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1.
Glycoconj J ; 38(3): 387-395, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33877489

RESUMO

Breast cancer is the most prevalent cancer in women. Early detection of this disease improves survival and therefore population screenings, based on mammography, are performed. However, the sensitivity of this screening modality is not optimal and new screening methods, such as blood tests, are being explored. Most of the analyses that aim for early detection focus on proteins in the bloodstream. In this study, the biomarker potential of total serum N-glycosylation analysis was explored with regard to detection of breast cancer. In an age-matched case-control setup serum protein N-glycan profiles from 145 breast cancer patients were compared to those from 171 healthy individuals. N-glycans were enzymatically released, chemically derivatized to preserve linkage-specificity of sialic acids and characterized by high resolution mass spectrometry. Logistic regression analysis was used to evaluate associations of specific N-glycan structures as well as N-glycosylation traits with breast cancer. In a case-control comparison three associations were found, namely a lower level of a two triantennary glycans and a higher level of one tetraantennary glycan in cancer patients. Of note, various other N-glycomic signatures that had previously been reported were not replicated in the current cohort. It was further evaluated whether the lack of replication of breast cancer N-glycomic signatures could be partly explained by the heterogenous character of the disease since the studies performed so far were based on cohorts that included diverging subtypes in different numbers. It was found that serum N-glycan profiles differed for the various cancer subtypes that were analyzed in this study.


Assuntos
Biomarcadores Tumorais/sangue , Neoplasias da Mama/sangue , Polissacarídeos/sangue , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Feminino , Humanos , Pessoa de Meia-Idade , Polissacarídeos/classificação , Polissacarídeos/metabolismo
2.
Cancer Med ; 9(22): 8519-8529, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32898301

RESUMO

BACKGROUND &AIMS: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer type with loco-regional spread that makes the tumor surgically unresectable. Novel diagnostic tools are needed to improve detection of PDAC and increase patient survival. In this study we explore serum protein N-glycan profiles from PDAC patients with regard to their applicability to serve as a disease biomarker panel. METHODS: Total serum N-glycome analysis was applied to a discovery set (86 PDAC cases/84 controls) followed by independent validation (26 cases/26 controls) using in-house collected serum specimens. Protein N-glycan profiles were obtained using ultrahigh resolution mass spectrometry and included linkage-specific sialic acid information. N-glycans were relatively quantified and case-control classification performance was evaluated based on glycosylation traits such as branching, fucosylation, and sialylation. RESULTS: In PDAC patients a higher level of branching (OR 6.19, P-value 9.21 × 10-11 ) and (antenna)fucosylation (OR 13.27, P-value 2.31 × 10-9 ) of N-glycans was found. Furthermore, the ratio of α2,6- vs α2,3-linked sialylation was higher in patients compared to healthy controls. A classification model built with three glycosylation traits was used for discovery (AUC 0.88) and independent validation (AUC 0.81), with sensitivity and specificity values of 0.85 and 0.71 for the discovery set and 0.75 and 0.72 for the validation set. CONCLUSION: Serum N-glycome analysis revealed glycosylation differences that allow classification of PDAC patients from healthy controls. It was demonstrated that glycosylation traits rather than single N-glycan structures obtained in this clinical glycomics study can serve as a basis for further development of a blood-based diagnostic test.


Assuntos
Biomarcadores Tumorais/sangue , Carcinoma Ductal Pancreático/sangue , Glicômica , Glicoproteínas/sangue , Neoplasias Pancreáticas/sangue , Idoso , Carcinoma Ductal Pancreático/diagnóstico , Estudos de Casos e Controles , Cromatografia Líquida de Alta Pressão , Feminino , Glicosilação , Humanos , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Neoplasias Pancreáticas/diagnóstico , Valor Preditivo dos Testes , Reprodutibilidade dos Testes
3.
Artigo em Inglês | MEDLINE | ID: mdl-32616483

