RESUMEN
Soluble guanylyl cyclase (sGC) is the receptor for nitric oxide and a highly sought-after therapeutic target for the management of cardiovascular diseases. New compounds that stimulate sGC show clinical promise, but where these stimulator compounds bind and how they function remains unknown. Here, using a photolyzable diazirine derivative of a novel stimulator compound, IWP-051, and MS analysis, we localized drug binding to the ß1 heme domain of sGC proteins from the hawkmoth Manduca sexta and from human. Covalent attachments to the stimulator were also identified in bacterial homologs of the sGC heme domain, referred to as H-NOX domains, including those from Nostoc sp. PCC 7120, Shewanella oneidensis, Shewanella woodyi, and Clostridium botulinum, indicating that the binding site is highly conserved. The identification of photoaffinity-labeled peptides was aided by a signature MS fragmentation pattern of general applicability for unequivocal identification of covalently attached compounds. Using NMR, we also examined stimulator binding to sGC from M. sexta and bacterial H-NOX homologs. These data indicated that stimulators bind to a conserved cleft between two subdomains in the sGC heme domain. L12W/T48W substitutions within the binding pocket resulted in a 9-fold decrease in drug response, suggesting that the bulkier tryptophan residues directly block stimulator binding. The localization of stimulator binding to the sGC heme domain reported here resolves the longstanding question of where stimulators bind and provides a path forward for drug discovery.
Asunto(s)
Bacterias/enzimología , Proteínas Bacterianas/química , Hemo/química , Mutación Missense , Guanilil Ciclasa Soluble/química , Sustitución de Aminoácidos , Bacterias/genética , Proteínas Bacterianas/genética , Sitios de Unión , Hemo/genética , Resonancia Magnética Nuclear Biomolecular , Dominios Proteicos , Guanilil Ciclasa Soluble/genéticaRESUMEN
Nitrile hydratases (NHases) possess a mononuclear iron or cobalt cofactor whose coordination environment includes rare post-translationally oxidized cysteine sulfenic and sulfinic acid ligands. This cofactor is located in the α-subunit at the interfacial active site of the heterodimeric enzyme. Unlike canonical NHases, toyocamycin nitrile hydratase (TNHase) from Streptomyces rimosus is a unique three-subunit member of this family involved in the biosynthesis of pyrrolopyrimidine antibiotics. The subunits of TNHase are homologous to the α- and ß-subunits of prototypical NHases. Herein we report the expression, purification, and characterization of the α-subunit of TNHase. The UV-visible, EPR, and mass spectra of the α-subunit TNHase provide evidence that this subunit alone is capable of synthesizing the active site complex with full post-translational modifications. Remarkably, the isolated post-translationally modified α-subunit is also catalytically active with the natural substrate, toyocamycin, as well as the niacin precursor 3-cyanopyridine. Comparisons of the steady state kinetic parameters of the single subunit variant to the heterotrimeric protein clearly show that the additional subunits impart substrate specificity and catalytic efficiency. We conclude that the α-subunit is the minimal sequence needed for nitrile hydration providing a simplified scaffold to study the mechanism and post-translational modification of this important class of catalysts.
