RESUMO
We have recently introduced multiple reaction monitoring (MRM) mass spectrometry as a novel tool for glycan biomarker research and discovery. Herein, we employ this technique to characterize the site-specific glycan alterations associated with primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC). Glycopeptides associated with disease severity were also identified. Multinomial regression modelling was employed to construct and validate multi-analyte diagnostic models capable of accurately distinguishing PBC, PSC, and healthy controls from one another (AUC = 0.93 ± 0.03). Finally, to investigate how disease-relevant environmental factors can influence glycosylation, we characterized the ability of bile acids known to be differentially expressed in PBC to alter glycosylation. We hypothesize that this could be a mechanism by which altered self-antigens are generated and become targets for immune attack. This work demonstrates the utility of the MRM method to identify diagnostic site-specific glycan classifiers capable of distinguishing even related autoimmune diseases from one another.
Assuntos
Autoimunidade , Colangite Esclerosante/imunologia , Cirrose Hepática Biliar/imunologia , Polissacarídeos/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Ácidos e Sais Biliares/sangue , Ácidos e Sais Biliares/imunologia , Biomarcadores/sangue , Estudos de Casos e Controles , Colangite Esclerosante/sangue , Colangite Esclerosante/diagnóstico , Diagnóstico Diferencial , Glicômica/métodos , Glicopeptídeos/sangue , Glicopeptídeos/imunologia , Glicosilação , Humanos , Cirrose Hepática Biliar/sangue , Cirrose Hepática Biliar/diagnóstico , Polissacarídeos/sangue , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
Alteration of immunoglobulin glycosylation correlates with inflammatory diseases and infectious diseases including parasitic infections. Immunoglobulin glycosylation patterns may be implicated in disease development and have also been proposed as diagnostic tools for several diseases. Previous studies have reported the immunoglobulin profiles in experimental animals and in patients infected with the carcinogenic human liver fluke, Opisthorchis viverrini. However, the N-glycosylation profiles of immunoglobulins and their subclass-specific glycoforms in opisthorchiasis patients have never been elucidated. Here, N-glycosylation patterns of immunoglobulins and their subclass-specific glycoforms in sera of O. viverrini-infected patients were investigated using triple quadrupole mass spectrometry coupled with multiple reaction monitoring. Peptide fragmentation was utilized to quantify the immunoglobulin glycoforms normalized to the unique peptide of each subclass. Overall, serum levels of IgG and IgA in O. viverrini patients were significantly increased compared to uninfected controls. Twenty-seven glycoforms were detected based on analysis of detached glycans in all immunoglobulin subclasses. The abundance of immunoglobulin glycopeptides in serum of opisthorchiasis patients deviated significantly from controls. Immunoglobulin glycosylation patterns were associated with both pro- and anti-inflammatory properties. In conclusion, O. viverrini infection alters the serum immunoglobulin glycosylation profile and these changes could distinguish between O. viverrini-infected individuals and healthy controls. SIGNIFICANCE: We demonstrated that both quantities and glycoforms of serum immunoglobulin subclasses were altered in Opisthorchis viverrini-infected individuals as investigated by the QqQ-MS-MRM method. Patterns of immunoglobulin with a specific glycoform might contribute to immune responses to O. viverrini infection.
Assuntos
Opistorquíase , Opisthorchis , Animais , Glicosilação , Humanos , Imunoglobulinas , Espectrometria de MassasRESUMO
Alterations in the human glycome have been associated with cancer and autoimmunity. Thus, constructing a site-specific map of the human glycome for biomarker research and discovery has been a highly sought-after objective. However, due to analytical barriers, comprehensive site-specific glycoprofiling is difficult to perform. To develop a platform to detect easily quantifiable, site-specific, disease-associated glycan alterations for clinical applications, we have adapted the multiple reaction monitoring mass spectrometry method for use in glycan biomarker research. The adaptations allow for highly precise site-specific glycan monitoring with minimum sample prep. Using this technique, we successfully mapped out the relative abundances of the most common 159 glycopeptides in the plasma of 97 healthy volunteers. This plasma glycome map revealed 796 significant (FDR < 0.05) site-specific inter-protein and intra-protein glycan associations, of which the vast majority were previously unknown. Since age and gender are relevant covariants in biomarker research, these variables were also characterized. 13 glycopeptides were found to be associated with gender and 41 to be associated with age. Using just five age-associated glycopeptides, a highly accurate age prediction model was constructed and validated (r2 = 0.62 ± 0.12). The human plasma site-specific glycan map described herein has utility in applications ranging from glycan biomarker research and discovery to the development of novel glycan-altering interventions.
