RESUMO
Chemokine-GAG interactions are crucial to facilitate chemokine immobilization, resulting in the formation of chemokine gradients that guide cell migration. Here we demonstrate chromatographic isolation and purification of two heparin hexasaccharide isomers that interact with the oligomeric chemokine Monocyte Chemoattractant Protein-1 (MCP-1)/CCL2 with different binding affinities. The sequences of these two hexasaccharides were deduced from unique MS/MS product ions and HPLC compositional analysis. Ion mobility mass spectrometry (IM-MS) showed that the two isolated oligosaccharides have different conformations and both displayed preferential binding for one of the two distinct conformations known for MCP-1 dimers. A significant shift in arrival time distribution of close to 70 Å2 was observed, indicating a more compact protein:hexasaccharide conformation. Clear differences in the MS spectra between bound and unbound protein allowed calculation of Kd values from the resulting data. The structural difference between the two hexasaccharides was defined as the differential location of a single sulfate at either C-6 of glucosamine or C-2 of uronic acid in the reducing disaccharide, resulting in a 200-fold difference in binding affinity for MCP-1. These data indicate sequence specificity for high affinity binding, supporting the view that sulfate position, and not simply the number of sulfates, is important for heparan sulfate protein binding.
Assuntos
Quimiocina CCL2/análise , Heparina/química , Oligossacarídeos/química , Cromatografia Líquida de Alta Pressão , Humanos , Isomerismo , Espectrometria de Massas em TandemRESUMO
The complexity of heparin and heparan sulfate saccharides makes their purification, including many isomeric structures, very challenging and is a bottleneck for structure-activity studies. High-resolution separations have been achieved by strong anion exchange (SAX) chromatography on Propac PA1 and cetyltrimethylammonium (CTA)-C18 silica columns; however, these entail subsequent desalting methodologies and consequent sample losses and are incompatible with orthogonal chromatography methodologies and, in particular, mass spectrometry. Here, we present the CTA-SAX purification of heparin oligosaccharides using volatile salt (VS) buffer. In VSCTA-SAX, the use of ammonium bicarbonate buffer for elution improves resolution through both weaker dissociation and conformational coordination of the ammonium across the sulfate groups. Using ion mobility mass spectrometry, we demonstrate that isomeric structures have different structural conformations, which makes chromatographic separation achievable. Resolution of such structures is improved compared to standard SAX methods, and in addition, VSCTA-SAX provides an orthogonal method to isolate saccharides with higher purity. Because ammonium bicarbonate is used, the samples can be evaporated rather than desalted, preventing substantial sample loss and allowing more effective subsequent analysis by electrospray mass spectrometry. We conclude that VSCTA-SAX is a powerful new tool that helps address the difficult challenge of heparin/heparan sulfate saccharide separation and will enhance structure-activity studies.
Assuntos
Heparina/química , Oligossacarídeos/isolamento & purificação , Compostos Orgânicos Voláteis/química , Compostos de Cetrimônio/química , Cromatografia por Troca Iônica , Espectrometria de Mobilidade Iônica , Oligossacarídeos/química , Sais/química , EstereoisomerismoRESUMO
Trehalose glycolipids are found in many bacteria in the suborder Corynebacterineae, but methyl-branched acyltrehaloses are exclusive to virulent species such as the human pathogen Mycobacterium tuberculosis. In M. tuberculosis, the acyltransferase PapA3 catalyzes the formation of diacyltrehalose (DAT), but the enzymes responsible for downstream reactions leading to the final product, polyacyltrehalose (PAT), have not been identified. The PAT biosynthetic gene locus is similar to that of another trehalose glycolipid, sulfolipid 1. Recently, Chp1 was characterized as the terminal acyltransferase in sulfolipid 1 biosynthesis. Here we provide evidence that the homologue Chp2 (Rv1184c) is essential for the final steps of PAT biosynthesis. Disruption of chp2 led to the loss of PAT and a novel tetraacyltrehalose species, TetraAT, as well as the accumulation of DAT, implicating Chp2 as an acyltransferase downstream of PapA3. Disruption of the putative lipid transporter MmpL10 resulted in a similar phenotype. Chp2 activity thus appears to be regulated by MmpL10 in a relationship similar to that between Chp1 and MmpL8 in sulfolipid 1 biosynthesis. Chp2 is localized to the cell envelope fraction, consistent with its role in DAT modification and possible regulatory interactions with MmpL10. Labeling of purified Chp2 by an activity-based probe was dependent on the presence of the predicted catalytic residue Ser141 and was inhibited by the lipase inhibitor tetrahydrolipstatin (THL). THL treatment of M. tuberculosis resulted in selective inhibition of Chp2 over PapA3, confirming Chp2 as a member of the serine hydrolase superfamily. Efforts to produce in vitro reconstitution of acyltransferase activity using straight-chain analogues were unsuccessful, suggesting that Chp2 has specificity for native methyl-branched substrates.
