RESUMO
Birnessite is ubiquitous in the natural environment where heavy metals are retained and easily transformed. The surface properties and structure of birnessite change with the changes in external environmental conditions, which also affects the fate of heavy metals. Clarifying the effect and mechanism of the birnessite phase transition process on heavy metals is the key to taking effective measures to prevent and control heavy metal pollution. Therefore, the four transformation pathways of birnessite are summarized first in this review. Second, the relationship between transformation pathways and environmental conditions is proposed. These relevant environmental conditions include abiotic (e.g., co-existing ions, pH, oxygen pressure, temperature, electric field, light, aging, pressure) and biotic factors (e.g., microorganisms, biomolecules). The phase transformation is achieved by the key intermediate of Mn(III) through interlayer-condensation, folding, neutralization-disproportionation, and dissolution-recrystallization mechanisms. The AOS (average oxidation state) of Mn and interlayer spacing are closely correlated with the phase transformation of birnessite. Last but not least, the mechanisms of heavy metals immobilization in the transformation process of birnessite are summed up. They involve isomorphous substitution, redox, complexation, hydration/dehydration, etc. The transformation of birnessite and its implication on heavy metals will be helpful for understanding and predicting the behavior of heavy metals and the crucial phase of manganese oxides/hydroxides in natural and engineered environments.
Assuntos
Manganês , Metais Pesados , Manganês/química , Adsorção , Metais Pesados/química , Óxidos/química , Compostos de Manganês/química , OxirreduçãoRESUMO
In non-ferrous metal smelting, the problem of gaseous arsenic in high-sulfur flue gas is difficult to solve. Now we have developed oxygen-enriched amorphous iron manganese oxide (AFMBO) based on the unique superiority of iron-manganese oxide for arsenic capture to realize the effective control of gaseous arsenic in the non-ferrous smelting flue gas. The experimental results show that the arsenic adsorption capacity of AFMBO is up to 102.7 mg/g, which has surpassed most of the current adsorbents. In particular, AFMBO can effectively capture gaseous arsenic even at 12% v/v SO2 concentrations (88.45 mg/g). Moreover, the spent AFMBO possesses pronounced magnetic characteristics that make it easier to separate from dust, which is conducive to reducing the secondary environmental risk of arsenic. In terms of mechanism study, various characterization methods are used to explain the important role of lattice oxygen and adsorbed oxygen in the capture process of gaseous arsenic. Moreover, the reason for the efficient arsenic removal performance of AFMBO is also reasonably explained at the microscopic level. This study provides ideas and implications for gaseous arsenic pollution control research.
Assuntos
Arsênio , Manganês , Gases , Óxidos , Ferro , OxigênioRESUMO
Deep eutectic solvents (DESs) as novel green solvents are potential options to replace inorganic acids for hydrometallurgy. Compared with inorganic acids, the physicochemical properties of DESs and their applications in recycling of spent lithium-ion batteries were summarized. The viscosity, metal solubility, toxicological properties and biodegradation of DESs depend on the hydrogen bond donor (HBD) and acceptor (HBA). The viscosity of ChCl-based DESs increased according to the HBD in the following order: alcohols < carboxylic acids < sugars < inorganic salts. The strongly coordinating HBDs increased the solubility of metal oxide via surface complexation reactions followed by ligand exchange for chloride in the bulk solvent. Interestingly, the safety and degradability of DESs reported in the literature are superior to those of inorganic acids. Both DESs and inorganic acids have excellent metal leaching efficiencies (>99%). However, the reaction kinetics of DESs are 2-3 orders of magnitude slower than those of inorganic acids. A significant advantage of DESs is that they can be regenerated and recycled multiple times after recovering metals by electrochemical deposition or precipitation. In the future, the development of efficient and selective DESs still requires a lot of attention.
