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
Lageveen-Kammeijer, G.S.M.; Kuster, B.; Reusch, D.; Wuhrer, M. 2021
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... Show moreMany 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. 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
Early detection of prostate cancer may lead to the overdiagnosis and overtreatment of patients as well as missing significant cancers. The current diagnostic approach uses elevated serum... Show moreEarly detection of prostate cancer may lead to the overdiagnosis and overtreatment of patients as well as missing significant cancers. The current diagnostic approach uses elevated serum concentrations of prostate-specific antigen (PSA) as an indicator of risk. However, this test has been widely criticized as it shows poor specificity and sensitivity. In order to improve early detection and diagnosis, several studies have investigated whether different PSA proteoforms are correlated to prostate cancer. Until now, studies and methodologies for the comprehensive characterization of PSA proteoforms from biofluids are scarce. For this purpose, we developed an intact protein assay to analyze PSA by capillary electrophoresis-electrospray ionization-mass spectrometry after affinity purification from patients? urine. Here, we determined six proteolytic cleavage variants. In regard to glycosylation, tri-, di-, mono- and non-sialylated complex-type N-glycans were found on non-cleaved PSA, as well as the non-glycosylated variant. The performance of the intact protein assay was assessed using a pooled sample, obtaining an inter-day variability of 15%. Furthermore, urinary patient samples were analyzed by intact protein analysis and a bottom-up approach (glycopeptide analysis). This combined approach revealed complimentary information on both levels, demonstrating the benefit of using two orthogonal techniques to provide a thorough profile of urinary PSA.Significance: The detection of clinically relevant prostate cancer requires a more specific and sensitive biomarker and, in this case, several PSA proteoforms may be able to aid or improve the current PSA test. However, a comprehensive analysis of the intact PSA proteoform profile is still lacking. This study investigated the PSA proteoforms present in urine and, in particular, determined the relative contribution of cleaved PSA and noncleaved PSA forms to the total glycosylation profile. Importantly, intact protein analysis did not require further sample treatment before being measured by CE-ESI-MS. Furthermore, its glycosylation was also assessed in a bottom-up approach to provide complementary information. Overall, these results represent an important basis for future characterization and biomarker studies. Show less
With 28 potential N-glycosylation sites, human carcinoembryonic antigen (CEA) bears an extreme amount of N-linked glycosylation, and approximately 60% of its molecular mass can be attributed to its... Show moreWith 28 potential N-glycosylation sites, human carcinoembryonic antigen (CEA) bears an extreme amount of N-linked glycosylation, and approximately 60% of its molecular mass can be attributed to its carbohydrates. CEA is often overexpressed and released by many solid tumors, including colorectal carcinomas. CEA displays an impressive heterogeneity and variability in sugar content; however, site-specific distribution of carbohydrate structures has not been reported so far. The present study investigated CEA samples purified from human colon carcinoma and human liver metastases and enabled the characterization of 21 out of 28 potential N-glycosylation sites with respect to their occupancy. The coverage was achieved by a multienzymatic digestion approach with specific enzymes, such as trypsin, endoproteinase Glu-C, and the nonspecific enzyme, Pronase, followed by analysis using sheathless CE-MS/MS. In total, 893 different N-glycopeptides and 128 unique N-glycan compositions were identified. Overall, a great heterogeneity was found both within (micro) and in between (macro) individual N-glycosylation sites. Moreover, notable differences were found on certain N-glycosylation sites between primary adenocarcinoma and metastatic tumor in regard to branching, bisection, sialylation, and fucosylation. Those features, if further investigated in a targeted manner, may pave the way toward improved diagnostics and monitoring of colorectal cancer progression and recurrence. Raw mass spectrometric data and Skyline processed data files that support the findings of this study are available in the MassIVE repository with the identifier MSV000086774 [DOI: 10.25345/C5Z50X]. Show less
Wang, W.; Kaluza, A.; Nouta, J.; Nicolardi, S.; Ferens-Sieczkowska, M.; Wuhrer, M.; ... ; Haan, N. de 2021