Asunto(s)
Proteínas Bacterianas/metabolismo , Hidroliasas/metabolismo , Actinomycetales/enzimología , Proteínas Bacterianas/química , Catálisis , Hidroliasas/química , Cinética , Procesamiento Proteico-Postraduccional , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Piridinas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Soluble guanylyl/guanylate cyclase (sGC) converts GTP to cGMP after binding nitric oxide, leading to smooth muscle relaxation and vasodilation. Impaired sGC activity is common in cardiovascular disease, and sGC stimulatory compounds are vigorously sought. sGC is a 150 kDa heterodimeric protein with two H-NOX domains (one with heme, one without), two PAS domains, a coiled-coil domain, and two cyclase domains. Binding of NO to the sGC heme leads to proximal histidine release and stimulation of catalytic activity. To begin to understand how binding leads to activation, we examined truncated sGC proteins from Manduca sexta (tobacco hornworm) that bind NO, CO, and stimulatory compound YC-1 but lack the cyclase domains. We determined the overall shape of truncated M. sexta sGC using analytical ultracentrifugation and small-angle X-ray scattering (SAXS), revealing an elongated molecule with dimensions of 115 Å × 90 Å × 75 Å. Binding of NO, CO, or YC-1 had little effect on shape. Using chemical cross-linking and tandem mass spectrometry, we identified 20 intermolecular contacts, allowing us to fit homology models of the individual domains into the SAXS-derived molecular envelope. The resulting model displays a central parallel coiled-coil platform upon which the H-NOX and PAS domains are assembled. The ß1 H-NOX and α1 PAS domains are in contact and form the core signaling complex, while the α1 H-NOX domain can be removed without a significant effect on ligand binding or overall shape. Removal of 21 residues from the C-terminus yields a protein with dramatically increased proximal histidine release rates upon NO binding.
Asunto(s)
Reactivos de Enlaces Cruzados/química , Guanilato Ciclasa/química , Guanilato Ciclasa/metabolismo , Manduca/enzimología , Receptores Citoplasmáticos y Nucleares/química , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Monóxido de Carbono/metabolismo , Indazoles/metabolismo , Manduca/química , Manduca/metabolismo , Espectrometría de Masas , Modelos Moleculares , Óxido Nítrico/metabolismo , Unión Proteica , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Dispersión del Ángulo Pequeño , Guanilil Ciclasa Soluble , Homología Estructural de Proteína , Ultracentrifugación , Difracción de Rayos XRESUMEN
Ixodes scapularis Say, 1821 larvae were fed on mice and allowed to molt under laboratory conditions. A liquid chromatography-tandem mass spectrometry-based proteomic study was conducted to identify the type of mammalian proteins present in the derived nymphal ticks at different time intervals after molting. Albumin was present for 85 d; transferrin was present for 29 d; and, more importantly, hemoglobin remained detectable for up to 309 d postmolting. Peptides of actin, keratin, and tubulin are highly similar between mouse and tick, and therefore, unambiguous assignment of these proteins to different species was not possible. Establishing a time line for the persistence of hemoglobin, one of the most abundant blood proteins, at detectable levels in ticks after the bloodmeal and molting advances our efforts to use this protein to identify the host species.
Asunto(s)
Proteínas Sanguíneas/metabolismo , Ixodes/fisiología , Ratones/parasitología , Secuencia de Aminoácidos , Animales , Cromatografía Liquida , Femenino , Ixodes/crecimiento & desarrollo , Larva/crecimiento & desarrollo , Larva/fisiología , Ratones/metabolismo , Ninfa/crecimiento & desarrollo , Ninfa/fisiología , Espectrometría de Masas en Tándem , Factores de TiempoRESUMEN