RESUMO

INTRODUCTION: Recent studies revealed N-glycosylation signatures of type 2 diabetes, inflammation and cardiovascular risk factors. Most people with diabetes use medication to reduce cardiovascular risk. The association of these medications with the plasma N-glycome is largely unknown. We investigated the associations of metformin, statin, ACE inhibitor/angiotensin II receptor blocker (ARB), sulfonylurea (SU) derivatives and insulin use with the total plasma N-glycome in type 2 diabetes. RESEARCH DESIGN AND METHODS: After enzymatic release from glycoproteins, N-glycans were measured by matrix-assisted laser desorption/ionization mass spectrometry in the DiaGene (n=1815) and Hoorn Diabetes Care System (n=1518) cohorts. Multiple linear regression was used to investigate associations with medication, adjusted for clinical characteristics. Results were meta-analyzed and corrected for multiple comparisons. RESULTS: Metformin and statins were associated with decreased fucosylation and increased galactosylation and sialylation in glycans unrelated to immunoglobulin G. Bisection was increased within diantennary fucosylated non-sialylated glycans, but decreased within diantennary fucosylated sialylated glycans. Only few glycans were associated with ACE inhibitor/ARBs, while none associated with insulin and SU derivative use. CONCLUSIONS: We conclude that metformin and statins associate with a total plasma N-glycome signature in type 2 diabetes. Further studies are needed to determine the causality of these relations, and future N-glycomic research should consider medication a potential confounder.


Assuntos
Diabetes Mellitus Tipo 2 , Inibidores de Hidroximetilglutaril-CoA Redutases , Metformina , Antagonistas de Receptores de Angiotensina , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Proteínas Sanguíneas , Diabetes Mellitus Tipo 2/tratamento farmacológico , Glicosilação , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Metformina/uso terapêutico
4.
Talanta ; 205: 120104, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31450448

RESUMO

Body fluid N-glycome analysis as well as glyco-proteoform profiling of existing protein biomarkers potentially provides a stratification layer additional to quantitative, diagnostic protein levels. For clinical omics applications, the collection of a dried blood spot (DBS) is increasingly pursued as an alternative to sampling milliliters of peripheral blood. Here we evaluate DBS cards as a blood collection strategy for protein N-glycosylation analysis aiming for high-throughput clinical applications. A protocol for facile N-glycosylation profiling from DBS is developed that includes sialic acid linkage differentiation. This protocol is based on a previously established total plasma N-glycome mass spectrometry (MS) method, with adjustments for the analysis of DBS specimens. After DBS-punching and protein solubilization N-glycans are released, followed by chemical derivatization of sialic acids and MS-measurement of N-glycan profiles. With this method, more than 80 different glycan structures are identified from a DBS, with RSDs below 10% for the ten most abundant glycans. N-glycan profiles of finger-tip blood and venous blood are compared and short-term stability of DBS is demonstrated. This method for fast N-glycosylation profiling of DBS provides a minimally invasive alternative to conventional serum and plasma protein N-glycosylation workflows. With simplified blood sampling this DBS approach has vast potential for clinical glycomics applications.


Assuntos
Teste em Amostras de Sangue Seco/métodos , Glicômica/métodos , Polissacarídeos/sangue , Humanos , Polissacarídeos/química , Ácidos Siálicos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos
5.
Anal Chem ; 90(20): 11955-11961, 2018 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-30230816

RESUMO

High-throughput mass spectrometry (MS) glycomics is an emerging field driven by technological advancements including sample preparation and data processing. Previously, we reported an automated protocol for the analysis of N-glycans released from plasma proteins that included sialic acid derivatization with linkage-specificity, namely, ethylation of α2,6-linked sialic acid residues and lactone formation of α2,3-linked sialic acids. In the current study, each step in this protocol was further optimized. Method improvements included minimizing the extent of side-reaction during derivatization, an adjusted glycan purification strategy and mass analysis of the released N-glycans by ultrahigh resolution matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance MS. The latter resolved peak overlap and simplified spectral alignment due to high mass measurement precision. Moreover, this resulted in more confident glycan assignments and improved signal-to-noise for low-abundant species. The performance of the protocol renders high-throughput applications feasible in the exciting field of clinical glycomics.


