RESUMEN
Defective autophagy is strongly associated with chronic inflammation. Loss-of-function of the core autophagy gene Atg16l1 increases risk for Crohn's disease in part by enhancing innate immunity through myeloid cells such as macrophages. However, autophagy is also recognized as a mechanism for clearance of certain intracellular pathogens. These divergent observations prompted a re-evaluation of ATG16L1 in innate antimicrobial immunity. In this study, we found that loss of Atg16l1 in myeloid cells enhanced the killing of virulent Shigella flexneri (S.flexneri), a clinically relevant enteric bacterium that resides within the cytosol by escaping from membrane-bound compartments. Quantitative multiplexed proteomics of murine bone marrow-derived macrophages revealed that ATG16L1 deficiency significantly upregulated proteins involved in the glutathione-mediated antioxidant response to compensate for elevated oxidative stress, which simultaneously promoted S.flexneri killing. Consistent with this, myeloid-specific deletion of Atg16l1 in mice accelerated bacterial clearance in vitro and in vivo. Pharmacological induction of oxidative stress through suppression of cysteine import enhanced microbial clearance by macrophages. Conversely, antioxidant treatment of macrophages permitted S.flexneri proliferation. These findings demonstrate that control of oxidative stress by ATG16L1 and autophagy regulates antimicrobial immunity against intracellular pathogens.
Asunto(s)
Proteínas Relacionadas con la Autofagia/deficiencia , Autofagia , Disentería Bacilar/microbiología , Inmunidad Innata , Macrófagos/microbiología , Estrés Oxidativo , Proteoma , Proteómica , Shigella flexneri/patogenicidad , Animales , Proteínas Relacionadas con la Autofagia/genética , Células Cultivadas , Modelos Animales de Enfermedad , Disentería Bacilar/inmunología , Disentería Bacilar/metabolismo , Interacciones Huésped-Patógeno , Mediadores de Inflamación/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Viabilidad Microbiana , Shigella flexneri/inmunología , Shigella flexneri/metabolismo , VirulenciaRESUMEN
Multiplexed quantitative analyses of complex proteomes enable deep biological insight. While a multitude of workflows have been developed for multiplexed analyses, the most quantitatively accurate method (SPS-MS3) suffers from long acquisition duty cycles. We built a new, real-time database search (RTS) platform, Orbiter, to combat the SPS-MS3 method's longer duty cycles. RTS with Orbiter eliminates SPS-MS3 scans if no peptide matches to a given spectrum. With Orbiter's online proteomic analytical pipeline, which includes RTS and false discovery rate analysis, it was possible to process a single spectrum database search in less than 10 ms. The result is a fast, functional means to identify peptide spectral matches using Comet, filter these matches, and more efficiently quantify proteins of interest. Importantly, the use of Comet for peptide spectral matching allowed for a fully featured search, including analysis of post-translational modifications, with well-known and extensively validated scoring. These data could then be used to trigger subsequent scans in an adaptive and flexible manner. In this work we tested the utility of this adaptive data acquisition platform to improve the efficiency and accuracy of multiplexed quantitative experiments. We found that RTS enabled a 2-fold increase in mass spectrometric data acquisition efficiency. Orbiter's RTS quantified more than 8000 proteins across 10 proteomes in half the time of an SPS-MS3 analysis (18 h for RTS, 36 h for SPS-MS3).
Asunto(s)
Proteoma , Proteómica , Bases de Datos Factuales , Espectrometría de Masas , PéptidosRESUMEN
Proteins are essential agents of biological processes. To date, large-scale profiling of cell line collections including the Cancer Cell Line Encyclopedia (CCLE) has focused primarily on genetic information whereas deep interrogation of the proteome has remained out of reach. Here, we expand the CCLE through quantitative profiling of thousands of proteins by mass spectrometry across 375 cell lines from diverse lineages to reveal information undiscovered by DNA and RNA methods. We observe unexpected correlations within and between pathways that are largely absent from RNA. An analysis of microsatellite instable (MSI) cell lines reveals the dysregulation of specific protein complexes associated with surveillance of mutation and translation. These and other protein complexes were associated with sensitivity to knockdown of several different genes. These data in conjunction with the wider CCLE are a broad resource to explore cellular behavior and facilitate cancer research.
