RESUMO
We describe deep analysis of the human proteome in less than 1 h. We achieve this expedited proteome characterization by leveraging state-of-the-art sample preparation, chromatographic separations, and data analysis tools, and by using the new Orbitrap Astral mass spectrometer equipped with a quadrupole mass filter, a high-field Orbitrap mass analyzer, and an asymmetric track lossless (Astral) mass analyzer. The system offers high tandem mass spectrometry acquisition speed of 200 Hz and detects hundreds of peptide sequences per second within data-independent acquisition or data-dependent acquisition modes of operation. The fast-switching capabilities of the new quadrupole complement the sensitivity and fast ion scanning of the Astral analyzer to enable narrow-bin data-independent analysis methods. Over a 30-min active chromatographic method consuming a total analysis time of 56 min, the Q-Orbitrap-Astral hybrid MS collects an average of 4319 MS1 scans and 438,062 tandem mass spectrometry scans per run, producing 235,916 peptide sequences (1% false discovery rate). On average, each 30-min analysis achieved detection of 10,411 protein groups (1% false discovery rate). We conclude, with these results and alongside other recent reports, that the 1-h human proteome is within reach.
Assuntos
Proteoma , Proteômica , Espectrometria de Massas em Tandem , Humanos , Proteoma/análise , Proteômica/métodos , Fatores de TempoRESUMO
The growing trend toward high-throughput proteomics demands rapid liquid chromatography-mass spectrometry (LC-MS) cycles that limit the available time to gather the large numbers of MS/MS fragmentation spectra required for identification. Orbitrap analyzers scale performance with acquisition time and necessarily sacrifice sensitivity and resolving power to deliver higher acquisition rates. We developed a new mass spectrometer that combines a mass-resolving quadrupole, the Orbitrap, and the novel Asymmetric Track Lossless (Astral) analyzer. The new hybrid instrument enables faster acquisition of high-resolution accurate mass (HRAM) MS/MS spectra compared with state-of-the-art mass spectrometers. Accordingly, new proteomics methods were developed that leverage the strengths of each HRAM analyzer, whereby the Orbitrap analyzer performs full scans with a high dynamic range and resolution, synchronized with the Astral analyzer's acquisition of fast and sensitive HRAM MS/MS scans. Substantial improvements are demonstrated over previous methods using current state-of-the-art mass spectrometers.
RESUMO
Adaptive behaviour necessitates the formation of memories for fearful events, but also that these memories can be extinguished. Effective extinction prevents excessive and persistent reactions to perceived threat, as can occur in anxiety and 'trauma- and stressor-related' disorders1. However, although there is evidence that fear learning and extinction are mediated by distinct neural circuits, the nature of the interaction between these circuits remains poorly understood2-6. Here, through a combination of in vivo calcium imaging, functional manipulations, and slice physiology, we show that distinct inhibitory clusters of intercalated neurons (ITCs) in the mouse amygdala exert diametrically opposed roles during the acquisition and retrieval of fear extinction memory. Furthermore, we find that the ITC clusters antagonize one another through mutual synaptic inhibition and differentially access functionally distinct cortical- and midbrain-projecting amygdala output pathways. Our findings show that the balance of activity between ITC clusters represents a unique regulatory motif that orchestrates a distributed neural circuitry, which in turn regulates the switch between high- and low-fear states. These findings suggest that the ITCs have a broader role in a range of amygdala functions and associated brain states that underpins the capacity to adapt to salient environmental demands.