Assuntos
Aciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Lipídeos/biossíntese , Lipídeos/química , Mycobacterium tuberculosis/metabolismo , Trealose/análogos & derivados , Aciltransferases/genética , Proteínas de Bactérias/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/fisiologia , Lactonas/farmacologia , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Orlistate , Estrutura Terciária de ProteínaRESUMO
In plants, the vascular system, specifically the phloem, functions in delivery of small RNA (sRNA) to exert epigenetic control over developmental and defense-related processes. Although the importance of systemic sRNA delivery has been established, information is currently lacking concerning the nature of the protein machinery involved in this process. Here, we show that a PHLOEM SMALL-RNA BINDING PROTEIN 1 (PSRP1) serves as the basis for formation of an sRNA ribonucleoprotein complex (sRNPC) that delivers sRNA (primarily 24 nt) to sink organs. Assembly of this complex is facilitated through PSRP1 phosphorylation by a phloem-localized protein kinase, PSRPK1. During long-distance transport, PSRP1-sRNPC is stable against phloem phosphatase activity. Within target tissues, phosphatase activity results in disassembly of PSRP1-sRNPC, a process that is probably required for unloading cargo sRNA into surrounding cells. These findings provide an insight into the mechanism involved in delivery of sRNA associated with systemic gene silencing in plants.
Assuntos
Cucurbita/genética , Proteínas de Plantas/metabolismo , RNA Interferente Pequeno , Sequência de Aminoácidos , Dados de Sequência Molecular , Floema/metabolismo , Fosforilação , Proteínas de Plantas/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismoRESUMO
Here we report ion mobility mass spectrometry (IMMS) separation and tandem mass spectrometry (MS(2)) sequencing methods used to analyze and differentiate six synthetically produced heparin/heparan sulfate (HS)-like octasaccharide (dp8) isomeric structures. These structures are isomeric with regard to either glucuronic acid (GlcA) or iduronic acid (IdoA) residues at various positions. IMMS analysis showed that a fully GlcA structure exhibited a more compact conformation, whereas the fully IdoA structure was more extended. Interestingly, the change from IdoA to GlcA in specific locations resulted in strong conformational distortions. MS(2) of the six isomers showed very different spectra with unique sets of diagnostic product ions. Analysis of MS(2) product ion spectra suggests that the GlcA group correlated with the formation of a glycosidic product ion under lower energy conditions. This resulted in an earlier product ion formation and more intense product ions. Importantly, this knowledge enabled a complete sequencing of the positions of GlcA and IdoA in each of the four positions located in each unique dp8 structure.
Assuntos
Ácido Glucurônico/química , Heparitina Sulfato/química , Ácido Idurônico/química , Polissacarídeos/química , Análise de Sequência/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Sítios de Ligação , Heparitina Sulfato/análise , IsomerismoRESUMO
Eukaryotic translation initiation factors are the principal molecular effectors regulating the process converting nucleic acid to functional protein. Commonly referred to as eIFs (eukaryotic initiation factors), this suite of proteins is comprised of at least 25 individual subunits that function in a coordinated, regulated, manner during mRNA translation. Multiple facets of eIF regulation have yet to be elucidated; however, many of the necessary protein factors are phosphorylated. Herein, we have isolated, identified and quantified phosphosites from eIF2, eIF3, and eIF4G generated from log phase grown HeLa cell lysates. Our investigation is the first study to globally quantify eIF phosphosites and illustrates differences in abundance of phosphorylation between the residues of each factor. Thus, identification of those phosphosites that exhibit either high or low levels of phosphorylation under log phase growing conditions may aid researchers to concentrate their investigative efforts to specific phosphosites that potentially harbor important regulatory mechanisms germane to mRNA translation.