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Regulación Neoplásica de la Expresión Génica/genética , Neoplasias/metabolismo , Proteoma/metabolismo , Línea Celular Tumoral , Perfilación de la Expresión Génica/métodos , Humanos , Espectrometría de Masas/métodos , Inestabilidad de Microsatélites , Mutación/genética , Proteómica/métodosRESUMEN
Gas-phase fractionation enables better quantitative accuracy, improves signal-to-noise ratios, and increases sensitivity in proteomic analyses. However, traditional gas-phase enrichment, which relies upon a large continuous bin, results in suboptimal enrichment, as most chromatographic separations are not 100% orthogonal relative to the first MS dimension (MS1m/z). As such, ions with similar m/z values tend to elute at the same retention time, which prevents the partitioning of narrow precursor m/z distributions into a few large continuous gas-phase enrichment bins. To overcome this issue, we developed and tested the use of notched isolation waveforms, which simultaneously isolate multiple discrete m/z windows in parallel (e.g., 650-700 m/z and 800-850 m/z). By comparison to a canonical gas-phase fractionation method, notched waveforms do not require bin optimization via in silico digestion or wasteful sample injections to isolate multiple precursor windows. Importantly, the collection of all m/z bins simultaneously using the isolation waveform does not suffer from the sensitivity and duty cycle pitfalls inherent to sequential collection of multiple m/z bins. Applying a notched injection waveform provided consistent enrichment of precursor ions, which resulted in improved proteome depth with greater coverage of low-abundance proteins. Finally, using a reductive dimethyl labeling approach, we show that notched isolation waveforms increase the number of quantified peptides with improved accuracy and precision across a wider dynamic range.
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Proteoma , Proteómica , Fraccionamiento Químico , Iones , PéptidosRESUMEN
Quantitative proteomics employing isobaric reagents has been established as a powerful tool for biological discovery. Current workflows often utilize a dedicated quantitative spectrum to improve quantitative accuracy and precision. A consequence of this approach is a dramatic reduction in the spectral acquisition rate, which necessitates the use of additional instrument time to achieve comprehensive proteomic depth. This work assesses the performance and benefits of online and real-time spectral identification in quantitative multiplexed workflows. A Real-Time Search (RTS) algorithm was implemented to identify fragment spectra within milliseconds as they are acquired using a probabilistic score and to trigger quantitative spectra only upon confident peptide identification. The RTS-MS3 was benchmarked against standard workflows using a complex two-proteome model of interference and a targeted 10-plex comparison of kinase abundance profiles. Applying the RTS-MS3 method provided the comprehensive characterization of a 10-plex proteome in 50% less acquisition time. These data indicate that the RTS-MS3 approach provides dramatic performance improvements for quantitative multiplexed experiments.
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Péptidos/aislamiento & purificación , Proteoma/aislamiento & purificación , Proteómica/métodos , Algoritmos , Bases de Datos Factuales , Humanos , Péptidos/química , Proteoma/química , Espectrometría de Masas en Tándem , Flujo de TrabajoRESUMEN
Triggered by Offset, Multiplexed, Accurate mass, High resolution, and Absolute Quantitation (TOMAHAQ) is a recently introduced targeted proteomics method that combines peptide and sample multiplexing. TOMAHAQ assays enable sensitive and accurate multiplexed quantification by implementing an intricate data collection scheme that comprises multiple MSn scans, mass inclusion lists, and data-driven filters. Consequently, manual creation of TOMAHAQ methods can be time-consuming and error prone, while the resulting TOMAHAQ data may not be compatible with common mass spectrometry analysis pipelines. To address these concerns we introduce TomahaqCompanion, an open-source desktop application that enables rapid creation of TOMAHAQ methods and analysis of TOMAHAQ data. Starting from a list of peptide sequences, a user can perform each step of TOMAHAQ assay development including (1) generation of priming run target list, (2) analysis of priming run data, (3) generation of TOMAHAQ method file, and (4) analysis and export of quantitative TOMAHAQ data. We demonstrate the flexibility of TomahaqCompanion by creating a variety of methods testing TOMAHAQ parameters (e.g., number of SPS notches, run length, etc.). Lastly, we analyze an interference sample comprising heavy yeast peptides, a standard human peptide mixture, TMT11-plex, and super heavy TMT (shTMT) isobaric labels to demonstrate â¼10-200 attomol limit of quantification within a complex background using TOMAHAQ.