Assuntos
Solventes Eutéticos Profundos , Lítio , Fontes de Energia Elétrica , Ligação de Hidrogênio , Íons , Metais , Solventes/químicaRESUMO
The cathode powder is obtained by wet crushing and screening, and the leaching behavior of Li, Ni, Co, Cu, and Al is then investigated using a ternary leaching system composed of ammonia, ammonium sulfite, and ammonium bicarbonate. Ammonium sulfite is necessary as a reductant to improve the Li, Ni, and Co leaching efficiencies, and ammonium bicarbonate acts as a buffer in ammoniacal solutions. A detailed understanding of the selective leaching process is obtained by investigating the effects of parameters such as the leaching reagent composition, leaching time (0-300â¯min), temperature (40-90⯰C), solid-to-liquid ratio (10-50â¯g/L), and agitation speed (300-700â¯rpm). It is found that Ni and Cu could be almost fully leached out, while Al is hardly leached and Li(60.53%) and Co(80.99%) exhibit a moderate leaching efficiency. The results show that the optimum solid-liquid ratio of the leaching system is 20â¯g/L, and the increase of temperature and reaction time is beneficial to metal leaching. The leaching kinetics analysis shows that the chemical reaction control explains the leaching behavior of Li, Ni, and Co well. Therefore, this work may be beneficial for further recycling valuable metals from leaching solutions by introducing an extraction agent.
Assuntos
Amônia , Lítio , Fontes de Energia Elétrica , Metais , Compostos de Amônio Quaternário , Reciclagem , SulfitosRESUMO
Huntington disease (HD) is a dominantly inherited neurodegenerative disease caused by a polyglutamine expansion in the protein huntingtin (Htt). Striatal and cortical neuronal loss are prominent features of this disease. No disease-modifying treatments have been discovered for HD. To identify new therapeutic targets in HD, we screened a kinase inhibitor library for molecules that block mutant Htt cellular toxicity in a mouse HD striatal cell model, Hdh(111Q/111Q) cells. We found that diacylglycerol kinase (DGK) inhibitor II (R59949) decreased caspase-3/7 activity after serum withdrawal in striatal Hdh(111Q/111Q) cells. In addition, R59949 decreased the accumulation of a 513-amino acid N-terminal Htt fragment processed by caspase-3 and blocked alterations in lipid metabolism during serum withdrawal. To identify the diacylglycerol kinase mediating this effect, we knocked down all four DGK isoforms expressed in the brain (ß, γ, ε, and ζ) using siRNA. Only the knockdown of the family member, DGKε, blocked striatal Hdh(111Q/111Q)-mediated toxicity. We also investigated the significance of these findings in vivo. First, we found that reduced function of the Drosophila DGKε homolog significantly improves Htt-induced motor dysfunction in a fly model of HD. In addition, we find that the levels of DGKε are increased in the striatum of R6/2 HD transgenic mice when compared with littermate controls. Together, these findings indicate that increased levels of kinase DGKε contribute to HD pathogenesis and suggest that reducing its levels or activity is a potential therapy for HD.
Assuntos
Diacilglicerol Quinase/metabolismo , Doença de Huntington/metabolismo , Metabolismo dos Lipídeos , Mutação , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Transdução de Sinais , Animais , Caspase 3/genética , Caspase 3/metabolismo , Caspase 7/genética , Caspase 7/metabolismo , Linhagem Celular , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Diacilglicerol Quinase/genética , Humanos , Proteína Huntingtina , Doença de Huntington/genética , Doença de Huntington/patologia , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genéticaRESUMO
We used a lectin chromatography/MS-based approach to screen conditioned medium from a panel of luminal (less aggressive) and triple negative (more aggressive) breast cancer cell lines (n=5/subtype). The samples were fractionated using the lectins Aleuria aurantia (AAL) and Sambucus nigra agglutinin (SNA), which recognize fucose and sialic acid, respectively. The bound fractions were enzymatically N-deglycosylated and analyzed by LC-MS/MS. In total, we identified 533 glycoproteins, â¼90% of which were components of the cell surface or extracellular matrix. We observed 1011 glycosites, 100 of which were solely detected in ≥3 triple negative lines. Statistical analyses suggested that a number of these glycosites were triple negative-specific and thus potential biomarkers for this tumor subtype. An analysis of RNaseq data revealed that approximately half of the mRNAs encoding the protein scaffolds that carried potential biomarker glycosites were up-regulated in triple negative vs luminal cell lines, and that a number of genes encoding fucosyl- or sialyltransferases were differentially expressed between the two subtypes, suggesting that alterations in glycosylation may also drive candidate identification. Notably, the glycoproteins from which these putative biomarker candidates were derived are involved in cancer-related processes. Thus, they may represent novel therapeutic targets for this aggressive tumor subtype.