Assuntos
Tonsila do Cerebelo/citologia , Tonsila do Cerebelo/fisiologia , Medo/fisiologia , Estimulação Acústica , Animais , Aprendizagem da Esquiva , Condicionamento Clássico , Extinção Psicológica , Feminino , Masculino , Camundongos , Inibição Neural , Neurônios/fisiologiaRESUMO
Optogenetics has become a key method to interrogate the function of neural populations and circuits in the brain. This technique combines the targeted expression of light-activated proteins with subsequent manipulation of neural activity by light. Opsins such as Channelrhodopsin-2 (ChR2), which is a light-gated cation-channel, can be fused to or coexpressed with fluorescent proteins to allow for visualization and concurrent activation of neurons and their axonal projections. Via stereotaxic delivery of viral vectors, ChR2 can be constitutively or conditionally expressed in specific neurons in defined brain regions. Subsequently, identified axonal projections can be studied functionally ex vivo in combination with patch-clamp recordings in brain slices. This optogenetic mapping of neural circuitry has enabled the identification and characterization of novel synaptic connections and the detailed investigation of known anatomical connections previously not amenable with electrical stimulation techniques. Here, we describe a protocol for investigating functional properties of local and long-range connectivity in the brain using blue-light activated ChR2 variants and whole-cell patch-clamp recordings in acute brain slices.
Assuntos
Encéfalo/fisiologia , Neurônios/fisiologia , Optogenética/métodos , Animais , Encéfalo/metabolismo , Channelrhodopsins/metabolismo , Eletrofisiologia , Camundongos , Neurônios/metabolismo , Sinapses/metabolismo , Sinapses/fisiologiaRESUMO
Natural lipidomes represent a complex mixture of lipid molecular species with a variety of biological and signaling functions. Modern mass spectrometry (MS)-based analytical platforms are often used to resolve the complexity of natural lipidomes. The quantitative transfer of lipid molecular species in the gas phase during the electrospray ionization required for MS analysis might be challenged by lipid in-source fragmentation (ISF) hampering their accurate identification and quantification. Here we evaluated the effect of transmission radio frequency (RF) levels and ion transfer temperatures (ITTs) on the analysis of four different lipids (ceramide, cholesteryl ester, phosphatidylethanolamine, and triacylglyceride) ionized in positive ion mode on three different Orbitrap-based platforms. ITT and RF levels were ramped in a systematic way to determine the best settings, allowing the most sensitive detection accompanied by the lowest ISF of a lipid. The extent of the ISF was shown to depend on the configurations of the transmission devices (S-lens vs letterbox/ion funnel) at defined RF and ITT levels for each studied lipid class. We provide here the recommendations for reducing the extent of lipid ISF without a significant loss in sensitivity for Q Exactive HF, Q Exactive HF-X, and Orbitrap Fusion Lumos platforms.
Assuntos
Lipidômica/métodos , Lipídeos/análise , Lipídeos/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Ceramidas , Ésteres do Colesterol , Fosfatidiletanolaminas , Sensibilidade e EspecificidadeRESUMO
Over 1500 different lipids have been reported in human plasma at the sum composition level. Yet the number of unique lipids present is surely higher, once isomeric contributions from double bond location(s) and fatty acyl regiochemistry are considered. In order to resolve this ambiguity, herein, we describe the incorporation of ozone-induced dissociation (OzID) into data-independent shotgun lipidomics workflows on a high-resolution hybrid quadrupole-Orbitrap platform. In this configuration, [M + Na]+ ions generated by electrospray ionization of a plasma lipid extract were transmitted through the quadrupole in 1 Da segments. Reaction of mass-selected lipid ions with ozone in the octopole collision cell yielded diagnostic ions for each double bond position. The increased ozone concentration in this region significantly improved ozonolysis efficiency compared with prior implementations on linear ion-trap devices. This advancement translates into increased lipidome coverage and improvements in duty cycle for data-independent MS/MS analysis using shotgun workflows. Grouping all precursor ions with a common OzID neutral loss enables straightforward classification of the lipidome by unsaturation position (with respect to the methyl terminus). Two-dimensional maps obtained from this analysis provide a powerful visualization of structurally related lipids and lipid isomer families within plasma. Global profiling of lipid unsaturation in plasma extracts reveals that most unsaturated lipids are present as isomeric mixtures. These new insights provide a unique picture of underlying metabolism that could in the future provide novel indicators of health and disease.