Assuntos
Fatores Etários , Biomarcadores/sangue , Polissacarídeos/sangue , Fatores Sexuais , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas Sanguíneas , Feminino , Glicômica , Glicopeptídeos/sangue , Glicosilação , Voluntários Saudáveis , Humanos , Imunoglobulina G/sangue , Funções Verossimilhança , Masculino , Pessoa de Meia-Idade , Espectrometria de Massas por Ionização por Electrospray , Adulto JovemRESUMO
Glycosylation of antibodies, particularly in the Fc domain, critically modulate the ability of antibodies to bind to FcRs, maintaining immune quiescence to achieve a finely orchestrated immune response. The removal of sialic acid and galactose residues dramatically alters the physiological function of IgGs, and alterations of Ig glycosylation have been associated with several autoimmune disorders. However, Ig glycosylation has not been extensively studied in autoimmune cholangitis. We applied triple quadruple mass spectroscopy with subsequent multiple reaction monitoring to elucidate the profile, composition and linkage of sugar residues of antibody glycans in patients with primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC) and healthy controls (HC). Agalactosylated, HexNAc terminated IgG1 glycoforms were enriched in both PBC and PSC. Levels of IgM glycans at site N439 and fucosylated glycans in J chain, were significantly decreased in PBC compared to PSC and HC. PSC patients had decreased bisecting glycoforms and increased biantennary glycoforms on IgA compared to PBC. Importantly, our data demonstrate the association of distinct branching and composition patterns of Ig glycoforms with disease severity and liver cirrhosis, which highlight the importance of glycan biology as a potential mechanism and/or a disease specific signal of inflammation.
Assuntos
Autoanticorpos/metabolismo , Doenças Autoimunes/diagnóstico , Colangite Esclerosante/diagnóstico , Imunoglobulina G/metabolismo , Cirrose Hepática Biliar/diagnóstico , Adulto , Idoso , Autoanticorpos/sangue , Autoanticorpos/imunologia , Doenças Autoimunes/sangue , Doenças Autoimunes/imunologia , Biomarcadores/sangue , Biomarcadores/metabolismo , Estudos de Casos e Controles , Colangite Esclerosante/sangue , Colangite Esclerosante/imunologia , Feminino , Glicômica/métodos , Glicosilação , Voluntários Saudáveis , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Cirrose Hepática Biliar/sangue , Cirrose Hepática Biliar/imunologia , Masculino , Pessoa de Meia-Idade , Polissacarídeos/imunologia , Polissacarídeos/metabolismo , Índice de Gravidade de DoençaRESUMO
The recruitment of a bacterial consortium by the host is a strategy not limited to animals but is also used in plants. A maize aerial root mucilage has been found that harbors nitrogen fixing bacteria that are attracted to the carbohydrate rich environment. This synbiotic relationship is facilitated by a polysaccharide, whose complicated structure has been previously unknown. In this report, we present the characterization of the maize polysaccharide by employing new analytical strategies combining chemical depolymerization, oligosaccharide sequencing, and monosaccharide and glycosidic linkage quantitation. The mucilage contains a single heterogeneous polysaccharide composed of a highly fucosylated and xylosylated galactose backbone with arabinan and mannoglucuronan branches. This unique polysaccharide structure may select for the diazotrophic community by containing monosaccharides and linkages that correspond to the glycosyl hydrolases associated with the microbial community. The elucidation of this complicated structure illustrates the power of the analytical methods, which may serve as a general platform for polysaccharide analysis in the future.