Assuntos
Biomarcadores Tumorais/análise , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Cromatografia de Afinidade/métodos , Glicoproteínas/análise , Lectinas/química , Biomarcadores Tumorais/química , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Feminino , Glicoproteínas/química , Glicoproteínas/classificação , Glicoproteínas/metabolismo , Humanos , Lectinas/metabolismo , Espectrometria de Massas/métodos , Proteínas de Membrana/análise , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteoma/análise , Proteoma/químicaRESUMO
3-nitrotyrosine (3NT) is an oxidative posttranslational modification associated with many diseases. Determining the specific sites of this modification remains a challenge due to the low stoichiometry of 3NT modifications in biological samples. Mass spectrometry-based proteomics is a powerful tool for identifying 3NT modifications, however several reports identifying 3NT sites were later demonstrated to be incorrect, highlighting that both the accuracy and efficiency of these workflows need improvement. To advance our understanding of the chromatographic and spectral properties of 3NT-containing peptides we have adapted a straightforward, reproducible procedure to generate a large set of 3NT peptides by chemical nitration of a defined, commercially available 48 protein mixture. Using two complementary LC-MS/MS platforms, a QTOF (QSTAR Elite) and dual pressure ion trap mass spectrometer (LTQ Velos), we detected over 200 validated 3NT-containing peptides with significant overlap in the peptides detected by both systems. We investigated the LC-MS/MS properties for each peptide manually using defined criteria and then assessed their utility to confirm that the peptide was 3NT modified. This broad set of validated 3NT-containing peptides can be utilized to optimize mass spectrometric instrumentation and data mining strategies or further develop 3NT peptide enrichment strategies for this biologically important, oxidative posttranslational modification.
Assuntos
Espectrometria de Massas/métodos , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Tirosina/análogos & derivados , Sequência de Aminoácidos , Animais , Bovinos , Cromatografia Líquida/métodos , Misturas Complexas/química , Misturas Complexas/metabolismo , Interpretação Estatística de Dados , Estabilidade de Medicamentos , Humanos , Espectrometria de Massas/instrumentação , Espectrometria de Massas/estatística & dados numéricos , Modelos Biológicos , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Soroalbumina Bovina/análise , Soroalbumina Bovina/química , Soroalbumina Bovina/efeitos dos fármacos , Soroalbumina Bovina/metabolismo , Tirosina/metabolismo , Tirosina/farmacologiaRESUMO
Differences in the fertilization behavior of Xenopus borealis from X. laevis and X. tropicalis suggest differences in the glycosylation of the egg jellies. To test this assumption, O-linked glycans were chemically released from the egg jelly coat glycoproteins of X. borealis. Over 50 major neutral glycans were observed, and no anionic glycans were detected from the released O-glycan pool. Preliminary structures of â¼30 neutral oligosaccharides were determined using matrix-assisted laser desorption/ionization (MALDI) infrared multiphoton dissociation tandem mass spectrometry (MS). The mass fingerprint of a group of peaks for the core-2 structure of O-glycans was conserved in the tandem mass spectra and was instrumental in rapid and efficient structure determination. Among the 29 O-glycans, 22 glycans contain the typical core-2 structure, 3 glycans have the core-1 structure and 2 glycans contained a previously unobserved core structure with hexose at the reducing end. There were seven pairs of structural isomers observed in the major O-linked oligosaccharides. To further elucidate the structures of a dozen O-linked glycans, specific and targeted exoglycosidase digestions were carried out and the products were monitored with MALDI-MS. Reported here are the elucidated structures of O-linked oligosaccharides from glycoproteins of X. borealis egg jelly coats. The structural differences in O-glycans from jelly coats of X. borealis and its close relatives may provide a better understanding of the structure-function relationships and the role of glycans in the fertilization process within Xenopodinae.
