RESUMO
Various cancers such as colorectal cancer (CRC) are associated with alterations in protein glycosylation. CRC cell lines are frequently used to study these (glyco)biological changes and their mechanisms. However, differences between CRC cell lines with regard to their glycosylation have hitherto been largely neglected. Here, we comprehensively characterized the N-glycan profiles of 25 different CRC cell lines, derived from primary tumors and metastatic sites, in order to investigate their potential as glycobiological tumor model systems and to reveal glycans associated with cell line phenotypes. We applied an optimized, high-throughput membrane-based enzymatic glycan release for small sample amounts. Released glycans were derivatized to stabilize and differentiate between α2,3- and α2,6-linked N-acetylneuraminic acids, followed by N-glycosylation analysis by MALDI-TOF(/TOF)-MS. Our results showed pronounced differences between the N-glycosylation patterns of CRC cell lines. CRC cell line profiles differed from tissue-derived N-glycan profiles with regard to their high-mannose N-glycan content but showed a large overlap for complex type N-glycans, supporting their use as a glycobiological cancer model system. Importantly, we could show that the high-mannose N-glycans did not only occur as intracellular precursors but were also present at the cell surface. The obtained CRC cell line N-glycan features were not clearly correlated with mRNA expression levels of glycosyltransferases, demonstrating the usefulness of performing the structural analysis of glycans. Finally, correlation of CRC cell line glycosylation features with cancer cell markers and phenotypes revealed an association between highly fucosylated glycans and CDX1 and/or villin mRNA expression that both correlate with cell differentiation. Together, our findings provide new insights into CRC-associated glycan changes and setting the basis for more in-depth experiments on glycan function and regulation.
Assuntos
Neoplasias Colorretais/metabolismo , Glicômica/métodos , Proteínas de Homeodomínio/metabolismo , Proteínas dos Microfilamentos/metabolismo , Polissacarídeos/metabolismo , Células CACO-2 , Diferenciação Celular/genética , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Fucose/metabolismo , Regulação Neoplásica da Expressão Gênica , Glicosilação , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Células HCT116 , Células HT29 , Proteínas de Homeodomínio/genética , Humanos , Proteínas dos Microfilamentos/genética , Análise de Componente Principal , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em TandemRESUMO
Metabolite quantitative traits carry great promise for epidemiological studies, and their genetic background has been addressed using Genome-Wide Association Studies (GWAS). Thus far, the role of less common variants has not been exhaustively studied. Here, we set out a GWAS for metabolite quantitative traits in serum, followed by exome sequence analysis to zoom in on putative causal variants in the associated genes. 1H Nuclear Magnetic Resonance (1H-NMR) spectroscopy experiments yielded successful quantification of 42 unique metabolites in 2,482 individuals from The Erasmus Rucphen Family (ERF) study. Heritability of metabolites were estimated by SOLAR. GWAS was performed by linear mixed models, using HapMap imputations. Based on physical vicinity and pathway analyses, candidate genes were screened for coding region variation using exome sequence data. Heritability estimates for metabolites ranged between 10% and 52%. GWAS replicated three known loci in the metabolome wide significance: CPS1 with glycine (P-value â=â1.27×10-32), PRODH with proline (P-value â=â1.11×10-19), SLC16A9 with carnitine level (P-value â=â4.81×10-14) and uncovered a novel association between DMGDH and dimethyl-glycine (P-value â=â1.65×10-19) level. In addition, we found three novel, suggestively significant loci: TNP1 with pyruvate (P-value â=â1.26×10-8), KCNJ16 with 3-hydroxybutyrate (P-value â=â1.65×10-8) and 2p12 locus with valine (P-value â=â3.49×10-8). Exome sequence analysis identified potentially causal coding and regulatory variants located in the genes CPS1, KCNJ2 and PRODH, and revealed allelic heterogeneity for CPS1 and PRODH. Combined GWAS and exome analyses of metabolites detected by high-resolution 1H-NMR is a robust approach to uncover metabolite quantitative trait loci (mQTL), and the likely causative variants in these loci. It is anticipated that insight in the genetics of intermediate phenotypes will provide additional insight into the genetics of complex traits.