Assuntos
Bactérias Fixadoras de Nitrogênio/química , Polissacarídeos/análise , Zea mays/química , Configuração de Carboidratos , Cromatografia Líquida de Alta Pressão , Espectrometria de MassasRESUMO
Analysis of serum protein glycovariants has the potential to identify new biomarkers of human disease. However, the inability to rapidly quantify glycans in a site-specific fashion remains the major barrier to applying such biomarkers clinically. Advancements in sample preparation and glycopeptide quantification are thus needed to better bridge glycoscience with biomarker discovery research. We present here the successful utilization of several sample preparation techniques, including multienzyme digestion and glycopeptide enrichment, to increase the repertoire of glycopeptides that can be generated from serum glycoproteins. These techniques combined with glycopeptide retention time prediction and UHPLC-QqQ conditions optimization were then used to develop a dynamic multiple-reaction monitoring (dMRM)-based strategy to simultaneously monitor over 100 glycosylation sites across 50 serum glycoproteins. In total, the abundances of over 600 glycopeptides were simultaneously monitored, some of which were identified by utilizing theoretically predicted ion products and presumed m/ z values. The dMRM method was found to have good sensitivity. In the targeted dMRM mode, the limit of quantitation (LOQ) of nine standard glycoproteins reached femtomole levels with dynamic ranges spanning 3-4 orders of magnitude. The dMRM-based strategy also showed high reproducibility with regards to both instrument and sample preparation performance. The high coverage of the serum glycoproteins that can be quantitated to the glycopeptide level makes this method especially suitable for the biomarker discovery from large sample sets. We predict that, in the near future, biomarkers, such as these, will be deployed clinically, especially in the fields of cancer and autoimmunity.
Assuntos
Doenças Autoimunes/sangue , Glicoproteínas/sangue , Neoplasias/sangue , Proteômica , Doenças Autoimunes/diagnóstico , Biomarcadores/sangue , Glicosilação , Humanos , Neoplasias/diagnósticoRESUMO
Kifunensine, a potent and selective inhibitor of class I α-mannosidases, prevents α-mannosidases I from trimming mannose residues on glycoproteins, thus resulting in oligomannose-type glycans. We report for the first time that through one-time vacuum infiltration of kifunensine in plant tissue, N-linked glycosylation of a recombinant protein transiently produced in whole-plants shifted completely from complex-type to oligomannose-type. Fc-fused capillary morphogenesis protein 2 (CMG2-Fc) containing one N-glycosylation site on the Fc domain, produced in Nicotiana benthamiana whole plants, served as a model protein. The CMG2-Fc fusion protein was produced transiently through vacuum agroinfiltration, with and without kifunensine at a concentration of 5.4 µM in the agroinfiltration suspension. The CMG2-Fc N-glycan profile was determined using LC-MS/MS with a targeted dynamic multiple reaction monitoring (MRM) method. The CMG2-Fc expression level in the infiltrated plant tissue and the percentage of oligomannose-type N-glycans for kifunensine treated plants was 874 mg/kg leaf fresh weight (FW) and 98.2%, respectively, compared to 717 mg/kg leaf FW and 2.3% for untreated plants. Oligomannose glycans are amenable to in vitro enzymatic modification to produce more human-like N-glycan structures that are preferred for the production of HIV-1 viral vaccine and certain monoclonal antibodies. This method allows glycan modifications using a bioprocessing approach without compromising protein yield or modification of the primary sequence, and could be expanded to other small molecule inhibitors of glycan-processing enzymes. For recombinant protein targeted for secretion, kifunensine treatment allows collection of glycoform-modified target protein from apoplast wash fluid (AWF) with minimal plant-specific complex N-glycan at higher starting purity and concentration than in whole-leaf extract, thus simplifying the downstream processing.