The humoral immune response plays an important role in the clearance of Giardia lamblia. However, our knowledge about the specific antigens of G. lamblia that induce a protective immune response is limited. The purpose of this study was to identify and characterise the immunogenic proteins of G. lamblia in a mouse model. We generated monoclonal antibodies (moAbs) specific to G. lamblia (1B10, 2C9.D11, 3C10.E5, 3D10, 5G8.B5, 5F4, 4C7, 3C5 and 3C6) by fusing splenocytes derived from infected mice. Most of these moAbs recognised a band of ± 71 kDa (5G8 protein) and this protein was also recognised by serum from the infected mice. We found that the moAbs recognised conformational epitopes of the 5G8 protein and that this antigen is expressed on the cell surface and inside trophozoites. Additionally, antibodies specific to the 5G8 protein induced strong agglutination (> 70-90%) of trophozoites. We have thus identified a highly immunogenic antigen of G. lamblia that is recognised by the immune system of infected mice. In summary, this study describes the identification and partial characterisation of an immunogenic protein of G. lamblia. Additionally, we generated a panel of moAbs specific for this protein that will be useful for the biochemical and immunological characterisation of this immunologically interesting Giardia molecule.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/inmunología , Giardia lamblia/inmunología , Proteínas Protozoarias/inmunología , Animales , Western Blotting , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Giardiasis/inmunología , Giardiasis/parasitología , RatonesRESUMEN
The main objectives of this work were to obtain a more extensive coverage of the Burkholderia vietnamiensis proteome than previously reported and to identify virulence factors using tandem mass spectrometry. The proteome of B. vietnamiensis was precipitated into four fractions to as extracellular, intracellular, cell surface and cell wall proteins. Two different approaches were used to analyze the proteins. The first was a gel-based method where 1D SDS-PAGE was used for separation of the proteins prior to reverse phase liquid chromatography tandem mass spectrometry (LC-MS/MS). The second method used MudPIT analysis (Multi dimensional Protein Identification Technique), where proteins are digested and separated using cation exchange and reversed phase separations before the MS/MS analysis (LC/LC-MS/MS). Overall, gel-based LC-MS/MS analysis resulted in more protein identifications than the MudPIT analysis. Combination of the results lead to identification of more than 1200 proteins, approximately 16% of the proteins coded from the annotated genome of Burkholderia species. Several virulence factors were detected including flagellin, porin, peroxiredoxin and zinc proteases.
Asunto(s)
Proteínas Bacterianas/análisis , Proteínas Bacterianas/metabolismo , Burkholderia/química , Burkholderia/metabolismo , Proteoma/análisis , Proteómica/métodos , Cromatografía Liquida , Electroforesis en Gel de Poliacrilamida , Espectrometría de Masas en TándemRESUMEN
Pholcid spiders (Araneae: Pholcidae), officially "cellar spiders" but popularly known as "daddy long-legs," are renown for the potential of deadly toxic venom, even though venom composition and potency has never formally been studied. Here we detail the venom composition of male Physocyclus mexicanus using proteomic analyses and venom-gland transcriptomes ("venomics"). We also analyze the venom's potency on insects, and assemble available evidence regarding mammalian toxicity. The majority of the venom (51% of tryptic polypeptides and 62% of unique tryptic peptides) consists of proteins homologous to known venom toxins including enzymes (astacin metalloproteases, serine proteases and metalloendopeptidases, particularly neprilysins) and venom peptide neurotoxins. We identify 17 new groups of peptides (U1-17-PHTX) most of which are homologs of known venom peptides and are predicted to have an inhibitor cysteine knot fold; of these, 13 are confirmed in the proteome. Neprilysins (M13 peptidases), and astacins (M12 peptidases) are the most abundant venom proteins, respectively representing 15 and 11% of the individual proteins and 32 and 20% of the tryptic peptides detected in crude venom. Comparative evidence suggests that the neprilysin gene family is expressed in venoms across a range of spider taxa, but has undergone an expansion in the venoms of pholcids and may play a central functional role in these spiders. Bioassays of crude venoms on crickets resulted in an effective paralytic dose of 3.9 µg/g, which is comparable to that of crude venoms of Plectreurys tristis and other Synspermiata taxa. However, crickets exhibit flaccid paralysis and regions of darkening that are not observed after P. tristis envenomation. Documented bites on humans make clear that while these spiders can bite, the typical result is a mild sting with no long-lasting effects. Together, the evidence we present indicates pholcid venoms are a source of interesting new peptides and proteins, and effects of bites on humans and other mammals are inconsequential.