Assuntos
Exoma/genética , Estudo de Associação Genômica Ampla , Metaboloma/genética , Locos de Características Quantitativas/genética , Feminino , Predisposição Genética para Doença , Glicina/sangue , Humanos , Erros Inatos do Metabolismo , Fenótipo , Polimorfismo de Nucleotídeo Único , Ácido Pirúvico/sangue , Valina/sangueRESUMO
With the development of more sensitive hyphenation strategies for capillary electrophoresis-electrospray-mass spectrometry the technique has reemerged as technique with high separation power combined with high sensitivity in the analysis of peptides and protein digests. This review will discuss the newly developed hyphenation strategies for CE-ESI-MS and their application in bottom-up proteomics as well as the applications in the same time span, 2009 to present, using co-axial sheathliquid. Subsequently all separate aspects in the development of a CE-ESI-MS method for bottom-up proteomics shall be discussed, highlighting certain applications and discussing pros and cons of the various choices. The separation of peptides in a capillary electrophoresis system is discussed including the great potential for modeling of this migration of peptides due to the simple electrophoretic separation process. Furthermore, the technical aspects of method development are discussed, namely; background electrolyte choice, coating of the separation capillary and chosen loading method. Finally, conclusions and an outlook on future developments in the field of bottom-up proteomics by CE-ESI-MS will be provided.
Assuntos
Eletroforese Capilar/métodos , Mapeamento de Peptídeos/métodos , Proteoma/química , Proteoma/isolamento & purificação , Proteômica/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Algoritmos , Eletroforese Capilar/instrumentação , Manejo de Espécimes/instrumentação , Manejo de Espécimes/métodos , Espectrometria de Massas por Ionização por Electrospray/instrumentação , Integração de SistemasRESUMO
BACKGROUND: Dendritic cells (DCs) are the sentinels of the immune system. Upon recognition of a pathogen, they mature and migrate to draining lymph nodes to prime and polarize T cell responses. Although it is known that helminths and helminth-derived molecules condition DCs to polarize T helper (Th) cells towards Th2, the underlying mechanisms remain incompletely understood. OBJECTIVES: The aim of this study was to conduct a proteome analysis of helminth antigen-stimulated DCs in order to gain more insight into the cellular processes associated with their ability to polarize immune responses. METHODS: We analyzed the maturation and polarization of monocyte-derived DCs from 9 donors at 2 different time points after stimulation with different Th1- and Th2-polarizing pathogen-derived molecules. The samples were measured using liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry for relative quantitation. RESULTS: Lipopolysaccharide-induced maturation promoted the expression of proteins related to metabolic, cellular, and immune system processes. Th1-polarizing DCs, conditioned by IFN-γ during maturation, displayed accelerated maturation by differentially expressing cytoskeletal proteins and proteins involved in immune regulation. The stimulation of DCs with soluble egg antigens and omega-1 derived from Schistosoma mansoni, which are both Th2-inducing stimuli, increased 60S acidic ribosomal protein P2, and vesicle amine transferase 1 while decreasing the expression of proteins related to antigen processing and presentation. CONCLUSION: Our data indicate that not only proteins involved in the interaction between T cells and DCs at the level of the immunological synapse, but also those related to cellular metabolism and stress, may promote Th2 polarization.
Assuntos
Células Dendríticas/imunologia , Schistosoma mansoni/imunologia , Esquistossomose mansoni/imunologia , Células Th1/imunologia , Células Th2/imunologia , Animais , Apresentação de Antígeno , Antígenos de Helmintos/imunologia , Diferenciação Celular , Células Cultivadas , Proteínas do Ovo/imunologia , Humanos , Evasão da Resposta Imune , Interferon gama/imunologia , Lipopolissacarídeos/imunologia , ProteômicaRESUMO
Glycans present on glycoproteins and glycolipids of the major human parasite Schistosoma mansoni induce innate as well as adaptive immune responses in the host. To be able to study the molecular characteristics of schistosome infections it is therefore required to determine the expression profiles of glycans and antigenic glycan-motifs during a range of critical stages of the complex schistosome lifecycle. We performed a longitudinal profiling study covering schistosome glycosylation throughout worm- and egg-development using a mass spectrometry-based glycomics approach. Our study revealed that during worm development N-glycans with Galß1-4(Fucα1-3)GlcNAc (LeX) and core-xylose motifs were rapidly lost after cercariae to schistosomula transformation, whereas GalNAcß1-4GlcNAc (LDN)-motifs gradually became abundant and predominated in adult worms. LeX-motifs were present on glycolipids up to 2 weeks of schistosomula development, whereas glycolipids with mono- and multifucosylated LDN-motifs remained present up to the adult worm stage. In contrast, expression of complex O-glycans diminished to undetectable levels within days after transformation. During egg development, a rich diversity of N-glycans with fucosylated motifs was expressed, but with α3-core fucose and a high degree of multifucosylated antennae only in mature eggs and miracidia. N-glycan antennae were exclusively LDN-based in miracidia. O-glycans in the mature eggs were also diverse and contained LeX- and multifucosylated LDN, but none of these were associated with miracidia in which we detected only the Galß1-3(Galß1-6)GalNAc core glycan. Immature eggs also exhibited short O-glycan core structures only, suggesting that complex fucosylated O-glycans of schistosome eggs are derived primarily from glycoproteins produced by the subshell envelope in the developed egg. Lipid glycans with multifucosylated GlcNAc repeats were present throughout egg development, but with the longer highly fucosylated stretches enriched in mature eggs and miracidia. This global analysis of the developing schistosome's glycome provides new insights into how stage-specifically expressed glycans may contribute to different aspects of schistosome-host interactions.
