for the antenna carrying specifically linked sialic acids. In total, 208 N-glycans were characterized in human plasma, with 57 compositions showing multiple isomers.

High-Throughput Glycomic Methods.

Glycomics aims to identify the structure and function of the glycome, the complete set of oligosaccharides (glycans), produced in a given cell or organism, as well as to identify genes and other factors that govern glycosylation. This challenging endeavor requires highly robust, sensitive, and potentially automatable analytical technologies for the analysis of hundreds or thousands of glycomes in a timely manner (termed high-throughput glycomics). This review provides a historic overview as well as highlights recent developments and challenges of glycomic profiling by the most prominent high-throughput glycomic approaches, with N-glycosylation analysis as the focal point. It describes the current state-of-the-art regarding levels of characterization and most widely used technologies, selected applications of high-throughput glycomics in deciphering glycosylation process in healthy and disease states, as well as future perspectives.

Specific (sialyl-)Lewis core 2 O-glycans differentiate colorectal cancer from healthy colon epithelium.

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 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-)LewisX/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 α2-6-linked sialylation, (sulfo-)LewisX/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.

High Diversity of Glycosphingolipid Glycans of Colorectal Cancer Cell Lines Reflects the Cellular Differentiation Phenotype.

Colorectal cancer (CRC)-associated changes of protein glycosylation have been widely studied. In contrast, the expression of glycosphingolipid (GSL) patterns in CRC has, hitherto, remained largely unexplored. Even though GSLs are major carriers of cell surface carbohydrates, they are understudied due to their complexity and analytical challenges. In this study, we provide an in-depth analysis of GSL glycans of 22 CRC cell lines using porous graphitized carbon nano-liquid chromatography coupled with electrospray ionization-mass spectrometry. Our data revealed that the GSL expression varies among different cell line classifications, with undifferentiated cell lines showing high expression of blood group A, B, and H antigens while for colon-like cell lines the most prominent GSL glycans contained (sialyl)-LewisA/X and LewisB/Y antigens. Moreover, the GSL expression correlated with relevant glycosyltransferases that are involved in their biosynthesis as well as with transcription factors (TFs) implicated in colon differentiation. Additionally, correlations between certain glycosyltransferases and TFs at mRNA expression level were found, such as FUT3, which correlated with CDX1, ETS2, HNF1A, HNF4A, MECOM, and MYB. These TFs are upregulated in colon-like cell lines pointing to their potential role in regulating fucosylation during colon differentiation. In conclusion, our study reveals novel layers of potential GSL glycans regulation relevant for future research in colon differentiation and CRC.

Sialic Acid Derivatization of Fluorescently Labeled N-Glycans Allows Linkage Differentiation by Reversed-Phase Liquid Chromatography-Fluorescence Detection-Mass Spectrometry.

Sialic acids have diverse biological roles, ranging from promoting up to preventing protein and cellular recognition in health and disease. The various functions of these monosaccharides are owed, in part, to linkage variants, and as a result, linkage-specific analysis of sialic acids is an important aspect of glycomic studies. This has been addressed by derivatization strategies using matrix-assisted laser desorption/ionization mass spectrometry (MS) or sialidase digestion arrays followed by liquid chromatography (LC)-MS. Despite this, these approaches are unable to simultaneously provide unambiguous assignment of sialic acid linkages and assess further isomeric glycan features within a single measurement. Thus, for the first time, we present the combination of procainamide fluorescent labeling with sialic acid linkage-specific derivatization via ethyl esterification and amidation for the analysis of released plasma N-glycans using reversed-phase (RP)LC-fluorescence detection (FD)-MS. As a result, α2,3- and α2,6-sialylated N-glycans, with the same mass prior to derivatization, are differentiated based on retention time, precursor mass, and fragmentation spectra, and additional sialylated isomers were also separated. Furthermore, improved glycan coverage and protocol precision were found via the novel application using a combined FD-MS quantification approach. Overall, this platform achieved unambiguous assignment of N-glycan sialic acid linkages within a single RPLC-FD-MS measurement, and by improving their retention on RPLC, this technique can be used for future investigations of released N-glycans as an additional or orthogonal method to current analytical approaches.

The minimum information required for a glycomics experiment (MIRAGE): reporting guidelines for capillary electrophoresis.