RESUMEN
In this chapter we present a detailed protocol for the large-scale identification of proteins present in rice leaf and root tissue samples using 2D liquid chromatography-tandem mass spectrometry of protein extracts. This is performed using biphasic (strong cation exchange/reversed phase) columns with integral electrospray emitters operating at nanoliter flow rates, a technique known by the acronym Mudpit (for multidimensional protein identification technique). The protocol involves harvesting of leaves and roots from rice plants, preparing protein extracts from the harvested tissues, preparing proteolytic digests of the extracted proteins, making a biphasic capillary column with an integral electrospray emitter, performing two-dimensional chromatographic separation of peptides with data-dependent tandem mass spectrometry, and the use of database searching of the acquired tandem mass spectra to identify peptides and proteins. This protocol is adaptable for use with a wide variety of plant materials and can be used to identify large numbers of proteins present in a specific tissue, organ, organelle, or other subcellular fraction. In addition to the detailed protocol, we also present the results of a representative experiment showing the identification of more than 1000 distinct proteins from rice leaf and root samples in two Mupdit experiments.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Oryza/química , Proteínas de Plantas/análisis , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Acetona , Precipitación Química , Bases de Datos de Proteínas , Indicadores y Reactivos , Nanotecnología , Péptido Hidrolasas , Hojas de la Planta/química , Proteínas de Plantas/química , Proteínas de Plantas/aislamiento & purificación , Raíces de Plantas/química , Proteómica/instrumentación , Espectrometría de Masa por Ionización de Electrospray/instrumentación , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/instrumentación , Ácido TricloroacéticoRESUMEN
The aim of this project was to investigate the molecular mechanisms of shade-avoidance response in tomato ( Solanum lycopersicum) plants. Plants were grown in direct sunlight in ambient temperature and in an adjacent environment under shade cloth. Leaves were harvested, and protein expression differences were investigated using two-dimensional differential in-gel electrophoresis and nanoflow high-performance liquid chromatography-tandem mass spectrometry. Striking differences in plant physiology and protein expression were observed. Plants grown in the shade grew very tall but bore almost no fruit and displayed a dramatic reduction in the accumulation of Rubisco and a number of other metabolic enzymes. We have identified, quantified, and classified 59 protein features found to be up- or down-regulated as part of a shade-avoidance response in S. lycopersicum and correlated these with phenotypic data. A large group of proteins related to metabolism and respiration were greatly reduced in accumulation in shade-grown plants, and there was also evidence of significant proteolysis occurring. Four stress-related proteins appear to be constitutively expressed as a result of heat acclimation, while three distinct stress-related proteins appear to accumulate as part of the shade-avoidance response. The identification and functional classification of all 59 differentially accumulating proteins is presented and discussed.
Asunto(s)
Hojas de la Planta/química , Proteínas de Plantas/análisis , Solanum lycopersicum/química , Solanum lycopersicum/crecimiento & desarrollo , Luz Solar , AmbienteRESUMEN
Humoral and cellular immune responses play an important role during Giardia lamblia infection. Several Giardia proteins have been identified as immunogenic antigens based on their elicited humoral immune response. Poorly is known about Giardia antigens that stimulate a cellular immune response. The main purpose of this study was to isolate and partial characterize an immunogenic antigen (5G8) of G. lamblia. The 5G8 protein was isolated from G. lamblia trophozoite lysates by affinity chromatography using moAb 5G8-coupled CNBr-Sepharose. The isolated protein was analysed by electrospray tandem mass spectrometry (ESI-MS/MS), and by diverse bioinformatics tools (GiardiaDB, BLASTn, BLASTp and ExPASy). Additionally, several biochemical and immunological characteristics of the isolated protein were analysed. By ESI-MS/MS the amino acidic 5G8 sequence was deduced. The 5G8 antigen belongs to the VSP family proteins of G. lamblia. This protein is composed by one polypeptide chain (±71kDa). Using the algorithm SYFPHEITI, we identified candidate CD4+ T-cell epitopes from the 5G8 antigen, which can elicit cell-mediated immune responses. In this study, we have identified a G. lamblia protein that induces a strong immune response in infected mice. The biochemical and immunological characterization of the immunogenic 5G8 antigen may contribute to the rational design of a Giardia vaccine.