Assuntos
Antígenos de Protozoários/metabolismo , Glicômica/métodos , Estágios do Ciclo de Vida , Parasitos/metabolismo , Polissacarídeos/metabolismo , Schistosoma mansoni/crescimento & desenvolvimento , Schistosoma mansoni/metabolismo , Animais , Epitopos/metabolismo , Glicoproteínas/química , Glicoproteínas/metabolismo , Glicosilação , Humanos , Lipídeos/química , Óvulo/metabolismo , Parasitos/crescimento & desenvolvimento , Polissacarídeos/química , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
All human cells are covered by glycans, the carbohydrate units of glycoproteins, glycolipids, and proteoglycans. Most glycans are localized to cell surfaces and participate in events essential for cell viability and function. Glycosylation evolves during carcinogenesis, and therefore carcinoma-related glycan structures are potential cancer biomarkers. Colorectal cancer is one of the world's three most common cancers, and its incidence is rising. Novel biomarkers are essential to identify patients for targeted and individualized therapy. We compared the N-glycan profiles of five rectal adenomas and 18 rectal carcinomas of different stages by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. Paraffin-embedded tumor samples were deparaffinized, and glycans were enzymatically released and purified. We found differences in glycosylation between adenomas and carcinomas: monoantennary, sialylated, pauci-mannose, and small high-mannose N-glycan structures were more common in carcinomas than in adenomas. We also found differences between stage I-II and stage III carcinomas. Based on these findings, we selected two glycan structures: pauci-mannose and sialyl Lewis a, for immunohistochemical analysis of their tissue expression in 220 colorectal cancer patients. In colorectal cancer, poor prognosis correlated with elevated expression of sialyl Lewis a, and in advanced colorectal cancer, poor prognosis correlated with elevated expression of pauci-mannose. In conclusion, by mass spectrometry we found several carcinoma related glycans, and we demonstrate a method of transforming these results into immunohistochemistry, a readily applicable method to study biomarker expression in patient samples.
Assuntos
Adenoma/metabolismo , Carcinoma/metabolismo , Glicômica/métodos , Neoplasias Retais/metabolismo , Adenoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Asparagina/metabolismo , Antígeno CA-19-9 , Carcinoma/patologia , Neoplasias Colorretais/metabolismo , Progressão da Doença , Feminino , Glicosilação , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Oligossacarídeos/metabolismo , Polissacarídeos/metabolismo , Análise de Componente Principal , Neoplasias Retais/patologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Análise de SobrevidaRESUMO
OBJECTIVES: To understand the molecular features distinguishing anti-citrullinated protein antibodies (ACPA) from 'conventional' antibodies in rheumatoid arthritis (RA). METHODS: Serum of ACPA-positive RA patients was fractionated by size exclusion chromatography and analysed for the presence of ACPA-IgG by ELISA. ACPA-IgG and non-citrulline-specific IgG were affinity purified from serum, plasma and/or synovial fluid and analysed by gel electrophoresis. Electrophoresis bands were excised, enzymatically digested and analysed by mass spectrometry. Binding affinity to citrullinated antigens was measured by ELISA and imaging surface plasmon resonance using recombinant monoclonal ACPA with molecular modifications. RESULTS: In all donor samples studied (n=24), ACPA-IgG exhibited a 10-20â kDa higher molecular weight compared with non-autoreactive IgG. This feature also distinguished ACPA-IgG from antibodies against recall antigens or other disease-specific autoantibodies. Structural analysis revealed that a high frequency of N-glycans in the (hyper)variable domains of ACPA is responsible for this observation. In line with their localisation, these N-glycans were found to modulate binding avidity of ACPA to citrullinated antigens. CONCLUSIONS: The vast majority of ACPA-IgG harbour N-glycans in their variable domains. As N-linked glycosylation requires glycosylation consensus sites in the protein sequence and as these are lacking in the 'germline-counterparts' of identified variable domains, our data indicate that the N-glycosylation sites in ACPA variable domains have been introduced by somatic hypermutation. This finding also suggests that ACPA-hyperglycosylation confers a selective advantage to ACPA-producing B cells. This unique and completely novel feature of the citrulline-specific immune response in RA elucidates our understanding of the underlying B cell response.