Assuntos
Lipídeos/sangue , Lipídeos/química , Ozônio/química , Espectrometria de Massas em Tandem/métodos , Ésteres do Colesterol/sangue , Ésteres do Colesterol/química , Humanos , Espectrometria de Massas por Ionização por Electrospray/instrumentação , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/instrumentação , Fluxo de TrabalhoRESUMO
The aminophospholipids (APL), phosphatidylethanolamine (PE) and phosphatidylserine (PS) are widely present in cell membranes and lipoproteins. Glucose and reactive oxygen species (ROS), such as the hydroxyl radical (â¢OH), can react with APL leading to an array of oxidised, glycated and glycoxidised derivatives. Modified APL have been implicated in inflammatory diseases and diabetes, and were identified as signalling molecules regulating cell death. However, the biological relevance of these molecules has not been completely established, since they are present in very low amounts, and new sensitive methodologies are needed to detect them in biological systems. Few studies have focused on the characterisation of APL modifications using liquid chromatography-tandem mass spectrometry (LC-MS/MS), mainly using C5 or C18 reversed phase (RP) columns. In the present study, we propose a new analytical approach for the characterisation of complex mixtures of oxidised, glycated and glycoxidised PE and PS. This LC approach was based on a reversed-phase C30 column combined with high-resolution MS, and higher energy C-trap dissociation (HCD) MS/MS. C30 RP-LC separated short and long fatty acyl oxidation products, along with glycoxidised APL bearing oxidative modifications on the glucose moiety and the fatty acyl chains. Functional isomers (e.g. hydroxy-hydroperoxy-APL and tri-hydroxy-APL) and positional isomers (e.g. 9-hydroxy-APL and 13-hydroxy-APL) were also discriminated by the method. HCD fragmentation patterns allowed unequivocal structural characterisation of the modified APL, and are translatable into targeted MS/MS fingerprinting of the modified derivatives in biological samples.
Assuntos
Glucose/química , Glicerofosfatos/análise , Lisofosfolipídeos/análise , Fosfatidiletanolaminas/análise , Fosfatidilserinas/análise , Cromatografia de Fase Reversa/métodos , Glicerofosfatos/química , Glicosilação , Humanos , Lisofosfolipídeos/química , Oxirredução , Fosfatidiletanolaminas/química , Fosfatidilserinas/química , Soluções , Espectrometria de Massas em TandemRESUMO
Natural lipidomes are characterized by extremely high complexity and dynamic range of lipid concentrations. Furthermore, high diversity of lipid physicochemical properties requires high resolving powers for both chromatographic and mass spectrometric analytical platforms. Reverse-phase chromatography coupled with data-dependent MS/MS acquisition is one of the most popular techniques in untargeted lipidomics. Optimal method should provide good chromatographic separation and resolution, reproducibility, selectivity and sensitivity. Here, we developed and set-up a RPLC-MS/MS workflow capable of resolving complex mixtures of lipids in 32 min of analysis. Human blood plasma was chosen as a representative complex natural lipidome with large variance of lipid classes, species and lipid concentrations. Lipids were separated by RPLC on five different reverse phase columns with different types of stationary phase particles, size and chemistry. High mass accuracy MS analysis and data-dependent MS/MS analysis were performed using a Q Exactive™ HF Hybrid Quadrupole-Orbitrap™ Mass Spectrometer to identify individual lipid molecular species. This workflow was applied to evaluate the separation capability of each column and to identify the lipidomics profile in highly complex biological samples. As a result, we report more than 600 lipid species covering 18 lipid classes in human blood plasma and provide suggestions to the selection of the appropriate reverse phase column for the analysis of specific lipidomes.