Asunto(s)
Antígenos de Protozoos/química , Antígenos de Protozoos/inmunología , Giardia lamblia/inmunología , Secuencia de Aminoácidos , Animales , Antígenos de Protozoos/administración & dosificación , Antígenos de Protozoos/genética , Antígenos de Protozoos/aislamiento & purificación , Epítopos de Linfocito B/química , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito B/aislamiento & purificación , Epítopos de Linfocito T/química , Epítopos de Linfocito T/inmunología , Epítopos de Linfocito T/aislamiento & purificación , Inmunidad Celular , Inmunidad Humoral , Proteínas de la Membrana/genética , Ratones , Proteínas Protozoarias/administración & dosificación , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Proteínas Protozoarias/aislamiento & purificación , Proteínas Protozoarias/metabolismo , Espectrometría de Masas en Tándem , Trofozoítos/inmunologíaRESUMEN
Data produced from the MudPIT analysis of yeast (S. cerevisiae) and rice (O. sativa) were used to develop a technique to validate single-peptide protein identifications using complementary database search algorithms. This results in a considerable reduction of overall false-positive rates for protein identifications; the overall false discovery rates in yeast are reduced from near 25% to less than 1%, and the false discovery rate of yeast single-peptide protein identifications becomes negligible. This technique can be employed by laboratories utilizing a SEQUEST-based proteomic analysis platform, incorporating the XTandem algorithm as a complementary tool for verification of single-peptide protein identifications. We have achieved this using open-source software, including several data-manipulation software tools developed in our laboratory, which are freely available to download.
Asunto(s)
Algoritmos , Bases de Datos de Proteínas , Péptidos/análisis , Proteínas/análisis , Oryza/química , Péptidos/química , Proteínas de Plantas/análisis , Proteínas de Plantas/química , Proteínas/químicaRESUMEN
We describe in this communication a set of functional perl script utilities for use in peptide mass spectral database searching and proteomics experiments, known as the Wildcat Toolbox. These are all freely available for download from our laboratory Web site (http://proteomics.arizona.edu/toolbox.html) as a combined zip file, and can also be accessed via the Proteome Commons Web site (www.proteomecommons.org) in the tools section. We make them available to other potential users in the spirit of open source software development; we do not have the resources to provide any significant technical support for them, but we hope users will share both bugs and improvements with the community at large.
Asunto(s)
Biología Computacional/métodos , Espectrometría de Masas/métodos , Proteómica/métodos , Algoritmos , Animales , Sistemas de Administración de Bases de Datos , Bases de Datos de Proteínas , Humanos , Almacenamiento y Recuperación de la Información , Internet , Lenguajes de Programación , Análisis de Secuencia de Proteína , Programas InformáticosRESUMEN
Spider venoms are complex cocktails rich in peptides, proteins and organic molecules that collectively act to immobilize prey. Venoms of the primitive hunting spider, Plectreurys tristis, have numerous neurotoxic peptides called "plectoxins" (PLTX), a unique acylpolyamine called bis(agmatine)oxalamide, and larger unidentified protein components. These spiders also have unconventional multi-lobed venom glands. Inspired by these unusual characteristics and their phylogenetic position as Haplogynes, we have partially characterized the venome of P. tristis using combined transcriptomic and proteomic methods. With these analyses we found known venom neurotoxins U1-PLTX-Pt1a, U3-PLTX-Pt1a, and we discovered new groups of potential neurotoxins, expanding the U1- and ω-PLTX families and adding U4-through U9-PLTX as six new groups. The venom also contains proteins that are homologs of astacin metalloproteases that, combined with venom peptides, make up 94% of components detected in crude venom, while the remaining 6% is a single undescribed protein with unknown function. Other proteins detected in the transcriptome were found to be members of conserved gene families and make up 20% of the transcripts. These include cDNA sequences that match venom proteins from Mesobuthus and Hottentotta scorpions, Loxosceles and Dysdera spiders, and also salivary and secreted peptide sequences from Ixodes, Amblyomma and Rhipicephalus ticks. Finally, we show that crude venom has neurotoxic effects and an effective paralytic dose on crickets of 3.3µg/gm.