Assuntos
Artrite Reumatoide/imunologia , Autoanticorpos/metabolismo , Autoantígenos/imunologia , Citrulina/metabolismo , Glicosilação , Imunoglobulina G/imunologia , Polissacarídeos/metabolismo , Adulto , Idoso , Autoanticorpos/química , Autoantígenos/metabolismo , Cromatografia em Gel , Eletroforese , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoglobulina G/metabolismo , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Peso Molecular , Polissacarídeos/química , Ressonância de Plasmônio de Superfície , Líquido Sinovial/imunologiaRESUMO
Immunoglobulin G (IgG) formed during pregnancy against human platelet antigens (HPAs) of the fetus mediates fetal or neonatal alloimmune thrombocytopenia (FNAIT). Because antibody titer or isotype does not strictly correlate with disease severity, we investigated by mass spectrometry variations in the glycosylation at Asn297 in the IgG Fc because the composition of this glycan can be highly variable, affecting binding to phagocyte IgG-Fc receptors (FcγR). We found markedly decreased levels of core fucosylation of anti-HPA-1a-specific IgG1 from FNAIT patients (n = 48), but not in total serum IgG1. Antibodies with a low amount of fucose displayed higher binding affinity to FcγRIIIa and FcγRIIIb, but not to FcγRIIa, compared with antibodies with a high amount of Fc fucose. Consequently, these antibodies with a low amount of Fc fucose showed enhanced phagocytosis of platelets using FcγRIIIb(+) polymorphonuclear cells or FcγRIIIa(+) monocytes as effector cells, but not with FcγRIIIa(-) monocytes. In addition, the degree of anti-HPA-1a fucosylation correlated positively with the neonatal platelet counts in FNAIT, and negatively to the clinical disease severity. In contrast to the FNAIT patients, no changes in core fucosylation were observed for anti-HLA antibodies in refractory thrombocytopenia (post platelet transfusion), indicating that the level of fucosylation may be antigen dependent and/or related to the immune milieu defined by pregnancy.
Assuntos
Plaquetas/imunologia , Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/química , Isoanticorpos/química , Trombocitopenia Neonatal Aloimune/sangue , Trombocitopenia Neonatal Aloimune/imunologia , Anticorpos Monoclonais/química , Asparagina/química , Estudos de Coortes , Feminino , Fucose/química , Glucose/química , Glicosilação , Antígenos HLA/química , Humanos , Fragmentos Fc das Imunoglobulinas/sangue , Imunoglobulina G/sangue , Isoanticorpos/sangue , Espectrometria de Massas , Monócitos/citologia , Contagem de Plaquetas , Período Pós-Parto , Gravidez , Proteínas Recombinantes/química , Ressonância de Plasmônio de SuperfícieRESUMO
RATIONALE: Peptide tandem mass spectra can be analyzed by a number of means. They can be compared against predicted spectra of peptides derived from genome sequences, compared against previously acquired and identified spectra, or - sometimes - sequenced de novo. We recently introduced another method which compares spectra between liquid chromatography/tandem mass spectrometry (LC/MS/MS) datasets to determine the shared spectral content, and demonstrated how this can be applied in a molecular phylogenetic study using sera from human and non-human primates. We will here explore if such a method have other, serendipitous uses. METHODS: We used the existing compareMS2 algorithm without modification on a diverse set of experiments. RESULTS: First we conducted a small phylogenetic study, using (mammalian) bone samples to study old material, and human pathogens aiming to distinguish clinically important strains. Although not as straightforward as primate sera analysis, the method shows significant promise for all these applications. We also used the algorithm to compare 24 different protocols for extraction of proteins from muscle tissue. The results provided useful information in comparing protocols. Finally, we applied compareMS2 aiming for quality control of two traceable protein reference standards (troponin) used in clinical chemistry assays, by analysing the effect of storage conditions. CONCLUSIONS: The results illustrate a broad applicability of the metric based on shared tandem mass spectra between LC/MS/MS datasets for analysing protein digests in different types of experiments. There is no reason to assume that our instance of this method is optimal in any of these situations, as it makes limited or no use of accurate mass and chromatographic retention time. We propose that with further improvement and refinement, this type of analysis can be applied as a simple but informative first step in many pipelines for bottom-up tandem mass spectrometry data analysis in proteomics and other fields, comparing or analysing large numbers of samples or datasets.