An altered total seminal plasma glycosylation has been associated with male infertility, and the highly abundant seminal plasma glycoprotein prostate-specific antigen (PSA) plays an important role... Show moreAn altered total seminal plasma glycosylation has been associated with male infertility, and the highly abundant seminal plasma glycoprotein prostate-specific antigen (PSA) plays an important role in fertilization. However, the exact role of PSA glycosylation in male fertility is not clear. To understand the involvement of PSA glycosylation in the fertilization process, analytical methods are required to study the glycosylation of PSA from seminal plasma with a high glycoform resolution and in a protein-specific manner. In this study, we developed a novel, high-throughput PSA glycopeptide workflow, based on matrix-assisted laser desorption/ionization-mass spectrometry, allowing the discrimination of sialic acid linkage isomers via the derivatization of glycopeptides. The method was successfully applied on a cohort consisting of seminal plasma from infertile and fertile men (N = 102). Forty-four glycopeptides were quantified in all samples, showing mainly complex-type glycans with high levels of fucosylation and sialylation. In addition, N,N-diacetyllactosamine (LacdiNAc) motives were found as well as hybrid-type and high mannose-type structures. Our method showed a high intra- and interday repeatability and revealed no difference in PSA glycosylation between fertile and infertile men. Next to seminal plasma, the method is also expected to be of use for studying PSA glycopeptides derived from other biofluids and/or in other disease contexts. Show less
Drouin, N.; Mever, M. van; Zhang, W.; Tobolkina, E.; Ferre, S.; Servais, A.C.; ... ; Ramautar, R. 2020
Capillary zone electrophoresis-mass spectrometry (CE-MS) is a mature analytical tool for the efficient profiling of (highly) polar and ionizable compounds. However, the use of CE-MS in comparison... Show moreCapillary zone electrophoresis-mass spectrometry (CE-MS) is a mature analytical tool for the efficient profiling of (highly) polar and ionizable compounds. However, the use of CE-MS in comparison to other separation techniques remains underrepresented in metabolomics, as this analytical approach is still perceived as technically challenging and less reproducible, notably for migration time. The latter is key for a reliable comparison of metabolic profiles and for unknown biomarker identification that is complementary to high resolution MS/MS. In this work, we present the results of a Metabo-ring trial involving 16 CE-MS platforms among 13 different laboratories spanning two continents. The goal was to assess the reproducibility and identification capability of CE-MS by employing effective electrophoretic mobility (mu(eff)) as the key parameter in comparison to the relative migration time (RMT) approach. For this purpose, a representative cationic metabolite mixture in water, pretreated human plasma, and urine samples spiked with the same metabolite mixture were used and distributed for analysis by all laboratories. The mu(eff) was determined for all metabolites spiked into each sample. The background electrolyte (BGE) was prepared and employed by each participating lab following the same protocol. All other parameters (capillary, interface, injection volume, voltage ramp, temperature, capillary conditioning, and rinsing procedure, etc.) were left to the discretion of the contributing laboratories. The results revealed that the reproducibility of the mu(eff) for 20 out of the 21 model compounds was below 3.1% vs 10.9% for RMT, regardless of the huge heterogeneity in experimental conditions and platforms across the 13 laboratories. Overall, this Metabo-ring trial demonstrated that CE-MS is a viable and reproducible approach for metabolomics. Show less
Drouin, N.F.P.; Mever, M. van; Zhang, W.; Tobolkina, E.; Ferre, S.; Servais, A.C.; ... ; Ramautar, R. 2020
Capillary zone electrophoresis-mass spectrometry (CE-MS) is a mature analytical tool for the efficient profiling of (highly) polar and ionizable compounds. However, the use of CE-MS in comparison... Show moreCapillary zone electrophoresis-mass spectrometry (CE-MS) is a mature analytical tool for the efficient profiling of (highly) polar and ionizable compounds. However, the use of CE-MS in comparison to other separation techniques remains underrepresented in metabolomics, as this analytical approach is still perceived as technically challenging and less reproducible, notably for migration time. The latter is key for a reliable comparison of metabolic profiles and for unknown biomarker identification that is complementary to high resolution MS/MS. In this work, we present the results of a Metabo-ring trial involving 16 CE-MS platforms among 13 different laboratories spanning two continents. The goal was to assess the reproducibility and identification capability of CE-MS by employing effective electrophoretic mobility (mu(eff)) as the key parameter in comparison to the relative migration time (RMT) approach. For this purpose, a representative cationic metabolite mixture in water, pretreated human plasma, and urine samples spiked with the same metabolite mixture were used and distributed