Assuntos
Glicosídeo Hidrolases/metabolismo , Oligossacarídeos/química , Óvulo/química , Polissacarídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Xenopus/metabolismo , Animais , Configuração de Carboidratos , Sequência de Carboidratos , Glicosilação , Dados de Sequência Molecular , Oligossacarídeos/metabolismo , Filogenia , Polissacarídeos/metabolismo , Xenopus/crescimento & desenvolvimentoRESUMO
Glycans are cell-type-specific, posttranslational protein modifications that are modulated during developmental and disease processes. As such, glycoproteins are attractive biomarker candidates. Here, we describe a mass spectrometry-based workflow that incorporates lectin affinity chromatography to enrich for proteins that carry specific glycan structures. As increases in sialylation and fucosylation are prominent among cancer-associated modifications, we focused on Sambucus nigra agglutinin (SNA) and Aleuria aurantia lectin (AAL), lectins which bind sialic acid- and fucose-containing structures, respectively. Fucosylated and sialylated glycopeptides from human lactoferrin served as positive controls, and high-mannose structures from yeast invertase served as negative controls. The standards were spiked into Multiple Affinity Removal System (MARS) 14-depleted, trypsin-digested human plasma from healthy donors. Samples were loaded onto lectin columns, separated by HPLC into flow-through and bound fractions, and treated with peptide: N-glycosidase F to remove N-linked glycans. The deglycosylated peptide fractions were interrogated by ESI HPLC-MS/MS. We identified a total of 122 human plasma glycoproteins containing 247 unique glycosites. Importantly, several of the observed glycoproteins (e.g., cadherin 5 and neutrophil gelatinase-associated lipocalin) typically circulate in plasma at low nanogram per milliliter levels. Together, these results provide mass spectrometry-based evidence of the utility of incorporating lectin-separation platforms into cancer biomarker discovery pipelines.
Assuntos
Biomarcadores Tumorais/química , Cromatografia Líquida de Alta Pressão/métodos , Glicoproteínas/química , Lectinas/química , Polissacarídeos/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Biomarcadores Tumorais/sangue , Cromatografia de Afinidade/métodos , Bases de Dados Factuais , Feminino , Glicopeptídeos/química , Glicoproteínas/sangue , Glicoproteínas/metabolismo , Humanos , Masculino , Neoplasias/diagnóstico , Polissacarídeos/isolamento & purificação , Ligação Proteica , Tripsina/metabolismoRESUMO
BACKGROUND: Cancer has profound effects on gene expression, including a cell's glycosylation machinery. Thus, tumors produce glycoproteins that carry oligosaccharides with structures that are markedly different from the same protein produced by a normal cell. A single protein can have many glycosylation sites that greatly amplify the signals they generate compared with their protein backbones. CONTENT: In this article, we survey clinical tests that target carbohydrate modifications for diagnosing and treating cancer. We present the biological relevance of glycosylation to disease progression by highlighting the role these structures play in adhesion, signaling, and metastasis and then address current methodological approaches to biomarker discovery that capitalize on selectively capturing tumor-associated glycoforms to enrich and identify disease-related candidate analytes. Finally, we discuss emerging technologies--multiple reaction monitoring and lectin-antibody arrays--as potential tools for biomarker validation studies in pursuit of clinically useful tests. SUMMARY: The future of carbohydrate-based biomarker studies has arrived. At all stages, from discovery through verification and deployment into clinics, glycosylation should be considered a primary readout or a way of increasing the sensitivity and specificity of protein-based analyses.
Assuntos
Biomarcadores Tumorais/sangue , Glicoproteínas/sangue , Proteínas de Neoplasias/sangue , Neoplasias/sangue , Glicosilação , Humanos , Lectinas/química , Espectrometria de Massas/métodosRESUMO
Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the internal status of the cell. Glycans on tumor cells tend to have abundant sialic acid and fucose moieties. We propose that these cancer-associated glycan variants be exploited for biomarker development aimed at diagnosing early-stage disease. Accordingly, we developed a mass spectrometry-based workflow that incorporates chromatography on affinity matrices formed from lectins, proteins that bind specific glycan structures. The lectins Sambucus nigra (SNA) and Aleuria aurantia (AAL), which bind sialic acid and fucose, respectively, were covalently coupled to POROS beads (Applied Biosystems) and packed into PEEK columns for high pressure liquid chromatography (HPLC). Briefly, plasma was depleted of the fourteen most abundant proteins using a multiple affinity removal system (MARS-14; Agilent). Depleted plasma was trypsin-digested and separated into flow-through and bound fractions by SNA or AAL HPLC. The fractions were treated with PNGaseF to remove N-linked glycans, and analyzed by LC-MS/MS on a QStar Elite. Data were analyzed using Mascot software. The experimental design included positive controls-fucosylated and sialylated human lactoferrin glycopeptides-and negative controls-high mannose glycopeptides from Saccharomyces cerevisiae-that were used to monitor the specificity of lectin capture. Key features of this workflow include the reproducibility derived from the HPLC format, the positive identification of the captured and PNGaseF-treated glycopeptides from their deamidated Asn-Xxx-Ser/Thr motifs, and quality assessment using glycoprotein standards. Protocol optimization also included determining the appropriate ratio of starting material to column capacity, identifying the most efficient capture and elution buffers, and monitoring the PNGaseF-treatment to ensure full deglycosylation. Future directions include using this workflow to perform mass spectrometry-based discovery experiments on plasma from breast cancer patients and control individuals.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Glicopeptídeos/isolamento & purificação , Lectinas/química , Lectinas de Plantas/química , Proteínas Inativadoras de Ribossomos/química , Proteínas Sanguíneas/isolamento & purificação , Glicopeptídeos/sangue , Humanos , Lactoferrina/análiseRESUMO
The structural elucidation of oligosaccharides remains a major challenge. Mass spectrometry provides a rapid and convenient method for structural elucidation on the basis of tandem mass spectrometry. Ions are commonly selected and subjected to collision-induced dissociation (CID) to obtain structural information. However, a disadvantage of CID is the decrease in both the degree and efficiency of dissociation with increasing mass. In this chapter, we illustrate the use of infrared multiphoton dissociation (IRMPD) to obtain structural information for O- and N-linked oligosaccharides. The IRMPD and CID behaviors of oligosaccharides are compared.