Assuntos
Alcaloides/farmacologia , Inibidores Enzimáticos/farmacologia , Glicoproteínas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Vacinas contra a AIDS/biossíntese , Agrobacterium/genética , Agrobacterium/metabolismo , Anticorpos Monoclonais/biossíntese , Glicosilação/efeitos dos fármacos , HIV-1/imunologia , Humanos , Manose/metabolismo , Receptores de Peptídeos/metabolismo , Nicotiana/genética , Nicotiana/metabolismo , Nicotiana/microbiologia , Vácuo , alfa-Manosidase/antagonistas & inibidoresRESUMO
Recombinant butyrylcholinesterase produced in a metabolically regulated transgenic rice cell culture (rrBChE) was purified to produce a highly pure (95%), active form of enzyme. The developed downstream process uses common manufacturing friendly operations including tangential flow filtration, anion-exchange chromatography, and affinity chromatography to obtain a process recovery of 42% active rrBChE. The purified rrBChE was then characterized to confirm its comparability to the native human form of the molecule (hBChE). The recombinant and native enzyme demonstrated comparable enzymatic behavior and had an identical amino acid sequence. However, rrBChE differs in that it contains plant-type complex N-glycans, including an α-1,3 linked core fucose, and a ß-1,2 xylose, and lacking a terminal sialic acid. Despite this difference, rrBChE is demonstrated to be an effective stoichiometric bioscavenger for five different organophosphorous nerve agents in vitro. Together, the efficient downstream processing scheme and functionality of rrBChE confirm its promise as a cost-effective alternative to hBChE for prophylactic and therapeutic use.
Assuntos
Butirilcolinesterase/isolamento & purificação , Butirilcolinesterase/metabolismo , Oryza/enzimologia , Plantas Geneticamente Modificadas/enzimologia , Butirilcolinesterase/química , Cromatografia Líquida , Filtração , Glicosilação , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
High density lipoprotein (HDL) particles are believed to be protective due to their inverse correlation with the prevalence of cardiovascular diseases. However, recent studies show that in some conditions such as heart disease and diabetes, HDL particles can become dysfunctional. Great attention has been directed toward HDL particle composition because the relative abundances of HDL constituents determine HDL's functional properties. A key factor to consider when studying the structure and composition of plasma particles is the protein glycosylation. Here, we profile the O- and N-linked glycosylation of HDL associated-proteins including the truncated form of Apo CIII and their glycan heterogeneity in a site-specific manner. Apolipoprotein CIII, fetuin A, and alpha 1 antitrypsin are glycoproteins associated with lipoproteins and are implicated in many cardiovascular and other disease conditions. A targeted method (UHPLC-QQQ) was used to measure the glycoprotein concentrations and site-specific glycovariations of the proteins in human plasma and compared with HDL particles isolated from the same plasma samples. The proteins found in the plasma are differentially glycosylated compared to those isolated in HDL. The results of this study suggest that glycosylation may play a role in protein partitioning in the blood, with possible functional implications.