Assuntos
Ensaios de Triagem em Larga Escala , Lipidômica , Lipídeos/sangue , Cromatografia Líquida , Humanos , Espectrometria de MassasRESUMO
Lipid identification and quantification are essential objectives in comprehensive lipidomics studies challenged by the high number of lipids, their chemical diversity, and their dynamic range. In this work, we developed a tailored method for profiling and quantification combining (1) isotope dilution, (2) enhanced isomer separation by C30 fused-core reversed-phase material, and (3) parallel Orbitrap and ion trap detection by the Orbitrap Fusion Lumos Tribid mass spectrometer. The combination of parallelizable ion analysis without time loss together with different fragmentation techniques (HCD/CID) and an inclusion list led to higher quality in lipid identifications exemplified in human plasma and yeast samples. Moreover, we used lipidome isotope-labeling of yeast (LILY)-a fast and efficient in vivo labeling strategy in Pichia pastoris-to produce (nonradioactive) isotopically labeled eukaryotic lipid standards in yeast. We integrated the 13C lipids in the LC-MS workflow to enable relative and absolute compound-specific quantification in yeast and human plasma samples by isotope dilution. Label-free and compound-specific quantification was validated by comparison against a recent international interlaboratory study on human plasma SRM 1950. In this way, we were able to prove that LILY enabled quantification leads to accurate results, even in complex matrices. Excellent analytical figures of merit with enhanced trueness, precision and linearity over 4-5 orders of magnitude were observed applying compound-specific quantification with 13C-labeled lipids. We strongly believe that lipidomics studies will benefit from incorporating isotope dilution and LC-MSn strategies.
Assuntos
Cromatografia de Fase Reversa/métodos , Lipídeos/sangue , Espectrometria de Massas/métodos , Isótopos de Carbono/análise , Isótopos de Carbono/sangue , Humanos , Técnicas de Diluição do Indicador , Lipídeos/análise , Fluxo de Trabalho , Leveduras/químicaRESUMO
The skins of Madagascar poison frogs (Mantella) and certain Neotropical poison frogs (Epipedobates, Dendrobates) secrete the new bile acid tauromantellic acid (1), which was found in both wild-caught and captive-born frogs. This is the first molecule of endogenous origin detected in skin secretions from these taxa. Sucrose was also detected in secretions from wild-caught Mantella but not in captive-born frogs, suggesting a dietary origin.
Assuntos
Anuros , Sacarose Alimentar/análise , Alcaloides , Animais , Ácidos e Sais Biliares/análise , Madagáscar , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Pele/metabolismoRESUMO
Amphibian skin secretions are, for the most part, complex peptidomes. While many peptide components have been biologically- and structurally-characterised into discrete "families", some of which are analogues of endogenous vertebrate regulatory peptides, a substantial number are of unique structure and unknown function. Among the components of these secretory peptidomes is an array of protease inhibitors. Inhibitors of trypsin are of widespread occurrence in different taxa and are representative of many established structural classes, including Kunitz, Kazal and Bowman-Birk. However, few protease inhibitors with activity against other specific proteases have been described from this source. Here we report for the first time, the isolation and structural characterisation of an inhibitor of chymotrypsin of Kunitz-type from the skin secretion of the African hyperoliid frog, Kassina senegalensis. To this end, we employed a functional peptidomic approach. This scheme involves fractionation of the peptidome, functional end-point screening, structural characterisation of resultant actives followed by molecular cloning of biosynthetic precursor-encoding cDNA(s). The novel mature and active polypeptide identified consisted of 62 amino acid residues (average molecular mass 6776.24 Da), of which 6 were positionally-conserved cysteines. The P(1) position within the active site was occupied by a phenylalanyl residue. Bioinformatic analysis of the sequence using BLAST, revealed a structural similarity to Kunitz-type chymotrypsin inhibitors from other organisms, ranging from silkworms to snakes.