Assuntos
Automação , Proteínas Sanguíneas/química , Ácido N-Acetilneuramínico/química , Polissacarídeos/sangue , Glicômica , Glicosilação , Humanos , Espectrometria de Massas , Polissacarídeos/química
6.
J Am Chem Soc ; 140(12): 4232-4243, 2018 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-29470065

RESUMO

Noninvasive methods to modulate G protein-coupled receptors (GPCRs) with temporal and spatial precision are in great demand. Photopharmacology uses photons to control in situ the biological properties of photoswitchable small-molecule ligands, which bodes well for chemical biological precision approaches. Integrating the light-switchable configurational properties of an azobenzene into the ligand core, we developed a bidirectional antagonist toolbox for an archetypical family A GPCR, the histamine H3 receptor (H3R). From 16 newly synthesized photoswitchable compounds, VUF14738 (28) and VUF14862 (33) were selected as they swiftly and reversibly photoisomerize and show over 10-fold increased or decreased H3R binding affinities, respectively, upon illumination at 360 nm. Both ligands combine long thermal half-lives with fast and high photochemical trans-/ cis conversion, allowing their use in real-time electrophysiology experiments with oocytes to confirm dynamic photomodulation of H3R activation in repeated second-scale cycles. VUF14738 and VUF14862 are robust and fatigue-resistant photoswitchable GPCR antagonists suitable for spatiotemporal studies of H3R signaling.


Assuntos
Compostos Azo/farmacologia , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Compostos Azo/síntese química , Compostos Azo/química , Humanos , Ligantes , Estrutura Molecular , Processos Fotoquímicos , Fótons , Receptores Acoplados a Proteínas G/metabolismo
7.
Front Med (Lausanne) ; 4: 241, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29359131

RESUMO

Symptoms of rheumatoid arthritis (RA) improve during pregnancy, a phenomenon that was found to be associated with N-glycosylation changes of immunoglobulin G. Recent advances in high-throughput glycosylation analysis allow the assessment of the N-glycome of human sera as well. The aim of this study was to identify new protein N-glycosylation properties that associate with changes in RA disease activity during and after pregnancy. A longitudinal cohort of serum samples was collected during 285 pregnancies (32 control individuals and 253 RA patients). Per individual one sample was collected before conception, three during pregnancy, and three after delivery. Released serum protein N-glycans were measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) after employing chemical modification of the sialic acids to allow discrimination of sialic acid linkage isomers. Serum protein N-glycosylation showed strongly modified during pregnancy, with similar changes visible in control individuals and RA pregnancies. Namely, a decrease in bisection and an increase in galactosylation in diantennary glycans were found, as well as an increase in tri- and tetraantennary species and α2,3-linked sialylation thereof. The change in RA disease activity [DAS28(3)-CRP] proved negatively associated with the galactosylation of diantennary N-glycans, and positively with the sialylation of triantennary fucosylated species (A3FGS). While the protein source of the novel finding A3FGS is thus far unknown, its further study may improve our understanding of the etiology of RA disease severity.

8.
Anal Bioanal Chem ; 409(2): 359-378, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27888305

RESUMO

Reversed-phase chromatography is a method that is often used for glycan separation. For this, glycans are often derivatized with a hydrophobic tag to achieve retention on hydrophobic stationary phases. The separation and elution order of glycans in reversed-phase chromatography is highly dependent on the hydrophobicity of the tag and the contribution of the glycan itself to the retention. The contribution of the different monosaccharides to the retention strongly depends on the position and linkage, and isomer separation may be achieved. The influence of sialic acids and fucoses on the retention of glycans is still incompletely understood and deserves further study. Analysis of complex samples may come with incomplete separation of glycan species, thereby complicating reversed-phase chromatography with fluorescence or UV detection, whereas coupling with mass spectrometry detection allows the resolution of complex mixtures. Depending on the column properties, eluents, and run time, separation of isomeric and isobaric structures can be accomplished with reversed-phase chromatography. Alternatively, porous graphitized carbon chromatography and hydrophilic interaction liquid chromatography are also able to separate isomeric and isobaric structures, generally without the necessity of glycan labeling. Hydrophilic interaction liquid chromatography, porous graphitized carbon chromatography, and reversed-phase chromatography all serve different research purposes and thus can be used for different research questions. A great advantage of reversed-phase chromatography is its broad distribution as it is used in virtually every bioanalytical research laboratory, making it an attracting platform for glycan analysis. Graphical Abstract Glycan isomer separation by reversed phase liquid chromatography.

9.
J Proteome Res ; 14(9): 4080-6, 2015 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-26179816

RESUMO

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.


Assuntos
Automação/métodos , Glicoproteínas/sangue , Glicoproteínas/química , Ensaios de Triagem em Larga Escala/métodos , Ácido N-Acetilneuramínico/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Esterificação , Glicoproteínas/metabolismo , Glicosilação , Ácido N-Acetilneuramínico/química , Reprodutibilidade dos Testes
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