Assuntos
Apolipoproteína C-III/isolamento & purificação , Glicopeptídeos/isolamento & purificação , Lipoproteínas HDL/isolamento & purificação , Processamento de Proteína Pós-Traducional , alfa 1-Antitripsina/isolamento & purificação , alfa-2-Glicoproteína-HS/isolamento & purificação , Sequência de Aminoácidos , Apolipoproteína C-III/química , Apolipoproteína C-III/metabolismo , Sequência de Carboidratos , Cromatografia Líquida de Alta Pressão/métodos , Análise por Conglomerados , Glicopeptídeos/química , Glicopeptídeos/metabolismo , Glicosilação , Humanos , Lipoproteínas HDL/química , Lipoproteínas HDL/metabolismo , Espectrometria de Massas em Tandem , alfa 1-Antitripsina/química , alfa 1-Antitripsina/metabolismo , alfa-2-Glicoproteína-HS/química , alfa-2-Glicoproteína-HS/metabolismoRESUMO
The goal of this pilot study was to determine whether HDL glycoprotein composition affects HDL's immunomodulatory function. HDL were purified from healthy controls (n = 13), subjects with metabolic syndrome (MetS) (n = 13), and diabetic hemodialysis (HD) patients (n = 24). Concentrations of HDL-bound serum amyloid A (SAA), lipopolysaccharide binding protein (LBP), apolipoprotein A-I (ApoA-I), apolipoprotein C-III (ApoC-III), α-1-antitrypsin (A1AT), and α-2-HS-glycoprotein (A2HSG); and the site-specific glycovariations of ApoC-III, A1AT, and A2HSG were measured. Secretion of interleukin 6 (IL-6) in lipopolysaccharide-stimulated monocytes was used as a prototypical assay of HDL's immunomodulatory capacity. HDL from HD patients were enriched in SAA, LBP, ApoC-III, di-sialylated ApoC-III (ApoC-III2) and desialylated A2HSG. HDL that increased IL-6 secretion were enriched in ApoC-III, di-sialylated glycans at multiple A1AT glycosylation sites and desialylated A2HSG, and depleted in mono-sialylated ApoC-III (ApoC-III1). Subgroup analysis on HD patients who experienced an infectious hospitalization event within 60 days (HD+) (n = 12), vs. those with no event (HD-) (n = 12) showed that HDL from HD+ patients were enriched in SAA but had lower levels of sialylation across glycoproteins. Our results demonstrate that HDL glycoprotein composition, including the site-specific glycosylation, differentiate between clinical groups, correlate with HDL's immunomodulatory capacity, and may be predictive of HDL's ability to protect from infection.
Assuntos
Interleucina-6/metabolismo , Lipopolissacarídeos/imunologia , Lipoproteínas HDL/metabolismo , Monócitos/imunologia , Monócitos/metabolismo , Biomarcadores , Citocinas/metabolismo , Diabetes Mellitus/imunologia , Diabetes Mellitus/metabolismo , Glicopeptídeos/metabolismo , Glicoproteínas/metabolismo , Glicosilação , Humanos , Infecções/imunologia , Infecções/metabolismo , Lipoproteínas HDL/isolamento & purificação , Diálise RenalRESUMO
Anthrax toxin receptor-mediated drug development for blocking anthrax toxin action has received much attention in recent decades. In this study, we produced a secreted anthrax decoy fusion protein comprised of a portion of the human capillary morphogenesis gene-2 (CMG2) protein fused via a linker to the fragment crystallizable (Fc) domain of human immunoglobulin G1 in Nicotiana benthamiana plants using a transient expression system. Using the Cauliflower Mosaic Virus (CaMV) 35S promoter and co-expression with the p19 gene silencing suppressor, we were able to achieve a high level of recombinant CMG2-Fc-Apo (rCMG2-Fc-Apo) protein accumulation. Production kinetics were observed up to eight days post-infiltration, and maximum production of 826 mg/kg fresh leaf weight was observed on day six. Protein A affinity chromatography purification of the rCMG2-Fc-Apo protein from whole leaf extract and apoplast wash fluid showed the homodimeric form under non-reducing gel electrophoresis and mass spectrometry analysis confirmed the molecular integrity of the secreted protein. The N-glycosylation pattern of purified rCMG2-Fc-Apo protein was analysed; the major portion of N-glycans consists of complex type structures in both protein samples. The most abundant (>50%) N-glycan structure was GlcNAc2(Xyl)Man3(Fuc)GlcNAc2 in rCMG2-Fc-Apo recovered from whole leaf extract and apoplast wash fluid. High mannose N-glycan structures were not detected in the apoplast wash fluid preparation, which confirmed the protein secretion. Altogether, these findings demonstrate that high-level production of rCMG2-Fc-Apo can be achieved by transient production in Nicotiana benthamiana plants with apoplast targeting.