Assuntos
Espectrometria de Massas/métodos , Oligossacarídeos/química , Animais , Sequência de Carboidratos , Cromatografia Líquida de Alta Pressão/métodos , Humanos , Espectrometria de Massas/instrumentação , Modelos Biológicos , Conformação Molecular , Dados de Sequência Molecular , Oligossacarídeos/isolamento & purificação , Espectrofotometria Infravermelho/instrumentação , Espectrofotometria Infravermelho/métodosRESUMO
The complexity of glycans poses a major challenge for structure elucidation. Tandem mass spectrometry is currently an efficient and powerful technique for the structural characterization of glycans. Collision-induced dissociation (CID) is most commonly used, and involves first isolating the glycan ions of interest, translationally exciting them, and then striking them with inert target gas to fragment the precursor ions. The structural information of the glycan can be obtained from the fragment ions of the tandem MS spectra. In this chapter, sustained off-resonance irradiation-collision-induced dissociation (SORI-CID) implemented with matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI FT ICR MS) is demonstrated to be a useful analysis tool for structural elucidation of mucin-type O-glycans released from mucin glycoproteins. The mechanisms by which the glycans undergo fragmentations in the tandem mass analysis are also discussed.
Assuntos
Polissacarídeos/química , Espectrometria de Massas em Tandem/métodos , Animais , Sequência de Carboidratos , Humanos , Modelos Biológicos , Conformação Molecular , Dados de Sequência Molecular , Polissacarídeos/análise , Polissacarídeos/isolamento & purificaçãoRESUMO
Ovarian cancer is difficult to diagnose in women because symptoms of the disease are often not noticed until the disease has progressed to an advanced untreatable stage. Although a serum test, CA125, is currently available to assist with monitoring treatment of ovarian cancer, this test lacks the necessary specificity and sensitivity for early detection. Therefore, better biomarkers of ovarian cancer are needed. A glycoprotein analysis approach was undertaken using high resolution Fourier transform ion cyclotron resonance mass spectrometry to analyze glycosylated proteins present in the conditioned media of ovarian cancer cell lines and in sera obtained from ovarian cancer patients and normal controls. In this study, glycosylated proteins were separated by gel electrophoresis, and individual glycoproteins were selected for glycosylation analysis and protein identification. The attached glycans from each protein were released and profiled by mass spectrometry. Glycosylation of a mucin protein and a large glycosylated protein isolated from the ES2 ovarian cancer cell line was determined to consist of mostly O-linked glycans. Four prominent glycoproteins of approximate 517, 370, 250, 163 kDa from serum samples were identified as two forms of apolipoprotein B-100, fibronectin, and immunoglobulin A1, respectively. Mass spectrometric analysis of glycans isolated from apolipoprotein B-100 (517 kD) showed the presence of small, specific O-linked oligosaccharides. In contrast, analysis of fibronectin (250 kD) and immunoglobulin A1 (163 kD) produced N-linked glycan fragments in forms that were sufficiently different from the glycans obtained from the corresponding protein band present in the normal serum samples. This study shows that not only a single protein but several are aberrantly glycosylated, and those abnormal glycosylation changes can be detected and may ultimately serve as glycan biomarkers for ovarian cancer.