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Péptidos/análisis , Proteómica/métodos , Humanos , Programas Informáticos , Coloración y Etiquetado , Espectrometría de Masas en Tándem/métodos , Factores de Tiempo , Interfaz Usuario-Computador , LevadurasRESUMEN
Targeted protein quantification using liquid chromatography-tandem mass spectrometry with stable isotope-labeled standards is recognized as the gold standard of practice for protein quantification. Such assays, however, can only cover a limited number of proteins, and developing targeted methods for larger numbers of proteins requires substantial investment. Alternatively, large-scale global proteomic experiments along with computational methods such as the "total protein approach" (TPA) have the potential to provide extensive protein quantification. In this study, we compared the TPA-based quantitation of seven major hepatic uptake transporters in four human liver tissue samples using global proteomic data obtained from two multiplexed tandem mass tag experiments (performed in two independent laboratories) to the quantitative data from targeted proteomic assays. The TPA-based quantitation of these hepatic transporters [sodium-taurocholate cotransporting polypeptide (NTCP/SLC10A1), organic anion transporter 2 (OAT2/SLC22A7), OAT7/SLC22A9, organic anion-transporting polypeptide 1B1 (OATP1B1/SLCO1B1), OATP1B3/SLCO1B3, OATP2B1/SLCO2B1, and organic cation transporter (OCT1/SLC22A1)] showed good-to-excellent correlations (Pearson r = 0.74-1.00) to the targeted data. In addition, the values were similar to those measured by targeted proteomics with 71% and 86% of the data sets falling within 3-fold of the targeted data. A comparison of the TPA-based quantifications of enzyme abundances to available literature data showed that the majority of the enzyme quantifications fell within the reference data intervals. In conclusion, these results demonstrate the capability of multiplexed global proteomic experiments to detect differences in protein expression between samples and provide reasonable estimations of protein expression levels.
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Transporte Biológico/fisiología , Hígado/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Preparaciones Farmacéuticas/metabolismo , Cromatografía Liquida/métodos , Hepatocitos/metabolismo , Humanos , Proteómica/métodos , Espectrometría de Masas en Tándem/métodosRESUMEN
Nitrate (NO3-) and nitrite (NO2-) are known to be cardioprotective and to alter energy metabolism in vivo NO3- action results from its conversion to NO2- by salivary bacteria, but the mechanism(s) by which NO2- affects metabolism remains obscure. NO2- may act by S-nitrosating protein thiols, thereby altering protein activity. But how this occurs, and the functional importance of S-nitrosation sites across the mammalian proteome, remain largely uncharacterized. Here we analyzed protein thiols within mouse hearts in vivo using quantitative proteomics to determine S-nitrosation site occupancy. We extended the thiol-redox proteomic technique, isotope-coded affinity tag labeling, to quantify the extent of NO2--dependent S-nitrosation of proteins thiols in vivo Using this approach, called SNOxICAT (S-nitrosothiol redox isotope-coded affinity tag), we found that exposure to NO2- under normoxic conditions or exposure to ischemia alone results in minimal S-nitrosation of protein thiols. However, exposure to NO2- in conjunction with ischemia led to extensive S-nitrosation of protein thiols across all cellular compartments. Several mitochondrial protein thiols exposed to the mitochondrial matrix were selectively S-nitrosated under these conditions, potentially contributing to the beneficial effects of NO2- on mitochondrial metabolism. The permeability of the mitochondrial inner membrane to HNO2, but not to NO2-, combined with the lack of S-nitrosation during anoxia alone or by NO2- during normoxia places constraints on how S-nitrosation occurs in vivo and on its mechanisms of cardioprotection and modulation of energy metabolism. Quantifying S-nitrosated protein thiols now allows determination of modified cysteines across the proteome and identification of those most likely responsible for the functional consequences of NO2- exposure.