Assuntos
Quimotripsina/antagonistas & inibidores , Quimotripsina/metabolismo , Peptídeos/farmacologia , Pele/química , Anfíbios , Animais , Anuros , Peptídeos/química , Inibidores de Proteases/química , Inibidores de Proteases/farmacologiaRESUMO
Amphibian skin secretions are established sources of bioactive peptides. Here we describe the isolation, structural and pharmacological characterisation of a novel vasoconstrictor peptide from the skin secretion of the African hyperoliid frog, Kassina maculata, which exhibits no structural similarity to any known class of amphibian skin peptide. The peptide consists of 21 amino acid residues, FIKELLPHLSGIIDSVANAIK, and is C-terminally amidated. The provisional structure was obtained by MS/MS fragmentation using an Orbitrap mass spectrometer and L/I ambiguities were resolved following molecular cloning of biosynthetic precursor-encoding cDNA. A synthetic replicate of the peptide was found to possess weak antimicrobial and haemolytic activities but was exceptionally effective in constricting the smooth muscle of rat tail artery (EC(50) of 25 pM). In reflection of its exceptional potency in constricting rat arterial smooth muscle, the peptide was named kasstasin, a derivation of Kassina and "stasis" (stoppage of flow). These data illustrate the continuing potential of amphibian skin secretions to provide novel natural peptide templates for biological evaluation.
Assuntos
Proteínas de Anfíbios/química , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos/química , Ranidae/metabolismo , Pele/metabolismo , Vasoconstritores/química , Sequência de Aminoácidos , Proteínas de Anfíbios/farmacologia , Animais , Peptídeos Catiônicos Antimicrobianos/farmacologia , Sequência de Bases , Clonagem Molecular , DNA Complementar/química , Hemólise , Técnicas In Vitro , Masculino , Dados de Sequência Molecular , Músculo Liso Vascular/efeitos dos fármacos , Ratos , Ratos Wistar , Pele/química , Vasoconstritores/farmacologiaRESUMO
Over the past decade peptide sequencing by collision induced dissociation (CID) has become the method of choice in mass spectrometry-based proteomics. The development of alternative fragmentation techniques such as electron transfer dissociation (ETD) has extended the possibilities within tandem mass spectrometry. Recent advances in instrumentation allow peptide fragment ions to be detected with high speed and sensitivity (e.g., in a 2D or 3D ion trap) or at high resolution and high mass accuracy (e.g., an Orbitrap or a ToF). Here, we describe a comprehensive experimental comparison of using ETD, ion-trap CID, and beam type CID (HCD) in combination with either linear ion trap or Orbitrap readout for the large-scale analysis of tryptic peptides. We investigate which combination of fragmentation technique and mass analyzer provides the best performance for the analysis of distinct peptide populations such as N-acetylated, phosphorylated, and tryptic peptides with up to two missed cleavages. We found that HCD provides more peptide identifications than CID and ETD for doubly charged peptides. In terms of Mascot score, ETD FT outperforms the other techniques for peptides with charge states higher than 2. Our data shows that there is a trade-off between spectral quality and speed when using the Orbitrap for fragment ion detection. We conclude that a decision-tree regulated combination of higher-energy collisional dissociation (HCD) and ETD can improve the average Mascot score.
Assuntos
Peptídeos/genética , Proteômica/métodos , Análise de Sequência de Proteína/métodos , Espectrometria de Massas em Tandem/métodos , Linhagem Celular , HumanosRESUMO
From defensive skin secretions acquired from two species of African hyperoliid frogs, Kassina maculata and Kassina senegalensis, we have isolated two structurally related, C-terminally amidated tridecapeptides of novel primary structure that exhibit a broad spectrum of biological activity. In reflection of their structural novelty and species of origin, we named the peptides kassorin M (FLEGLLNTVTGLLamide; 1387.8 Da) and kassorin S (FLGGILNTITGLLamide; 1329.8 Da), respectively. The primary structure and organisation of the biosynthetic precursors of kassorins M and S were deduced from cloned skin secretion-derived cDNA. Both open-reading frames encoded a single copy of kassorin M and S, respectively, located at the C-terminus. Kassorins display limited structural similarities to vespid chemotactic peptides (7/13 residues), temporin A (5/13 residues), the N-terminus of Lv-ranaspumin, a foam nest surfactant protein of the frog, Leptodactylus vastus, and an N-terminal domain of the equine sweat surfactant protein, latherin. Both peptides elicit histamine release from rat peritoneal mast cells. However, while kassorin S was found to possess antibacterial activity against Staphylococcus aureus, kassorin M was devoid of such activity. In contrast, kassorin M was found to contract the smooth muscle of guinea pig urinary bladder (EC(50) = 4.66 nM) and kassorin S was devoid of this activity. Kassorins thus represent the prototypes of a novel family of peptides from the amphibian innate immune system as occurring in defensive skin secretions.