Assuntos
Carboidratos/análise , Neoplasias Ovarianas/metabolismo , Proteômica , Linhagem Celular Tumoral , Eletroforese em Gel de Poliacrilamida , Feminino , Glicosilação , Humanos , Neoplasias Ovarianas/sangue , Neoplasias Ovarianas/patologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Posttranslational modifications such as glycosylation can play a fundamental role in signaling pathways that transform an ordinary cell into a malignant one. The development of a protocol to detect these changes in the preliminary stages of disease can lead to a sensitive and specific diagnostic for the early detection of malignancies such as ovarian cancer in which differential glycan patterns are linked to etiology and progression. Small variations in instrument parameters and sample preparation techniques are known to have significant influence on the outcome of an experiment. For an experiment to be effective and reproducible, these parameters must be optimized for the analyte(s) under study. We present a detailed examination of sample preparation and matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FT-ICR-MS) analysis of O-linked glycans globally cleaved from mucin glycoproteins. Experiments with stable isotope-labeled biomolecules allowed for the establishment of appropriate acquisition times and excitation voltages for MALDI-FT-ICR-MS of oligosaccharides. Quadrupole ion guide optimization studies with mucin glycans identified conditions for the comprehensive analysis of the entire mass range of O-linked carbohydrates in this glycoprotein. Separately optimized experimental parameters were integrated in a method that allowed for the effective study of O-linked glycans.
Assuntos
Mucinas/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Espectrometria de Massas em Tandem/métodos , Angiotensinas/química , Animais , Glicosilação , Oligossacarídeos/química , Reprodutibilidade dos Testes , SuínosRESUMO
Because the glycosylation of proteins is known to change in tumor cells during the development of breast cancer, a glycomics approach is used here to find relevant biomarkers of breast cancer. These glycosylation changes are known to correlate with increasing tumor burden and poor prognosis. Current antibody-based immunochemical tests for cancer biomarkers of ovarian (CA125), breast (CA27.29 or CA15-3), pancreatic, gastric, colonic, and carcinoma (CA19-9) target highly glycosylated mucin proteins. However, these tests lack the specificity and sensitivity for use in early detection. This glycomics approach to find glycan biomarkers of breast cancer involves chemically cleaving oligosaccharides (glycans) from glycosylated proteins that are shed or secreted by breast cancer tumor cell lines. The resulting free glycan species are analyzed by MALDI-FT-ICR MS. Further structural analysis of the glycans can be performed in FTMS through the use of tandem mass spectrometry with infrared multiphoton dissociation. Glycan profiles were generated for each cell line and compared. These methods were then used to analyze sera obtained from a mouse model of breast cancer and a small number of serum samples obtained from human patients diagnosed with breast cancer or patients with no known history of breast cancer. In addition to the glycosylation changes detected in mice as mouse mammary tumors developed, glycosylation profiles were found to be sufficiently different to distinguish patients with cancer from those without. Although the small number of patient samples analyzed so far is inadequate to make any legitimate claims at this time, these promising but very preliminary results suggest that glycan profiles may contain distinct glycan biomarkers that may correspond to glycan "signatures of cancer."
Assuntos
Biomarcadores Tumorais/sangue , Neoplasias da Mama/sangue , Neoplasias da Mama/química , Polissacarídeos/sangue , Adulto , Idoso , Animais , Linhagem Celular Tumoral , Meios de Cultivo Condicionados , Glicoproteínas/sangue , Glicoproteínas/química , Glicosilação , Humanos , Camundongos , Pessoa de Meia-Idade , Polissacarídeos/química , Análise de Componente Principal , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Frações Subcelulares , Espectrometria de Massas em Tandem , Fatores de TempoRESUMO
The structural elucidation of oligosaccharides remains a major challenge. Mass spectrometry provides a rapid and convenient method for structural elucidation based on tandem mass spectrometry. Ions commonly are selected and subjected to collision-induced dissociation (CID) to obtain structural information. Unfortunately, N-linked oligosaccharides are relatively large compounds and are not readily fragmented using CID. In this report, we illustrate the use of infrared multiphoton dissociation (IRMPD) to obtain structural information for large N-linked oligosaccharides. The IRMPD and CID behavior of oligosaccharides were compared for high-mannose-type oligosaccharides. Fragmentation that could not be obtained through conventional CID in Fourier transform ion cyclotron resonance mass spectrometry was observed with N-linked oligosaccharides. O-Linked and N-linked glycans of similarly large sizes were compared. It was found that internal cross-ring cleavages were observed only for N-linked oligosaccharides. The mannose branch points of N-linked oligosaccharides are apparently more susceptible to cross-ring cleavages.