RESUMEN
The fragmentation behavior of a set of model peptides containing proline, its four-membered ring analog azetidine-2-carboxylic acid (Aze), its six-membered ring analog pipecolic acid (Pip), an acyclic secondary amine residue N-methyl-alanine (NMeA), and the D stereoisomers of Pro and Pip has been determined using collision-induced dissociation in ESI-tandem mass spectrometers. Experimental results for AAXAA, AVXLG, AAAXA, AGXGA, and AXPAA peptides are presented, where X represents Pro, Aze, Pip, or NMeA. Aze- and Pro-containing peptides fragment according to the well-established "proline effect" through selective cleavage of the amide bond N-terminal to the Aze/Pro residue to give yn (+) ions. In contrast, Pip- and NMA-fragment through a different mechanism, the "pipecolic acid effect," selectively at the amide bond C-terminal to the Pip/NMA residue to give bn (+) ions. Calculations of the relative basicities of various sites in model peptide molecules containing Aze, Pro, Pip, or NMeA indicate that whereas the "proline effect' can in part be rationalized by the increased basicity of the prolyl-amide site, the "pipecolic acid effect" cannot be justified through the basicity of the residue. Rather, the increased flexibility of the Pip and NMeA residues allow for conformations of the peptide for which transfer of the mobile proton to the amide site C-terminal to the Pip/NMeA becomes energetically favorable. This argument is supported by the differing results obtained for AAPAA versus AA(D-Pro)AA, a result that can best be explained by steric effects. Fragmentation of pentapeptides containing both Pro and Pip indicate that the "pipecolic acid effect" is stronger than the "proline effect."
Asunto(s)
Fragmentos de Péptidos/química , Ácidos Pipecólicos/química , Prolina/química , Espectrometría de Masas en Tándem/métodos , Iones/químicaRESUMEN
The humoral immune response plays an important role in the clearance of Giardia lamblia. However, our knowledge about the specific antigens of G. lamblia that induce a protective immune response is limited. The purpose of this study was to identify and characterise the immunogenic proteins of G. lamblia in a mouse model. We generated monoclonal antibodies (moAbs) specific to G. lamblia (1B10, 2C9.D11, 3C10.E5, 3D10, 5G8.B5, 5F4, 4C7, 3C5 and 3C6) by fusing splenocytes derived from infected mice. Most of these moAbs recognised a band of ± 71 kDa (5G8 protein) and this protein was also recognised by serum from the infected mice. We found that the moAbs recognised conformational epitopes of the 5G8 protein and that this antigen is expressed on the cell surface and inside trophozoites. Additionally, antibodies specific to the 5G8 protein induced strong agglutination (> 70-90%) of trophozoites. We have thus identified a highly immunogenic antigen of G. lamblia that is recognised by the immune system of infected mice. In summary, this study describes the identification and partial characterisation of an immunogenic protein of G. lamblia. Additionally, we generated a panel of moAbs specific for this protein that will be useful for the biochemical and immunological characterisation of this immunologically interesting Giardia molecule.