Assuntos
Anuros/imunologia , Imunidade Inata/imunologia , Peptídeos/metabolismo , Pele/metabolismo , África , Sequência de Aminoácidos , Animais , Anti-Infecciosos/farmacologia , Sequência de Bases , Candida albicans/efeitos dos fármacos , Clonagem Molecular , DNA Complementar/genética , Cobaias , Técnicas In Vitro , Espectrometria de Massas , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Músculo Liso/efeitos dos fármacos , Peptídeos/química , Peptídeos/isolamento & purificação , Ratos , Alinhamento de Sequência , Pele/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Bexiga Urinária/efeitos dos fármacosRESUMO
Recently, software has become available to automate localization of phosphorylation sites from CID data and to assign associated confidence scores. We present an algorithm, SLoMo (Site Localization of Modifications), which extends this capability to ETD/ECD mass spectra. Furthermore, SLoMo caters for both high and low resolution data and allows for site-localization of any UniMod post-translational modification. SLoMo accepts input data from a variety of formats (e.g., Sequest, OMSSA). We validate SLoMo with high and low resolution ETD, ECD, and CID data.
Assuntos
Algoritmos , Processamento de Proteína Pós-Traducional/fisiologia , Proteoma/metabolismo , Análise de Sequência de ProteínaRESUMO
Isocyanide-based multicomponent reactions of the Ugi- and Passerini-type have been valuable tools for the rapid exploration of the novel fungicidal compound classes of phenylglycinamides and mandelamides. Mandipropamid (6), which was discovered during this derivatisation, displays excellent activity against the economically important phytopathogens Phytophthora infestans (potato and tomato late blight) and Plasmopara viticola (grape downy mildew).
Assuntos
Amidas/síntese química , Amidas/farmacologia , Benzamidinas/síntese química , Benzamidinas/farmacologia , Ácidos Carboxílicos/síntese química , Ácidos Carboxílicos/farmacologia , Fungicidas Industriais/síntese química , Fungicidas Industriais/farmacologia , Amidas/química , Benzamidinas/química , Ácidos Carboxílicos/química , Fungicidas Industriais/química , Estrutura Molecular , Phytophthora/efeitos dos fármacos , Estereoisomerismo , Relação Estrutura-Atividade , Valina/químicaRESUMO
The basic question addressed in this study is how energy metabolism is adjusted to cope with iron deficiency in Chlamydomonas reinhardtii. To investigate the impact of iron deficiency on bioenergetic pathways, comparative proteomics was combined with spectroscopic as well as voltametric oxygen measurements to assess protein dynamics linked to functional properties of respiratory and photosynthetic machineries. Although photosynthetic electron transfer is largely compromised under iron deficiency, our quantitative and spectroscopic data revealed that the functional antenna size of photosystem II (PSII) significantly increased. Concomitantly, stress-related chloroplast polypeptides, like 2-cys peroxiredoxin and a stress-inducible light-harvesting protein, LhcSR3, as well as a novel light-harvesting protein and several proteins of unknown function were induced under iron-deprivation. Respiratory oxygen consumption did not decrease and accordingly, polypeptides of respiratory complexes, harboring numerous iron-sulfur clusters, were only slightly diminished or even increased under low iron. Consequently, iron-deprivation induces a transition from photoheterotrophic to primarily heterotrophic metabolism, indicating that a hierarchy for iron allocations within organelles of a single cell exists that is closely linked with the metabolic state of the cell.