Assuntos
Cromatografia Líquida/métodos , Biologia Molecular/métodos , Filogenia , Espectrometria de Massas em Tandem/métodos , Animais , Biomarcadores/análise , HumanosRESUMO
An essential and so far unresolved factor influencing the evolution of cancer and the clinical management of patients is intratumour clonal and phenotypic heterogeneity. However, the de novo identification of tumour subpopulations is so far both a challenging and an unresolved task. Here we present the first systematic approach for the de novo discovery of clinically detrimental molecular tumour subpopulations. In this proof-of-principle study, spatially resolved, tumour-specific mass spectra were acquired, using matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry, from tissues of 63 gastric carcinoma and 32 breast carcinoma patients. The mass spectra, representing the proteomic heterogeneity within tumour areas, were grouped by a corroborated statistical clustering algorithm in order to obtain segmentation maps of molecularly distinct regions. These regions were presumed to represent different phenotypic tumour subpopulations. This was confirmed by linking the presence of these tumour subpopulations to the patients' clinical data. This revealed several of the detected tumour subpopulations to be associated with a different overall survival of the gastric cancer patients (p = 0.025) and the presence of locoregional metastases in patients with breast cancer (p = 0.036). The procedure presented is generic and opens novel options in cancer research, as it reveals microscopically indistinct tumour subpopulations that have an adverse impact on clinical outcome. This enables their further molecular characterization for deeper insights into the biological processes of cancer, which may finally lead to new targeted therapies.
Assuntos
Neoplasias da Mama/patologia , Tumores do Estroma Gastrointestinal/patologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Algoritmos , Neoplasias da Mama/mortalidade , Análise por Conglomerados , Feminino , Tumores do Estroma Gastrointestinal/mortalidade , Humanos , Masculino , Fenótipo , Proteômica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
A fully automated workflow was developed and validated for simultaneous quantification of the cardiovascular disease risk markers apolipoproteins A-I (apoA-I) and B-100 (apoB-100) in clinical sera. By coupling of stable-isotope standards and capture by anti-peptide antibodies (SISCAPA) for enrichment of proteotypic peptides from serum digests to matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS detection, the standardized platform enabled rapid, liquid chromatography-free quantification at a relatively high throughput of 96 samples in 12h. The average imprecision in normo- and triglyceridemic serum pools was 3.8% for apoA-I and 4.2% for apoB-100 (4 replicates over 5 days). If stored properly, the MALDI target containing enriched apoA-1 and apoB-100 peptides could be re-analyzed without any effect on bias or imprecision for at least 7 days after initial analysis. Validation of the workflow revealed excellent linearity for daily calibration with external, serum-based calibrators (R(2) of 0.984 for apoA-I and 0.976 for apoB-100 as average over five days), and absence of matrix effects or interference from triglycerides, protein content, hemolysates, or bilirubins. Quantification of apoA-I in 93 normo- and hypertriglyceridemic clinical sera showed good agreement with immunoturbidimetric analysis (slope = 1.01, R(2) = 0.95, mean bias = 4.0%). Measurement of apoB-100 in the same clinical sera using both methods, however, revealed several outliers in SISCAPA-MALDI-TOF-MS measurements, possibly as a result of the lower MALDI-TOF-MS signal intensity (slope = 1.09, R(2) = 0.91, mean bias = 2.0%). The combination of analytical performance, rapid cycle time and automation potential validate the SISCAPA-MALDI-TOF-MS platform as a valuable approach for standardized and high-throughput quantification of apoA-I and apoB-100 in large sample cohorts.
Assuntos
Apolipoproteína A-I/sangue , Apolipoproteína B-100/sangue , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Anticorpos Monoclonais , Apolipoproteína A-I/imunologia , Apolipoproteína B-100/imunologia , Biomarcadores/sangue , Calibragem , Humanos , Fluxo de TrabalhoRESUMO
Bacterial secreted proteins constitute a biologically important subset of proteins involved in key processes related to infection such as adhesion, colonization, and dissemination. Bacterial extracellular proteases, in particular, have attracted considerable attention, as they have been shown to be indispensable for bacterial virulence. Here, we analyzed the extracellular subproteome of Clostridium difficile and identified a hypothetical protein, CD2830, as a novel secreted metalloprotease. Following the identification of a CD2830 cleavage site in human HSP90ß, a series of synthetic peptide substrates was used to identify the favorable CD2830 cleavage motif. This motif was characterized by a high prevalence of proline residues. Intriguingly, CD2830 has a preference for cleaving Pro-Pro bonds, unique among all hitherto described proteases. Strikingly, within the C. difficile proteome two putative adhesion molecules, CD2831 and CD3246, were identified that contain multiple CD2830 cleavage sites (13 in total). We subsequently found that CD2830 efficiently cleaves CD2831 between two prolines at all predicted cleavage sites. Moreover, native CD2830, secreted by live cells, cleaves endogenous CD2831 and CD3246. These findings highlight CD2830 as a highly specific endoproteinase with a preference for proline residues surrounding the scissile bond. Moreover, the efficient cleavage of two putative surface adhesion proteins points to a possible role of CD2830 in the regulation of C. difficile adhesion.