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Modelos Animales de Enfermedad , Mitocondrias Cardíacas/metabolismo , Isquemia Miocárdica/metabolismo , Miocardio/metabolismo , Nitritos/metabolismo , Procesamiento Proteico-Postraduccional , Regulación hacia Arriba , Marcadores de Afinidad/metabolismo , Animales , Cardiotónicos/farmacología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Cisteína/metabolismo , Femenino , Corazón/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Mitocondrias Cardíacas/efectos de los fármacos , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/metabolismo , Dilatación Mitocondrial/efectos de los fármacos , Isquemia Miocárdica/tratamiento farmacológico , Nitratos/farmacología , Nitritos/farmacología , Nitrosación/efectos de los fármacos , Compuestos de Potasio/farmacología , Proteómica/métodos , Ratas Wistar , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Brown adipose tissue (BAT) mitochondria exhibit high oxidative capacity and abundant expression of both electron transport chain components and uncoupling protein 1 (UCP1). UCP1 dissipates the mitochondrial proton motive force (Δp) generated by the respiratory chain and increases thermogenesis. Here we find that in mice genetically lacking UCP1, cold-induced activation of metabolism triggers innate immune signaling and markers of cell death in BAT. Moreover, global proteomic analysis reveals that this cascade induced by UCP1 deletion is associated with a dramatic reduction in electron transport chain abundance. UCP1-deficient BAT mitochondria exhibit reduced mitochondrial calcium buffering capacity and are highly sensitive to mitochondrial permeability transition induced by reactive oxygen species (ROS) and calcium overload. This dysfunction depends on ROS production by reverse electron transport through mitochondrial complex I, and can be rescued by inhibition of electron transfer through complex I or pharmacologic depletion of ROS levels. Our findings indicate that the interscapular BAT of Ucp1 knockout mice exhibits mitochondrial disruptions that extend well beyond the deletion of UCP1 itself. This finding should be carefully considered when using this mouse model to examine the role of UCP1 in physiology.
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Aclimatación/fisiología , Tejido Adiposo Pardo/metabolismo , Frío , Transporte de Electrón , Proteína Desacopladora 1/deficiencia , Animales , Calcio/metabolismo , Femenino , Masculino , Ratones , Ratones Noqueados , Mitocondrias/metabolismo , Mitocondrias/patología , Especies Reactivas de Oxígeno/metabolismo , Proteína Desacopladora 1/genéticaRESUMEN
The physiology of a cell can be viewed as the product of thousands of proteins acting in concert to shape the cellular response. Coordination is achieved in part through networks of protein-protein interactions that assemble functionally related proteins into complexes, organelles, and signal transduction pathways. Understanding the architecture of the human proteome has the potential to inform cellular, structural, and evolutionary mechanisms and is critical to elucidating how genome variation contributes to disease. Here we present BioPlex 2.0 (Biophysical Interactions of ORFeome-derived complexes), which uses robust affinity purification-mass spectrometry methodology to elucidate protein interaction networks and co-complexes nucleated by more than 25% of protein-coding genes from the human genome, and constitutes, to our knowledge, the largest such network so far. With more than 56,000 candidate interactions, BioPlex 2.0 contains more than 29,000 previously unknown co-associations and provides functional insights into hundreds of poorly characterized proteins while enhancing network-based analyses of domain associations, subcellular localization, and co-complex formation. Unsupervised Markov clustering of interacting proteins identified more than 1,300 protein communities representing diverse cellular activities. Genes essential for cell fitness are enriched within 53 communities representing central cellular functions. Moreover, we identified 442 communities associated with more than 2,000 disease annotations, placing numerous candidate disease genes into a cellular framework. BioPlex 2.0 exceeds previous experimentally derived interaction networks in depth and breadth, and will be a valuable resource for exploring the biology of incompletely characterized proteins and for elucidating larger-scale patterns of proteome organization.