Assuntos
Fatores de Iniciação em Eucariotos/metabolismo , Processamento de Proteína Pós-Traducional , Células HeLa , Humanos , FosforilaçãoRESUMO
Mycobacterium tuberculosis possesses unique cell-surface lipids that have been implicated in virulence. One of the most abundant is sulfolipid-1 (SL-1), a tetraacyl-sulfotrehalose glycolipid. Although the early steps in SL-1 biosynthesis are known, the machinery underlying the final acylation reactions is not understood. We provide genetic and biochemical evidence for the activities of two proteins, Chp1 and Sap (corresponding to gene loci rv3822 and rv3821), that complete this pathway. The membrane-associated acyltransferase Chp1 accepts a synthetic diacyl sulfolipid and transfers an acyl group regioselectively from one donor substrate molecule to a second acceptor molecule in two successive reactions to yield a tetraacylated product. Chp1 is fully active in vitro, but in M. tuberculosis, its function is potentiated by the previously identified sulfolipid transporter MmpL8. We also show that the integral membrane protein Sap and MmpL8 are both essential for sulfolipid transport. Finally, the lipase inhibitor tetrahydrolipstatin disrupts Chp1 activity in M. tuberculosis, suggesting an avenue for perturbing SL-1 biosynthesis in vivo. These data complete the SL-1 biosynthetic pathway and corroborate a model in which lipid biosynthesis and transmembrane transport are coupled at the membrane-cytosol interface through the activity of multiple proteins, possibly as a macromolecular complex.
Assuntos
Aciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Glicolipídeos/biossíntese , Mycobacterium tuberculosis/metabolismo , Fatores de Virulência/biossíntese , Acilação/fisiologia , Aciltransferases/antagonistas & inibidores , Aciltransferases/genética , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Transporte Biológico Ativo/efeitos dos fármacos , Transporte Biológico Ativo/fisiologia , Inibidores Enzimáticos/farmacologia , Glicolipídeos/genética , Lactonas/farmacologia , Mycobacterium tuberculosis/genética , Orlistate , Fatores de Virulência/genéticaRESUMO
Chemokines, 8 kDa proteins implicated in leukocyte migration via oligomerization, bind to glycosaminoglycans (GAGs) during the inflammation response as a means to regulate chemokine migration. Structural characterization of chemokines non-covalently bound to GAGs provides physiologically meaningful data in regard to routine inmmunosurveillance and disease response. In order to analyze the structures resulting from the GAG:chemokine interaction, we employed ion mobility mass spectrometry (IMMS) to analyze monocyte chemoattractant protein-1 (MCP-1), a CC chemokine, and interleukin-8 (IL-8), a CXC chemokine, along with their individual interactions with GAG heparin octasaccharides. We show that MCP-1 and IL-8 are physiologically present as a dimer, with MCP-1 having two variants of its dimeric form and IL-8 having only one. We also show that the MCP-1 dimer adopts two conformations, one extended and one compact, when bound to a dodecasulfated heparin octasaccharide. Binding of MCP-1 to heparin octasaccharide isomers of varying sulfation patterns results in similar arrival time distribution values, which suggests minimal distinguishing features among the resultant complexes. Additionally, tandem mass spectrometry (MS/MS) showed that the binding of MCP-1 to a heparin octasaccharide has different dissociation patterns when compared with the corresponding IL-8 bound dimer. Overall, IMMS and MS/MS were used to better define the structural tendencies and differences associated with CC and CXC dimers when associated with GAG octasaccharides.