Assuntos
Oligossacarídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Animais , Configuração de Carboidratos , Bovinos , Galinhas , Manose/químicaRESUMO
A glycomic approach is developed to identify oligosaccharide markers for ovarian cancer by rapidly profiling globally released oligosaccharides. Glycoproteins shed by cancer cells are found in the supernatant (or conditioned media) of cultured cells. In this approach, shed glycoproteins are profiled for their oligosaccharide content using beta-elimination conditions. Changes in glycosylation are monitored by matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR-MS). Because shed glycoproteins would also be found in serum, similar glycan profiling was performed to observe potential oligosaccharide markers. Oligosaccharide profiling data on a limited set of patient and normal serum samples were studied to determine potential glycan markers in ovarian cancer. We were able to demonstrate the presence of at least 15 unique serum glycan markers in all patients but absent in normal individuals. To determine the structure of the glycan biomarkers, a number of the ions were isolated and further analyzed using infrared multiphoton dissociation (IRMPD). One major advantage of this approach is that glycans are examined directly from patient sera without the need to obtain cancer biopsy specimens or to purify glycosylated proteins from these specimens.
Assuntos
Biomarcadores Tumorais/sangue , Proteínas Sanguíneas/análise , Oligossacarídeos/sangue , Neoplasias Ovarianas/sangue , Biomarcadores Tumorais/química , Linhagem Celular Tumoral , Meios de Cultivo Condicionados/análise , Ciclotrons , Feminino , Humanos , Oligossacarídeos/química , Valor Preditivo dos Testes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Glycosylation is one of the most important classes of post-translational protein modifications, but the identification of glycans is difficult because of their branched structures and numerous isomers. We describe an algorithm called CartoonistTwo that proposes structures for O-linked glycans by automatically analyzing fragmentation mass spectra. CartoonistTwo improves upon previous glycan identification software primarily in its scoring function, which can more successfully distinguish among a number of similar structures. CartoonistTwo was designed and tested with FTICR mass spectra, and includes automatic recalibration and peak selection especially tuned for such data, yet it can be easily adapted to fragmentation spectra (MS2 or MSn) from other instrument types. On a validated test set of 34 SORI-CID MSn FTICR spectra from Xenopus egg jelly, CartoonistTwo gave the manually determined structural assignment either the first or second highest score over 90% of the time. And for over 50% of these spectra, CartoonistTwo selected a unique highest scoring structure that agreed with the manually determined one.
Assuntos
Polissacarídeos/análise , Algoritmos , Animais , Feminino , Glicosilação , Espectrometria de Massas , Óvulo/química , XenopusRESUMO
Fredericamycin (FDM) A, a pentadecaketide featuring two sets of peri-hydroxy tricyclic aromatic moieties connected through a unique chiral spiro carbon center, exhibits potent cytotoxicity and has been studied as a new type of anticancer drug lead because of its novel molecular architecture. The fdm gene cluster was localized to 33-kb DNA segment of Streptomyces griseus ATCC 49344, and its involvement in FDM A biosynthesis was proven by gene inactivation, complementation, and heterologous expression experiments. The fdm cluster consists of 28 open reading frames (ORFs), encoding a type II polyketide synthase (PKS) and tailoring enzymes as well as several regulatory and resistance proteins. The FDM PKS features a KSalpha subunit with heretofore unseen tandem cysteines at its active site, a KSbeta subunit that is distinct phylogenetically from KSbeta of hexa-, octa-, or decaketide PKSs, and a dedicated phosphopantetheinyl transferase. Further study of the FDM PKS could provide new insight into how a type II PKS controls chain length in aromatic polyketide biosynthesis. The availability of the fdm genes, in vivo characterization of the fdm cluster in S. griseus, and heterologous expression of the fdm cluster in Streptomyces albus set the stage to investigate FDM A biosynthesis and engineer the FDM biosynthetic machinery for the production of novel FDM A analogues.