Asunto(s)
Animales , Ratones , Anticuerpos Monoclonales/inmunología , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/inmunología , Giardia lamblia/inmunología , Proteínas Protozoarias/inmunología , Western Blotting , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Giardiasis/inmunología , Giardiasis/parasitologíaRESUMEN
We have investigated the use of a variety of different techniques to identify as many proteins as possible in a yeast lysate, with the aim of investigating the overlap and complementarity of data from different approaches. A standard lysate was prepared from log phase yeast (Saccharomyces cerevisiae). This was then subjected to analysis via five different approaches aimed at identifying as many proteins as possible using an ion trap mass spectrometer. The total number of non-redundant protein identifications from each experiment was: 524 proteins by 2-D (SCX/C18) nanoflow liquid chromatography-liquid chromatography tandem mass spectrometry (nanoLC-LC MS/MS (MudPIT)); 381 proteins by nanoLC-MS/MS with gas phase fractionation by mass range selection; 390 proteins by nanoLC-MS/MS with gas phase fractionation by ion abundance selection; 898 proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation of proteins, in-gel digestion, and nanoLC-MS/MS of gel slices; and 422 proteins by isoelectric focusing of proteins, in-gel digestion and nanoLC-MS/MS of gel slices. The total number of non-redundant protein identifications in the five experiments was 1204. Combining only the two best experiments, the SDS-PAGE gel slices and the Mudpit, produces 1024 proteins identified, more than 85% of the total. Clearly, combining a Mudpit analysis with an SDS-PAGE gel slice experiment gives the greatest amount of protein identification information from a limited amount of sample.
Asunto(s)
Electroforesis en Gel de Poliacrilamida , Cromatografía de Gases y Espectrometría de Masas , Focalización Isoeléctrica , Proteómica , Proteínas de Saccharomyces cerevisiae/análisis , Fraccionamiento Químico , Cromatografía Líquida de Alta Presión , Espectrometría de Masas , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/crecimiento & desarrollo , Tripsina/farmacologíaRESUMEN
MS/MS techniques in proteomics make possible the identification of proteins from organisms with little or no genome sequence information available. Peptide sequences are obtained from tandem mass spectra by matching peptide mass and fragmentation information to protein sequence information from related organisms, including unannotated genome sequence data. This peptide identification data can then be grouped and reconstructed into protein data. In this study, we have used this approach to study protein secretion by Aspergillus flavus, a filamentous fungus for which very little genome sequence information is available. A. flavus is capable of degrading the flavonoid rutin (quercetin 3-O-glycoside), as the only source of carbon via an extracellular enzyme system. In this continuing study, a proteomic analysis was used to identify secreted proteins from A. flavus when grown on rutin. The growth media glucose and potato dextrose were used to identify differentially expressed secreted proteins. The secreted proteins were analyzed by 1- and 2-DE and MS/MS. A total of 51 unique A. flavus secreted proteins were identified from the three growth conditions. Ten proteins were unique to rutin-, five to glucose- and one to potato dextrose-grown A. flavus. Sixteen secreted proteins were common to all three media. Fourteen identifications were of hypothetical proteins or proteins of unknown functions. To our knowledge, this is the first extensive proteomic study conducted to identify the secreted proteins from a filamentous fungus.
Asunto(s)
Aspergillus flavus/metabolismo , Proteínas/química , Carbono/metabolismo , Bases de Datos de Proteínas , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Proteínas Fúngicas/química , Genes Fúngicos , Glucosa/química , Focalización Isoeléctrica , Espectrometría de Masas , Proteínas/metabolismo , Proteómica/métodos , Rutina/metabolismo , Factores de TiempoRESUMEN
Fragmentation at the Xxx-Pro bond was analyzed for a group of peptide mass spectra that were acquired in a Finnigan ion trap mass spectrometer and were generated from proteins digested by enzymes and identified by the Sequest algorithm. Cleavage with formation of a + b + y ions occurred more readily at the Xxx-Pro bond than at other locations in these peptides, and the importance of this cleavage varied by the identity of Xxx. The most abundant Xxx-Pro relative bond cleavage ratios were observed when Xxx was Val, His, Asp, Ile, and Leu, whereas the least abundant cleavage ratios occurred when Xxx was Gly or Pro. Rationalization for these cleavage ratios at Xxx-Pro may include contribution of the Asp or His side chain to enhanced cleavage or the conformation of Pro, Gly, and the aliphatic residues Val, Ile, and Leu at the Xxx location in the Xxx-Pro bond. Although unusual fragmentation behavior has been noted for Pro-containing peptides, this analysis suggests that fragmentation at the Xxx-Pro bond is predictable and that this information may be used to improve the identification of proteins if it is incorporated into peptide sequencing algorithms.