Assuntos
Proteínas de Bactérias/metabolismo , Clostridioides difficile/enzimologia , Proteínas de Membrana/genética , Metaloproteases/metabolismo , Prolina/metabolismo , Sinais Direcionadores de Proteínas , Proteínas de Bactérias/genética , Domínio Catalítico , Infecções por Clostridium/parasitologia , Evolução Molecular , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Metaloproteases/química , Metaloproteases/genética , Modelos Moleculares , Filogenia , Proteoma/análiseRESUMO
The biological and clinical relevance of glycosylation is becoming increasingly recognized, leading to a growing interest in large-scale clinical and population-based studies. In the past few years, several methods for high-throughput analysis of glycans have been developed, but thorough validation and standardization of these methods is required before significant resources are invested in large-scale studies. In this study, we compared liquid chromatography, capillary gel electrophoresis, and two MS methods for quantitative profiling of N-glycosylation of IgG in the same data set of 1201 individuals. To evaluate the accuracy of the four methods we then performed analysis of association with genetic polymorphisms and age. Chromatographic methods with either fluorescent or MS-detection yielded slightly stronger associations than MS-only and multiplexed capillary gel electrophoresis, but at the expense of lower levels of throughput. Advantages and disadvantages of each method were identified, which should inform the selection of the most appropriate method in future studies.
Assuntos
Ensaios de Triagem em Larga Escala/métodos , Imunoglobulina G/genética , Espectrometria de Massas/métodos , Polissacarídeos/genética , Adulto , Cromatografia Líquida , Eletroforese Capilar , Glicosilação , Humanos , Interações Hidrofóbicas e Hidrofílicas , Polimorfismo Genético , Polissacarídeos/isolamento & purificaçãoRESUMO
Genetic variants influencing the transcriptome have been extensively studied. However, the impact of the genetic factors on the human proteome is largely unexplored, mainly due to lack of suitable high-throughput methods. Here we present unique and comprehensive identification of genetic variants affecting the human plasma protein profile by combining high-throughput and high-resolution mass spectrometry (MS) with genome-wide SNP data. We identified and quantified the abundance of 1,056 tryptic-digested peptides, representing 163 proteins in the plasma of 1,060 individuals from two population-based cohorts. The abundance level of almost one-fifth (19%) of the peptides was found to be heritable, with heritability ranging from 0.08 to 0.43. The levels of 60 peptides from 25 proteins, 15% of the proteins studied, were influenced by cis-acting SNPs. We identified and replicated individual cis-acting SNPs (combined P value ranging from 3.1 × 10(-52) to 2.9 × 10(-12)) influencing 11 peptides from 5 individual proteins. These SNPs represent both regulatory SNPs and nonsynonymous changes defining well-studied disease alleles such as the ε4 allele of apolipoprotein E (APOE), which has been shown to increase risk of Alzheimer's disease. Our results show that high-throughput mass spectrometry represents a promising method for large-scale characterization of the human proteome, allowing for both quantification and sequencing of individual proteins. Abundance and peptide composition of a protein plays an important role in the etiology, diagnosis, and treatment of a number of diseases. A better understanding of the genetic impact on the plasma proteome is therefore important for evaluating potential biomarkers and therapeutic agents for common diseases.
Assuntos
Alelos , Proteínas Sanguíneas , Polimorfismo de Nucleotídeo Único , Proteoma , Adolescente , Adulto , Biomarcadores/sangue , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Estudos de Coortes , Feminino , Marcadores Genéticos , Estudo de Associação Genômica Ampla , Humanos , Masculino , Pessoa de Meia-Idade , Proteoma/genética , Proteoma/metabolismoRESUMO
Glycosylation of immunoglobulin G (IgG) influences IgG effector function by modulating binding to Fc receptors. To identify genetic loci associated with IgG glycosylation, we quantitated N-linked IgG glycans using two approaches. After isolating IgG from human plasma, we performed 77 quantitative measurements of N-glycosylation using ultra-performance liquid chromatography (UPLC) in 2,247 individuals from four European discovery populations. In parallel, we measured IgG N-glycans using MALDI-TOF mass spectrometry (MS) in a replication cohort of 1,848 Europeans. Meta-analysis of genome-wide association study (GWAS) results identified 9 genome-wide significant loci (P<2.27 × 10(-9)) in the discovery analysis and two of the same loci (B4GALT1 and MGAT3) in the replication cohort. Four loci contained genes encoding glycosyltransferases (ST6GAL1, B4GALT1, FUT8, and MGAT3), while the remaining 5 contained genes that have not been previously implicated in protein glycosylation (IKZF1, IL6ST-ANKRD55, ABCF2-SMARCD3, SUV420H1, and SMARCB1-DERL3). However, most of them have been strongly associated with autoimmune and inflammatory conditions (e.g., systemic lupus erythematosus, rheumatoid arthritis, ulcerative colitis, Crohn's disease, diabetes type 1, multiple sclerosis, Graves' disease, celiac disease, nodular sclerosis) and/or haematological cancers (acute lymphoblastic leukaemia, Hodgkin lymphoma, and multiple myeloma). Follow-up functional experiments in haplodeficient Ikzf1 knock-out mice showed the same general pattern of changes in IgG glycosylation as identified in the meta-analysis. As IKZF1 was associated with multiple IgG N-glycan traits, we explored biomarker potential of affected N-glycans in 101 cases with SLE and 183 matched controls and demonstrated substantial discriminative power in a ROC-curve analysis (area under the curve = 0.842). Our study shows that it is possible to identify new loci that control glycosylation of a single plasma protein using GWAS. The results may also provide an explanation for the reported pleiotropy and antagonistic effects of loci involved in autoimmune diseases and haematological cancer.