Assuntos
Quimiocina CCL2/metabolismo , Heparina/metabolismo , Interleucina-8/metabolismo , Quimiocina CCL2/química , Heparina/química , Humanos , Interleucina-8/química , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Espectrometria de Massas em TandemRESUMO
Heparan sulfate (HS) is one of the most complex and informative biopolymers found on the cell surface or in the extracellular matrix as either free HS fragments or constituents of HS proteoglycans (HSPGs). Analysis of free HS and HSPG sugar chains in human serum at the disaccharide level has great potential for early disease diagnosis and prognosis; however, the low concentration of HS in human serum, together with the complexity of the serum matrix, limits the information on HS. In this study, we present and validate the development of a new sensitive method for in-depth compositional analysis of free HS and HSPG sugar chains. This protocol involved several steps including weak anion exchange chromatography, ultrafiltration, and solid-phase extraction for enhanced detection prior to LC-MS/MS analysis. Using this protocol, a total of 51 serum samples from 26 premenopausal and 25 postmenopausal women were analyzed. Statistically significant differences in heparin/HS disaccharide profiles were observed. The proportion of N-acetylation and N-sulfation in both free HS and HSPG sugar chains were significantly different between pre- and postmenopausal women, indicating changes in N-deacetylase/N-sulfotransferases (NDSTs), the enzymes involved in the initial step of the biosynthetic pathway. Differences in the proportion of 6-O-sulfation suggest that 6-O-sulfotransferase and/or 6-O-sulfatase enzymes may also be implicated.
Assuntos
Heparitina Sulfato/sangue , Pós-Menopausa/sangue , Pré-Menopausa/sangue , Proteoglicanas/sangue , Sulfatases/sangue , Sulfotransferases/sangue , Adulto , Idoso , Vias Biossintéticas/fisiologia , Feminino , Heparitina Sulfato/análise , Humanos , Pessoa de Meia-Idade , Proteoglicanas/análise , Sulfatases/análise , Sulfotransferases/análiseRESUMO
Tyrosine O-sulfation is a post-translational modification catalyzed by two tyrosylprotein sulfotransferases (TPST-1 and TPST-2) in the trans-Golgi network. Tpst2-deficient mice have male infertility, sperm motility defects, and possible abnormalities in sperm-egg membrane interactions. Studies here show that compared with wild-type sperm, fewer Tpst2-null sperm bind to the egg membrane, but more of these bound sperm progress to membrane fusion. Similar outcomes were observed with wild-type sperm treated with the anti-sulfotyrosine antibody PSG2. The increased extent of sperm-egg fusion is not due to a failure of Tpst2-null sperm to trigger establishment of the egg membrane block to polyspermy. Anti-sulfotyrosine staining of sperm showed localization similar to that of IZUMO1, a sperm protein that is essential for gamete fusion, but we detected little to no tyrosine sulfation of IZUMO1 and found that IZUMO1 expression and localization were normal in Tpst2-null sperm. Turning to a discovery-driven approach, we used mass spectrometry to characterize sperm proteins that associated with PSG2. This identified ADAM6, a member of the A disintegrin and A metalloprotease (ADAM) family; members of this protein family are associated with multiple sperm functions. Subsequent studies revealed that Tpst2-null sperm lack ADAM6 and ADAM3. Loss of ADAM3 is strongly associated with male infertility and is observed in knockouts of male germ line-specific endoplasmic reticulum-resident chaperones, raising the possibility that TPST-2 may function in quality control in the secretory pathway. These data suggest that TPST-2-mediated tyrosine O-sulfation participates in regulating the sperm surface proteome or membrane order, ultimately affecting male fertility.