Asunto(s)
Péptidos/química , Prolina/química , Secuencia de Aminoácidos , Cromatografía de Gases y Espectrometría de Masas , Datos de Secuencia Molecular , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
A series of gas-phase experiments and extensive theoretical modeling was done on the family of singly protonated peptides AARAA, Ac-AARAA, and AARAA-OMe. (AARAA)H(+) underwent extensive H/D exchange with D(2)O, whereas the other two peptides with blocked termini did not, implying that a salt bridge was involved in the H/D exchange process. Ion mobility measurements and complementary molecular modeling unambiguously identified the 300 K structures of all three protonated peptides as charge solvation structures, not salt bridges. High-level density functional theory calculations indicated the global minimum of (AARAA)H(+) was a charge solvation structure with the lowest-energy salt bridge structure 4.8 kcal/mol higher in energy. Uptake of the first five water molecules of hydration at 260 K showed near identical propensities for all three peptides consistent with a common structural motif. Quantitative measurements of Delta H degrees and Delta S degrees for the first two waters of hydration were very similar for all three peptides, again suggestive of a common structure. A detailed search of the potential energy surface for the singly hydrated (AARAA)H(+) using molecular mechanics and density functional theory approaches indicated a charge solvation structure was the global minimum, but now the lowest-energy salt bridge structure was only 1.8 kcal/mol higher in energy. Importantly, a low-energy transition state connecting the charge solvation and the salt bridge structures was found where the D(2)O molecule facilitated H/D exchange via the relay mechanism. This "relay" transition state was 7 kcal/mol below the (AARAA)H(+) + D(2)O asymptotic energy, suggesting that facile H/D exchange could occur in this system. There was no equivalent low-lying relay mechanism transition state for the (Ac-AARAA)H(+) and (AARAA-OMe)H(+) peptides, consistent with the fact that H/D exchange was not observed. Hence, the combined experimental and theoretical methods confirmed that a salt bridge was involved in the H/D exchange by D(2)O of (AARAA)H(+), but it existed only as a kinetic intermediate, not as a global minimum structure. These findings suggest that caution must be observed in drawing structural conclusions from H/D exchange only. A prescription is given here for understanding both the structural and H/D exchange mechanistic aspects of bare and singly hydrated peptides.
Asunto(s)
Oligopéptidos/química , Agua/química , Medición de Intercambio de Deuterio , Enlace de Hidrógeno , Cinética , Espectrometría de Masas , Modelos MolecularesRESUMEN
Collision-induced dissociation (CID) is a common ion activation technique used to energize mass-selected peptide ions during tandem mass spectrometry. Characteristic fragment ions form from the cleavage of amide bonds within a peptide undergoing CID, allowing the inference of its amino acid sequence. The statistical characterization of these fragment ions is essential for improving peptide identification algorithms and for understanding the complex reactions taking place during CID. An examination of 1465 ion trap spectra from doubly charged tryptic peptides reveals several trends important to understanding this fragmentation process. While less abundant than y ions, b ions are present in sufficient numbers to aid sequencing algorithms. Fragment ions exhibit a characteristic series-specific relationship between their masses and intensities. Each residue influences fragmentation at adjacent amide bonds, with Pro quantifiably enhancing cleavage at its N-terminal amide bond and His increasing the formation of b ions at its C-terminal amide bond. Fragment ions corresponding to a formal loss of ammonia appear preferentially in peptides containing Gln and Asn. These trends are partially responsible for the complexity of peptide tandem mass spectra.