The Minimum Information Required for a Glycomics Experiment (MIRAGE) is an initiative to standardize the reporting of glycoanalytical methods and to assess their reproducibility. To date, the MIRAGE Commission has published several reporting guidelines that describe what information should be provided for sample preparation methods, mass spectrometry methods, liquid chromatography analysis, exoglycosidase digestions, glycan microarray methods, and nuclear magnetic resonance methods. Here, we present the first version of reporting guidelines for glyco(proteo)mics analysis by capillary electrophoresis (CE) for standardized and high-quality reporting of experimental conditions in the scientific literature. The guidelines cover all aspects of a glyco(proteo)mics CE experiment including sample preparation, CE operation mode (CZE, CGE, CEC, MEKC, cIEF, cITP), instrument configuration, capillary separation conditions, detection, data analysis, and experimental descriptors. These guidelines are linked to other MIRAGE guidelines and are freely available through the project website https://www.beilstein-institut.de/en/projects/mirage/guidelines/#ce_analysis (doi:10.3762/mirage.7).

High-Mannose N-Glycans as Malignant Progression Markers in Early-Stage Colorectal Cancer.

The increase incidence of early colorectal cancer (T1 CRC) last years is mainly due to the introduction of population-based screening for CRC. T1 CRC staging based on histological criteria remains challenging and there is high variability among pathologists in the scoring of these criteria. It is crucial to unravel the biology behind the progression of adenoma into T1 CRC. Glycomic studies have reported extensively on alterations of the N-glycomic pattern in CRC; therefore, investigating these alterations may reveal new insights into the development of T1 CRC. We used matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI) to spatially profile the N-glycan species in a cohort of pT1 CRC using archival formalin-fixed and paraffin-embedded (FFPE) material. To generate structural information on the observed N-glycans, CE-ESI-MS/MS was used in conjunction with MALDI-MSI. Relative intensities and glycosylation traits were calculated based on a panel of 58 N-glycans. Our analysis showed pronounced differences between normal epithelium, dysplastic, and carcinoma regions. High-mannose-type N-glycans were higher in the dysplastic region than in carcinoma, which correlates to increased proliferation of the cells. We observed changes in the cancer invasive front, including higher expression of α2,3-linked sialic acids which followed the glycosylation pattern of the carcinoma region.

Glycosphingolipid-Glycan Signatures of Acute Myeloid Leukemia Cell Lines Reflect Hematopoietic Differentiation.

Aberrant expression of certain glycosphingolipids (GSLs) is associated with the differentiation of acute myeloid leukemia (AML) cells. However, the expression patterns of GSLs in AML are still poorly explored because of their complexity, the presence of multiple isomeric structures, and tedious analytical procedures. In this study, we performed an in-depth GSL glycan analysis of 19 AML cell lines using porous graphitized carbon liquid chromatography-mass spectrometry revealing strikingly different GSL glycan profiles between the various AML cell lines. The cell lines of the M6 subtype showed a high expression of gangliosides with α2,3-sialylation and Neu5Gc, while the M2 and M5 subtypes were characterized by high expression of (neo)lacto-series glycans and Lewis A/X antigens. Integrated analysis of glycomics and available transcriptomics data revealed the association of GSL glycan abundances with the transcriptomics expression of certain glycosyltransferases (GTs) and transcription factors (TFs). In addition, correlations were found between specific GTs and TFs. Our data reveal TFs GATA2, GATA1, and RUNX1 as candidate inducers of the expression of gangliosides and sialylation via regulation of the GTs ST3GAL2 and ST8SIA1. In conclusion, we show that GSL glycan expression levels are associated with hematopoietic AML classifications and TF and GT gene expression. Further research is needed to dissect the regulation of GSL expression and its role in hematopoiesis and associated malignancies.

High sensitivity glycomics in biomedicine.

Many analytical challenges in biomedicine arise from the generally high heterogeneity and complexity of glycan- and glycoconjugate-containing samples, which are often only available in minute amounts. Therefore, highly sensitive workflows and detection methods are required. In this review mass spectrometric workflows and detection methods are evaluated for glycans and glycoproteins. Furthermore, glycomic methodologies and innovations that are tailored for enzymatic treatments, chemical derivatization, purification, separation, and detection at high sensitivity are highlighted. The discussion is focused on the analysis of mammalian N-linked and GalNAc-type O-linked glycans.

Integrated N- and O-Glycomics of Acute Myeloid Leukemia (AML) Cell Lines.

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 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.

Dopant-Enriched Nitrogen Gas for Enhanced Electrospray Ionization of Released Glycans in Negative Ion Mode.

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. 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.