Assuntos
Proteínas ADAM/metabolismo , Fusão de Membrana/fisiologia , Glicoproteínas de Membrana/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Interações Espermatozoide-Óvulo/fisiologia , Espermatozoides/enzimologia , Sulfotransferases/metabolismo , Proteínas ADAM/genética , Animais , Membrana Celular/genética , Membrana Celular/metabolismo , Epididimo/citologia , Epididimo/enzimologia , Feminino , Regulação da Expressão Gênica/fisiologia , Imunoglobulinas/genética , Imunoglobulinas/metabolismo , Infertilidade Masculina/enzimologia , Infertilidade Masculina/genética , Masculino , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Proteoma/genética , Proteoma/metabolismo , Espermatozoides/citologia , Sulfotransferases/genéticaRESUMO
Mucopolysaccharide (MPS) diseases are characterized by accumulation of glycosaminoglycans (GAGs) due to deficiencies in lysosomal enzymes responsible for GAG breakdown. Using a murine model of MPSI Hurler (MPSIH), we have quantified the heparan sulfate (HS) accumulation resulting from α-l-iduronidase (Idua) deficiency. HS levels were significantly increased in liver and brain tissue from 12-week-old Idua(-/-) mice by 87- and 20-fold, respectively. In addition, HS chains were shown to contain significantly increased N-, 2-O-, and 6-O-sulfation. Disaccharide compositional analyses also uncovered an HS disaccharide uniquely enriched in MPSIH, representing the terminal iduronic acid residue capping the non-reducing end of the HS chain, where no further degradation can occur in the absence of Idua. Critically, we identified that excess HS, some of which is colocalized to the Golgi secretory pathway, acts as a positive regulator of HS-sulfation, increasing the N-sulfotransferase activity of HS-modifying N-deacetylase/N-sulfotransferase enzymes. This mechanism may have severe implications during disease progression but, now identified, could help direct improved therapeutic strategies.
Assuntos
Complexo de Golgi/metabolismo , Heparitina Sulfato/metabolismo , Iduronidase , Mucopolissacaridose I/enzimologia , Sulfotransferases/metabolismo , Animais , Modelos Animais de Doenças , Complexo de Golgi/genética , Heparitina Sulfato/genética , Humanos , Ácido Idurônico/metabolismo , Camundongos , Camundongos Knockout , Mucopolissacaridose I/genética , Sulfotransferases/genéticaRESUMO
Mycobacteria, including the pathogen Mycobacterium tuberculosis, use the non-mammalian disaccharide trehalose as a precursor for essential cell-wall glycolipids and other metabolites. Here we describe a strategy for exploiting trehalose metabolic pathways to label glycolipids in mycobacteria with azide-modified trehalose (TreAz) analogues. Subsequent bioorthogonal ligation with alkyne-functionalized probes enabled detection and visualization of cell-surface glycolipids. Characterization of the metabolic fates of four TreAz analogues revealed unique labeling routes that can be harnessed for pathway-targeted investigation of the mycobacterial trehalome.
Assuntos
Mycobacterium/metabolismo , Trealose/química , Trealose/metabolismo , Alcinos/química , Azidas/química , Corantes Fluorescentes/química , Glicolipídeos/metabolismoRESUMO
The stoichiometry of protein phosphorylation significantly impacts protein function. The development of quantitative techniques in mass spectrometry has generated the ability to systematically monitor the regulation levels of various proteins. This study reports an integrated methodology using cerium oxide nanoparticles and isobaric tandem mass tag (TMT) labeling to assess absolute stoichiometries of protein phosphorylation. This protocol was designed to directly measure the dephosphorylation levels for a known phosphorylation site, therefore allowing for quantification of phosphosites. Both the accuracy and precision of the method were verified using standard peptides and protein tryptic digests. This novel method was then applied to quantify phosphorylations on eukaryotic initiation factor 3H (eIF3H), a protein integral to overall eukaryotic protein translation initiation. To date, this is the first report of assessment of protein phosphorylation quantification on eIF3.