for analysis by all laboratories. The mu(eff) was determined for all metabolites spiked into each sample. The background electrolyte (BGE) was prepared and employed by each participating lab following the same protocol. All other parameters (capillary, interface, injection volume, voltage ramp, temperature, capillary conditioning, and rinsing procedure, etc.) were left to the discretion of the contributing laboratories. The results revealed that the reproducibility of the mu(eff) for 20 out of the 21 model compounds was below 3.1% vs 10.9% for RMT, regardless of the huge heterogeneity in experimental conditions and platforms across the 13 laboratories. Overall, this Metabo-ring trial demonstrated that CE-MS is a viable and reproducible approach for metabolomics. 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
Changes in the abundance of antennary fucosylated glycans in human total plasma N-glycome (TPNG) have been associated with several diseases ranging from diabetes to various forms of cancer. However... Show moreChanges in the abundance of antennary fucosylated glycans in human total plasma N-glycome (TPNG) have been associated with several diseases ranging from diabetes to various forms of cancer. However, it is challenging to address this important part of the human glycome. Most commonly, time-consuming chromatographic separations are performed to differentially quantify core and antenna fucosylation. Obtaining sufficient resolution for larger, more complex glycans can be challenging. We introduce a matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) assay for the relative quantitation of antennary fucosylation in TPNG. N-linked glycans are released from plasma by PNGase F and further treated with a core fucosidase before performing a linkage-informative sialic acid derivatization. The core fucosylated glycans are thus depleted while the remaining antennary fucosylated glycans are quantitated. Simultaneous quantitation of alpha 2,3-linked sialic acids and antennary fucosylation allows an estimation of the sialyl-Lewis x motif. The approach is feasible using either ultrahigh-resolution Fourier-transform ion cyclotron resonance mass spectrometry or time-of-flight mass spectrometry. The assay was used to investigate changes of antennary fucosylation as clinically relevant marker in 14 colorectal cancer patients. In accordance with a previous report, we found elevated levels of antennary fucosylation pre-surgery which decreased after tumor resection. The assay has the potential for revealing antennary fucosylation signatures in various conditions including diabetes and different types of cancer. Show less
A broad-based interlaboratory study of glycosylation profiles of a reference and modified IgG antibody involving 103 reports from 76 laboratories.Glycosylation is a topic of intense current... Show moreA broad-based interlaboratory study of glycosylation profiles of a reference and modified IgG antibody involving 103 reports from 76 laboratories.Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods. Show less
Changes in the abundance of antennary fucosylated glycans in human total plasma N-glycome (TPNG) have been associated with several diseases ranging from diabetes to various forms of cancer. However... Show moreChanges in the abundance of antennary fucosylated glycans in human total plasma N-glycome (TPNG) have been associated with several diseases ranging from diabetes to various forms of cancer. However, it is challenging to address this important part of the human glycome. Most commonly, time-consuming chromatographic separations are performed to differentially quantify core and antenna fucosylation. Obtaining sufficient resolution for larger, more complex glycans can be challenging. We introduce a matrix-assisted laser desorption/ionization—mass spectrometry (MALDI-MS) assay for the relative quantitation of antennary fucosylation in TPNG. N-linked glycans are released from plasma by PNGase F and further treated with a core fucosidase before performing a linkage-informative sialic acid derivatization. The core fucosylated glycans are thus depleted while the remaining antennary fucosylated glycans are quantitated. Simultaneous quantitation of α2,3-linked sialic acids and antennary fucosylation allows an estimation of the sialyl-Lewis x motif. The approach is feasible using either ultrahigh-resolution Fourier-transform ion cyclotron resonance mass spectrometry or time-of-flight mass spectrometry. The assay was used to investigate changes of antennary fucosylation as clinically relevant marker in 14 colorectal cancer patients. In accordance with a previous report, we found elevated levels of antennary fucosylation pre-surgery which decreased after tumor resection. The assay has the potential for revealing antennary fucosylation signatures in various conditions including diabetes and different types of cancer. Show less
Lageveen-Kammeijer, G.S.M.; Haan, N. de; Mohaupt, P.; Wagt, S.; Filius, M.; Nouta, J.; ... ; Wuhrer, M. 2019