Assuntos
Doenças Autoimunes , Pleiotropia Genética , Glicosiltransferases/genética , Neoplasias Hematológicas , Imunoglobulina G , Animais , Doenças Autoimunes/genética , Doenças Autoimunes/metabolismo , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Glicosilação , Glicosiltransferases/sangue , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/metabolismo , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/genética , Camundongos , Camundongos Knockout , Esclerose Múltipla/genéticaRESUMO
Granulomatosis with polyangiitis (GPA) is associated with circulating immunoglobulin (Ig) G anti-proteinase 3 specific (anti-PR3) anti-neutrophil cytoplasm antibodies (ANCA), which activate cytokine primed neutrophils via Fcgamma receptors. ANCA are class switched IgG antibodies implying T cell help in their production. Glycosylation of IgG Fc, under the control of T cell cytokines, determines the interaction between IgG and its receptors. Previous studies have reported aberrant glycosylation of Ig Fc in GPA patients. We investigated whether aberrant Fc glycosylation was present on anti-PR3 ANCA as well as whole IgG subclass preparations compared to healthy controls and whether this correlated with Birmingham vasculitis activity scores (BVAS), serum cytokines, and time to remission. Here, IgG Fc glycosylation of GPA patients and controls and anti-PR3 ANCA Fc glycosylation were determined by mass spectrometry of glycopeptides. IgG1 and IgG2 subclasses from GPA patients showed reduced galactosylation, sialylation, and bisection compared to healthy controls. Anti-PR3 IgG1 ANCA Fc galactosylation, sialylation, and bisection were reduced compared to total IgG1 in GPA. Galactosylation of anti-PR3 ANCA Fc correlated with inflammatory cytokines and time to remission but not BVAS. Bisection of anti-PR3 ANCA Fc correlated with BVAS. Total IgG1 and anti-PR3 IgG1 Fc galactosylation were weakly correlated, while bisection of IgG1 and anti-PR3 showed no correlation. Our data indicate that aberrant ANCA galactosylation may be driven in an antigen-specific manner.
Assuntos
Autoanticorpos/metabolismo , Granulomatose com Poliangiite/metabolismo , Imunoglobulina G/metabolismo , Mieloblastina/imunologia , Adulto , Autoanticorpos/imunologia , Citocinas/sangue , Glicosilação , Granulomatose com Poliangiite/imunologia , Humanos , Imunoglobulina G/imunologia , Espectrometria de Massas , Pessoa de Meia-Idade , Vasculite/patologiaRESUMO
An accurate mass measurement of a known protein provides information on potential amino acid deletions and post-translational modifications. Although this field is dominated by strategies based on electrospray ionization, mass spectrometry (MS) methods using matrix-assisted laser desorption/ionization (MALDI) have the advantage of yielding predominantly singly charged precursor ions, thus avoiding peak overlap from different charge states of multiple species. Such MALDI-MS methods require mass measurement at ultrahigh resolution, which is provided by Fourier transform ion cyclotron resonance (FTICR) mass analyzers. Recently, using a MALDI-FTICR-MS platform equipped with a 15 T magnet, we reported on the mass analysis of intact human serum peptides and small proteins with isotopic resolution up to â¼15 kDa and identified new proteoforms from an accurate measurement of mass distances. In the current study, we have used this FTICR system after an upgrade with a novel dynamically harmonized ICR cell, i.e., ParaCell, for mapping isotopically resolved intact proteins up to about 17 kDa and performed top-down MALDI in-source decay (ISD) analysis. Standard proteins myoglobin (m/z-value 16,950) and ribonuclease B (m/z-value 14,900) were measured with resolving powers of 62,000 and 61,000, respectively. Furthermore, it will be shown that (singly charged) MALDI-ISD fragment ions can be measured at isotopic resolution up to m/z-value 12,000 (e.g., resolving power 39,000 at m/z-value 12,000) providing more reliable identifications. Moreover, examples are presented of pseudo-MS(3) experiments on ISD fragment ions from RNase B by collisional-induced dissociation (CID).