Assuntos
Imunoglobulina G/genética , Nicotiana/genética , Plantas Geneticamente Modificadas/genética , Receptores de Peptídeos/genética , Sequência de Aminoácidos , Antraz/metabolismo , Antraz/microbiologia , Bacillus anthracis/metabolismo , Biotecnologia , Caulimovirus/genética , Clonagem Molecular , Descoberta de Drogas , Glicosilação , Humanos , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Regiões Promotoras Genéticas , Receptores de Peptídeos/química , Receptores de Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Human milk plays a substantial role in the child growth, development and determines their nutritional and health status. Despite the importance of the proteins and glycoproteins in human milk, very little quantitative information especially on their site-specific glycosylation is known. As more functions of milk proteins and other components continue to emerge, their fine-detailed quantitative information is becoming a key factor in milk research efforts. The present work utilizes a sensitive label-free MRM method to quantify seven milk proteins (α-lactalbumin, lactoferrin, secretory immunoglobulin A, immunoglobulin G, immunoglobulin M, α1-antitrypsin, and lysozyme) using their unique peptides while at the same time, quantifying their site-specific N-glycosylation relative to the protein abundance. The method is highly reproducible, has low limit of quantitation, and accounts for differences in glycosylation due to variations in protein amounts. The method described here expands our knowledge about human milk proteins and provides vital details that could be used in monitoring the health of the infant and even the mother. Graphical Abstract The glycopeptides EICs generated from QQQ.
Assuntos
Análise de Alimentos/métodos , Proteínas do Leite/análise , Proteínas do Leite/química , Leite Humano/química , Glicosilação , Humanos , Espectrometria de MassasRESUMO
Protein glycosylation and other post-translational modifications are involved in potentially all aspects of human growth and development. Defective glycosylation has adverse effects on human physiological conditions and accompanies many chronic and infectious diseases. Altered glycosylation can occur at the onset and/or during tumor progression. Identifying these changes at early disease stages may aid in making decisions regarding treatments, as early intervention can greatly enhance survival. This review highlights some of the efforts being made to identify N- and O-glycosylation profile shifts in cancer using mass spectrometry. The analysis of single or panels of potential glycoprotein cancer markers are covered. Other emerging technologies such as global glycan release and site-specific glycosylation analysis and quantitation are also discussed. Graphical Abstract Steps involved in the biomarker discovery.
Assuntos
Biomarcadores , Glicoproteínas/análise , Neoplasias/diagnóstico , Polissacarídeos/análise , Glicoproteínas/química , Humanos , Espectrometria de Massas , Polissacarídeos/química , Processamento de Proteína Pós-TraducionalRESUMO
The stereochemistry of the hexuronic acid residues of the structure of glycosaminoglycans (GAGs) is a key feature that affects their interactions with proteins and other biological functions. Electron based tandem mass spectrometry methods, in particular electron detachment dissociation (EDD), have been able to distinguish glucuronic acid (GlcA) from iduronic acid (IdoA) residues in some heparan sulfate tetrasaccharides by producing epimer-specific fragments. Similarly, the relative abundance of glycosidic fragment ions produced by collision-induced dissociation (CID) or EDD has been shown to correlate with the type of hexuronic acid present in chondroitin sulfate GAGs. The present work examines the effect of charge state and degree of sodium cationization on the CID fragmentation products that can be used to distinguish GlcA and IdoA containing chondroitin sulfate A and dermatan sulfate chains. The cross-ring fragments (2,4)A(n) and (0,2)X(n) formed within the hexuronic acid residues are highly preferential for chains containing GlcA, distinguishing it from IdoA. The diagnostic capability of the fragments requires the selection of a molecular ion and fragment ions with specific ionization characteristics, namely charge state and number of ionizable protons. The ions with the appropriate characteristics display diagnostic properties for all the chondroitin sulfate and dermatan sulfate chains (degree of polymerization of 4-10) studied.