Assuntos
Cério/química , Nanopartículas Metálicas/química , Fosfopeptídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Fator de Iniciação 3 em Eucariotos/metabolismo , FosforilaçãoRESUMO
Unique to ion mobility mass spectrometry (IM-MS) is the ability to provide collision cross section (CCS) data and the capacity to delineate any dissociation and/or unfolding of protein complexes. The strong correlation of the experimentally determined CCS with theory is indicative of the retention of native structure in the gas phase, which in turn, qualifies as a means in evaluating the IM-MS data. The assessment of IM-MS data, however, is currently impeded due to the lack of appropriate structural coordinates to use as input in the in silico calculation of theory. To address this issue, this study involves the use of rapid protein threading predictor (RAPTOR) to generate tertiary structures of closely related monomeric chemokines (MCP-1, MCP-3, MCP-4, and eotaxin) and, subsequently, utilize these models to estimate the theoretical values. Experimental CCS of both the model proteins and chemokines correlate well with theory generated by RAPTOR. All conformations for z = 5+ of chemokines fall within theoretical limits. Of the four chemokines, MCP-4 with z = 6+ appears to adopt an extended conformation, while eotaxin gradually unfolds, and the extended structures of MCP-1 and MCP-3 increase in abundance upon activation. Combining RAPTOR with IM-MS and collision-induced dissociation (CID) enables us to interrogate the conformations of homologous proteins with very similar tertiary structures.
Assuntos
Quimiocinas/química , Espectrometria de Massas , Estatística como Assunto/métodos , Algoritmos , Quimiocinas/metabolismo , Glicosaminoglicanos/metabolismo , Modelos Moleculares , Nanotecnologia , Estabilidade Proteica , Estrutura Terciária de Proteína , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
Heparin is a linear sulfated polysaccharide widely used in medicine because of its anticoagulant properties. The various sulfation and/or acetylation patterns on heparin impart different degrees of conformational change around the glycosidic bonds and subsequently alter its function as an anticoagulant, anticancer, or antiviral drug. Characterization of these structures is important for eventual elucidation of its function but presents itself as an analytical challenge due to the inherent heterogeneity of the carbohydrates. Heparin octasaccharide structural isomers of various sulfation patterns were investigated using ion mobility mass spectrometry (IMMS). In addition to distinguishing the isomers, we report the preparation and tandem mass spectrometry analysis for multiple sulfated or acetylated oligosaccharides. Herein, our data indicate that heparin octasaccharide isomers were separated on the basis of their structural conformations in the ion mobility cell. Subsequent to this separation, isomers were further distinguished using product ions resulting from tandem mass spectrometry. Overall, IMMS analysis was used to successfully characterize and separate individual isomers and subsequently measure their conformations.
Assuntos
Heparina/química , Sulfatos/química , Espectrometria de Massas em Tandem , Dissacarídeos/análise , Íons/química , Isomerismo , Conformação Molecular , Oligossacarídeos/químicaRESUMO
Eukaryotic translation requires a suite of proteins known as eukaryotic initiation factors (eIFs). These molecular effectors oversee the highly regulated initiation phase of translation. Essential to eukaryotic translation initiation is the protein eIF2, a heterotrimeric protein composed of the individually distinct subunits eIF2α, eIF2ß, and eIF2γ. The ternary complex, formed when eIF2 binds to GTP and Met-tRNA(i), is responsible for shuttling Met-tRNA(i) onto the awaiting 40S ribosome. As a necessary component for translation initiation, much attention has been given to the phosphorylation of eIF2α. Despite several previous investigations into eIF2 phosphorylation, most have centered on α- or ß-subunit phosphorylation and little is known regarding γ-subunit phosphorylation. Herein, we report eight sites of phosphorylation on the largest eIF2 subunit with seven novel phosphosite identifications via high resolution mass spectrometry. Of the eight sites identified, three are located in either the switch regions or nucleotide binding pocket domain. In addition, we have identified a possible kinase of eIF2, protein kinase C (PKC), which is capable of phosphorylating threonine 66 (thr-66) on the intact heterotrimer. These findings may shed new light on the regulation of ternary complex formation and alternate molecular effectors involved in this process prior to 80S ribosome formation and subsequent translation elongation and termination.