Assuntos
Chlamydomonas reinhardtii/metabolismo , Metabolismo Energético , Ferro/metabolismo , Proteínas de Algas/genética , Proteínas de Algas/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Chlamydomonas reinhardtii/genética , Clorofila/metabolismo , Primers do DNA/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Dados de Sequência Molecular , Complexo de Proteína do Fotossistema II/genética , Complexo de Proteína do Fotossistema II/metabolismo , Proteômica/métodos , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Fluorescência , Espectrometria de Massas em TandemRESUMO
Novel types of anti-oomycetic compounds have been designed and prepared. The synthetic approach to these mandelamides is outlined. Biological data demonstrate their high efficacy against important plant diseases such as tomato and potato late blight (Phytophthora infestans De Bary) and grape downy mildew (Plasmopara viticola Berliner & de Toni). Structure-activity relationship studies are discussed. The new development product mandipropamid is presented.
Assuntos
Ácidos Mandélicos , Oomicetos , Benzenoacetamidas/síntese química , Benzenoacetamidas/química , Fungicidas Industriais/síntese química , Fungicidas Industriais/química , Ácidos Mandélicos/síntese química , Ácidos Mandélicos/química , Testes de Sensibilidade Microbiana , Estrutura Molecular , PhytophthoraRESUMO
Electron capture dissociation (ECD) offers many benefits for the analysis of peptides and proteins, and consequently shows great potential for the field of proteomics. Recent developments have reduced the time scale required for ECD to milliseconds resulting in the technique's compatibility with on-line separation techniques, e.g., HPLC. Here, we demonstrate incorporation of ECD into a high-throughput data-dependent LC-MS/MS approach for the analysis of proteomic samples. The approach is applied to analysis of the protein Fc-ROR2 isolated from chondrocytes and is the first example of LC-ECD-MS/MS of such a sample. Protein sequence coverage was 29%. Within that coverage, fifteen peptides were isolated and subjected to ECD. In most cases, the sequence tag generated by ECD was over 70% (in terms of the number of peptide backbone cleavages). The ECD data were searched against the nonredundant human NCBI database using the SEQUEST algorithm. Protein ROR2 was assigned, as was IgG (Fc domain). The results demonstrate the suitability of ECD as an integral technique in high-throughput proteomic strategies.
Assuntos
Espectrometria de Massas/métodos , Proteômica/métodos , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Algoritmos , Sequência de Aminoácidos , Condrócitos/metabolismo , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Bases de Dados como Assunto , Bases de Dados Genéticas , Elétrons , Eletroforese em Gel de Poliacrilamida , Humanos , Dados de Sequência Molecular , Peptídeos/química , Proteoma , Fatores de Tempo , Tripsina/farmacologiaRESUMO
Electrospray ionization (ESI) is extensively used in the analysis of biological compounds; yet some fundamental properties of this technique are not completely understood. It is widely recognized that care should be exercised when noncovalent complexes are being studied by ESI, since weak noncovalent binding can be broken or formed during the desolvation process. In the present work, spectra from the noncovalent complex, vancomycin/diacetyl-L-lysyl-D-alanyl-D-alanine, obtained from ESI and from nanoelectrospray ionization (nanoESI), have been compared. The results indicated that the milder desolvation conditions arising as a result of the smaller sizes of droplets produced in the nanoESI source attenuated effects upon weak bonds in the desolvation process. The association constant values calculated from the relative peak intensities suggest that, when using ESI, the analyzed noncovalent complex dissociated in the condensed phase during the spraying process. The influences of experimental parameters such as tip diameter and coating for nanoESI needles were investigated. Principal component analysis, a multivariate analysis method, was applied to achieve a better evaluation of the spectra obtained using different needle diameters and coatings for the analysis of the noncovalent complex vancomycin/diacetyl-L-lysyl-D-alanyl-D-alanine. It was found that 2-microm tip diameter resulted in more reproducible spectra than the larger tip diameters tested (6-20 microm).