Assuntos
Sulfatos de Condroitina/química , Cromatografia Líquida de Alta Pressão/métodos , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Ácidos Urônicos/análise , Ácidos Urônicos/química , Sulfatos de Condroitina/análise , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
High-field asymmetric waveform ion mobility spectrometry (FAIMS) is shown to be capable of resolving isomeric and isobaric glycosaminoglycan negative ions and to have great utility for the analysis of this class of molecules when combined with Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) and tandem mass spectrometry. Electron detachment dissociation (EDD) and other ion activation methods for tandem mass spectrometry can be used to determine the sites of labile sulfate modifications and for assigning the stereochemistry of hexuronic acid residues of glycosaminoglycans (GAGs). However, mixtures with overlapping mass-to-charge values present a challenge, as their precursor species cannot be resolved by a mass analyzer prior to ion activation. FAIMS is shown to resolve two types of mass-to-charge overlaps. A mixture of chondroitin sulfate A (CSA) oligomers with 4-10 saccharides units produces ions of a single mass-to-charge by electrospray ionization, as the charge state increases in direct proportion to the degree of polymerization for these sulfated carbohydrates. FAIMS is shown to resolve the overlapping charge. A more challenging type of mass-to-charge overlap occurs for mixtures of diastereomers. FAIMS is shown to separate two sets of epimeric GAG tetramers. For the epimer pairs, the complexity of the separation is reduced when the reducing end is alkylated, suggesting that anomers are also resolved by FAIMS. The resolved components were activated by EDD and the fragment ions were analyzed by FTICR-MS. The resulting tandem mass spectra were able to distinguish the two epimers from each other.
Assuntos
Glicosaminoglicanos/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Isomerismo , Modelos Moleculares , Análise de Componente Principal , Espectroscopia de Infravermelho com Transformada de FourierAssuntos
Oligossacarídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/tendências , Animais , Humanos , Espectrometria de Massas/métodos , Espectrometria de Massas/tendências , Oligossacarídeos/químicaRESUMO
The highly sulfated glycosaminoglycan oligosaccharides derived from heparin and heparan sulfate have been a highly intractable class of molecules to analyze by tandem mass spectrometry. Under the many methods of ion activation, this class of molecules generally exhibits SO3 loss as the most significant fragmentation pathway, interfering with the assignment of the location of sulfo groups in glycosaminoglycan chains. We report here a method that stabilizes sulfo groups and facilitates the complete structural analysis of densely sulfated (two or more sulfo groups per disaccharide repeat unit) heparin and heparan sulfate oligomers. This is achieved by complete removal of all ionizable protons, either by charging during electrospray ionization or by Na(+)/H(+) exchange. The addition of millimolar levels of NaOH to the sample solution facilitates the production of precursor ions that meet this criterion. This approach is found to work for a variety of heparin sulfate oligosaccharides derived from natural sources or produced by chemoenzymatic synthesis, with up to 12 saccharide subunits and up to 11 sulfo groups.
Assuntos
Heparina/química , Heparitina Sulfato/química , Sulfotransferases/química , Espectrometria de Massas em Tandem , Animais , Biocatálise , Configuração de Carboidratos , Sequência de Carboidratos , Heparina/biossíntese , Heparitina Sulfato/biossíntese , Dados de Sequência Molecular , Oligossacarídeos/biossíntese , Oligossacarídeos/química , Fosfoadenosina Fosfossulfato/química , Espectrometria de Massas por Ionização por Electrospray , Ésteres do Ácido Sulfúrico/química , Sus scrofaRESUMO
Glycosaminoglycans (GAGs) are a class of biologically important molecules, and their structural analysis is the target of considerable research effort. Advances in tandem mass spectrometry (MS/MS) have recently enabled the structural characterization of several classes of GAGs; however, the highly sulfated GAGs, such as heparins, have remained a relatively intractable class due their tendency to lose SO(3) during MS/MS, producing few sequence-informative fragment ions. The present work demonstrates for the first time the complete structural characterization of the highly sulfated heparin-based drug Arixtra. This was achieved by Na(+)/H(+) exchange to create a more ionized species that was stable against SO(3) loss, and that produced complete sets of both glycosidic and cross-ring fragment ions. MS/MS enables the complete structural determination of Arixtra, including the stereochemistry of its uronic acid residues, and suggests an approach for solving the structure of more complex, highly sulfated heparin-based drugs.