Assuntos
Fator de Iniciação 2 em Eucariotos/metabolismo , Proteína Quinase C/metabolismo , Proteômica/métodos , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Espectrometria de Massas/métodos , Modelos Biológicos , Fosforilação , Proteína Quinase C/química , Estrutura Terciária de Proteína , Proteoma , RNA de Transferência/metabolismo , Ribossomos/metabolismo , Treonina/químicaRESUMO
The analysis of heparan sulfate glycosaminoglycans (HSGAGs) variations in human serum at the disaccharide level has a great potential for disease diagnosis and prognosis. However, the lack of available analytical methodology for the compositional analysis of HSGAGs in human serum remains to be addressed to delineate the possible role of HSGAGs on the onset and/or progression of a disease. In this study, we have developed a method for the in-depth compositional analysis of the 12 heparin/HS-derived disaccharides from human serum using a combination of technologies--fractionation, exhaustive digestion, solid phase extraction, and LC-MS/MS. The method exhibits high recovery (72-110%) and good reproducibility (standard deviation of less than 5%) with a low limit of detection and quantification. Errors from the method validation were within 1.1%. Nondetectable non- or low-sulfated disaccharides in human serum were also detected using the optimized protocol. Further applying this method, the comprehensive analysis of HSGAGs compositions in human sera from female donors showed considerable variations in disaccharide patterns and compositions.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Dissacarídeos/sangue , Heparina/química , Heparitina Sulfato/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Adulto , Dissacarídeos/isolamento & purificação , Feminino , Heparina/sangue , Heparitina Sulfato/sangue , Humanos , Masculino , Extração em Fase Sólida/métodosRESUMO
Heparin interacts with many proteins and is involved in biological processes such as anticoagulation, angiogenesis, and antitumorigenic activities. These heparin-protein interactions can be influenced by the binding of various metal ions to these complexes. In particular, physiologically relevant metal cations influence heparin-protein conformations through electronic interactions inherent to this polyanion. In this study, we employed ion mobility mass spectrometry (IMMS) to observe conformational changes that occur in fully-sulfated heparin octasaccharides after the successive addition of metal ions. Our results indicate that binding of positive counter ions causes a decrease in collision cross section (CCS) measurements, thus promoting a more compact octasaccharide structure.
RESUMO
Mycothiol (MSH), the primary low-molecular weight thiol produced in mycobacteria, acts to protect the cell from oxidative stress and to maintain redox homeostasis, notably in the pathogenic Mycobacterium tuberculosis in the course of human infection. The mycothiol disulfide reductase (Mtr) enzyme reduces the oxidized form of mycothiol, mycothione (MSSM), back to MSH, however its role in bacterial viability is not clear. In this study, we sought to determine the MSH levels of wild-type (WT) and Mtr mutant mycobacteria during oxidative stress. We describe a rapid method for the relative quantification of MSH using high-sensitivity mass spectrometry (MS) with selected ion monitoring (SIM). This method uses only minimal sample cleanup, and does not require advanced chromatographic equipment or fluorescent compounds. MSH levels decreased in the Mtr mutant only upon treatment with peroxide, and the results were consistent between our method and previously-described thiol quantification methods. Our results indicate that our MS-based method is a useful, high-throughput alternative tool for the quantification of MSH from mycobacteria.
RESUMO
The eukaryotic initiation factor 3 (eIF3) plays an important role in translation initiation, acting as a docking site for several eIFs that assemble on the 40S ribosomal subunit. Here, we use mass spectrometry to probe the subunit interactions within the human eIF3 complex. Our results show that the 13-subunit complex can be maintained intact in the gas phase, enabling us to establish unambiguously its stoichiometry and its overall subunit architecture via tandem mass spectrometry and solution disruption experiments. Dissociation takes place as a function of ionic strength to form three stable modules eIF3(c:d:e:l:k), eIF3(f:h:m), and eIF3(a:b:i:g). These modules are linked by interactions between subunits eIF3b:c and eIF3c:h. We confirmed our interaction map with the homologous yeast eIF3 complex that contains the five core subunits found in the human eIF3 and supplemented our data with results from immunoprecipitation. These results, together with the 27 subcomplexes identified with increasing ionic strength, enable us to define a comprehensive interaction map for this 800-kDa species. Our interaction map allows comparison of free eIF3 with that bound to the hepatitis C virus internal ribosome entry site (HCV-IRES) RNA. We also compare our eIF3 interaction map with related complexes, containing evolutionarily conserved protein domains, and reveal the location of subunits containing RNA recognition motifs proximal to the decoding center of the 40S subunit of the ribosome.