Assuntos
Ciclotrons , Análise de Fourier , Mioglobina/química , Ribonucleases/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Sequência de Aminoácidos , Animais , Bovinos , Isótopos , Dados de Sequência Molecular , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/instrumentaçãoRESUMO
OBJECTIVE: To determine whether anticitrullinated protein antibodies (ACPA) exhibit specific changes in Fc glycosylation prior to the onset of arthritis. METHODS: Serum samples of patients with ACPA-positive arthralgia (n=183) were collected at baseline and at various time points of follow-up. 105 patients developed arthritis after a median of 12â months (IQR 6-24) and were classified as having either rheumatoid arthritis (RA, n=48) or undifferentiated arthritis (UA, n=57) based on the 1987 American College of Rheumatology (ACR) criteria. ACPA and total serum IgG were isolated by affinity purification and cleaved by trypsin. ACPA-IgG1 Fc-glycopeptides were subsequently analysed by nano-liquid chromatography mass spectrometry and compared to those of total IgG1. RESULTS: At baseline, ACPA-IgG1 and total IgG1 from arthralgia patients displayed similar Fc glycosylation patterns. By contrast, at the onset of arthritis, ACPA exhibited a decrease in galactose residues in RA patients, but not in UA patients. This decrease occurred around 3â months prior to diagnosis and was paralleled by an increase in systemic inflammation (erythrocyte sedimentation rate). Galactosylation of total IgG1 was also decreased in RA, but this did not precede the onset of arthritis. Interestingly, we additionally noted a higher degree of ACPA-IgG1 Fc core fucosylation at baseline as compared with total IgG1, which further increased prior to diagnosis. CONCLUSIONS: ACPA display significant changes in Fc galactosylation and fucosylation prior to the onset of RA. These changes towards a more pro-inflammatory phenotype could be involved in driving the disease process.
Assuntos
Artrite Reumatoide/metabolismo , Autoanticorpos/metabolismo , Fragmentos Fc das Imunoglobulinas/metabolismo , Peptídeos Cíclicos/imunologia , Polissacarídeos/metabolismo , Sintomas Prodrômicos , Adulto , Artrite/imunologia , Artrite/metabolismo , Artrite Reumatoide/imunologia , Autoanticorpos/imunologia , Estudos de Casos e Controles , Feminino , Fucose/metabolismo , Galactose/metabolismo , Glicosilação , Humanos , Inflamação , Masculino , Pessoa de Meia-Idade , FenótipoRESUMO
Standardization of body fluid sampling, processing and storage procedures is pivotal to ensure data quality in metabolomics studies. Yet, despite strict adherence to standard sampling guidelines, we detected variable levels of ethanol in the (1)H-NMR spectra of human cerebrospinal fluid (CSF) samples (range 9.2 × 10(-3)-10.0 mM). The presence of ethanol in all samples and the wide range of concentrations clearly indicated contamination of the samples of some sort, which affected the (1)H-NMR spectra quality and the interpretation. To determine where in the sampling protocol the ethanol contamination occurs, we performed a CSF sampling protocol simulation with 0.9 % NaCl (saline) instead of CSF and detected ethanol in all simulation samples. Ethanol diffusion through air during sampling and preparation stages appeared the only logical explanation. With a bench study, we showed that ethanol easily diffuses into ex vivo CSF samples via air transmission. Ethanol originated from routinely used skin disinfectants containing ethanol and from laboratory procedures. Ethanol affected the CSF sample matrix at concentrations above ~9.4 mM and obscured a significant part of the (1)H-NMR spectrum. CSF sample preparation for (1)H-NMR-based metabolomics analyses should therefore be carried out in a well-ventilated atmosphere with laminar flow, and use of ethanol should be avoided.
Assuntos
Líquido Cefalorraquidiano/química , Etanol/análise , Metabolômica , Espectroscopia de Prótons por Ressonância Magnética/métodos , Estudos de Coortes , HumanosRESUMO
Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging using 9-aminoacridine as the matrix leads to the detection of low mass metabolites and lipids directly from cancer tissues. These included lactate and pyruvate for studying the Warburg effect, as well as succinate and fumarate, metabolites whose accumulation is associated with specific syndromes. By using the pathway information present in the human metabolome database, it was possible to identify regions within tumor tissue samples with distinct metabolic signatures that were consistent with known tumor biology. We present a data analysis workflow for assessing metabolic pathways in their histopathological context.