Asunto(s)
Bases de Datos de Proteínas , Enfermedad , Mapeo de Interacción de Proteínas , Mapas de Interacción de Proteínas , Proteoma/metabolismo , Fenómenos Fisiológicos Celulares/genética , Genoma Humano , Humanos , Espacio Intracelular/metabolismo , Cadenas de Markov , Espectrometría de Masas , Anotación de Secuencia Molecular , Sistemas de Lectura Abierta , Proteoma/análisis , Proteoma/química , Proteoma/genéticaRESUMEN
Targeted mass spectrometry assays for protein quantitation monitor peptide surrogates, which are easily multiplexed to target many peptides in a single assay. However, these assays have generally not taken advantage of sample multiplexing, which allows up to ten analyses to occur in parallel. We present a two-dimensional multiplexing workflow that utilizes synthetic peptides for each protein to prompt the simultaneous quantification of >100 peptides from up to ten mixed sample conditions. We demonstrate that targeted analysis of unfractionated lysates (2 hr) accurately reproduces the quantification of fractionated lysates (72 hr analysis) while obviating the need for peptide detection prior to quantification. We targeted 131 peptides corresponding to 69 proteins across all 60 National Cancer Institute cell lines in biological triplicate, analyzing 180 samples in only 48 hr (the equivalent of 16 min/sample). These data further elucidated a correlation between the expression of key proteins and their cellular response to drug treatment.
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Ensayos Analíticos de Alto Rendimiento , Espectrometría de Masas , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Proteoma , Proteómica/métodos , Antibióticos Antineoplásicos/farmacología , Biomarcadores/metabolismo , Línea Celular Tumoral , Doxorrubicina/farmacología , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Factores de Tiempo , Factores de Transcripción/metabolismo , Flujo de TrabajoRESUMEN
Brown and beige adipose tissues can dissipate chemical energy as heat through thermogenic respiration, which requires uncoupling protein 1 (UCP1). Thermogenesis from these adipocytes can combat obesity and diabetes, encouraging investigation of factors that control UCP1-dependent respiration in vivo. Here we show that acutely activated thermogenesis in brown adipose tissue is defined by a substantial increase in levels of mitochondrial reactive oxygen species (ROS). Remarkably, this process supports in vivo thermogenesis, as pharmacological depletion of mitochondrial ROS results in hypothermia upon cold exposure, and inhibits UCP1-dependent increases in whole-body energy expenditure. We further establish that thermogenic ROS alter the redox status of cysteine thiols in brown adipose tissue to drive increased respiration, and that Cys253 of UCP1 is a key target. UCP1 Cys253 is sulfenylated during thermogenesis, while mutation of this site desensitizes the purine-nucleotide-inhibited state of the carrier to adrenergic activation and uncoupling. These studies identify mitochondrial ROS induction in brown adipose tissue as a mechanism that supports UCP1-dependent thermogenesis and whole-body energy expenditure, which opens the way to improved therapeutic strategies for combating metabolic disorders.
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Cisteína/química , Metabolismo Energético , Canales Iónicos/química , Canales Iónicos/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/química , Proteínas Mitocondriales/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Termogénesis , Tejido Adiposo Pardo/química , Tejido Adiposo Pardo/citología , Tejido Adiposo Pardo/metabolismo , Animales , Respiración de la Célula , Cisteína/genética , Cisteína/metabolismo , Metabolismo Energético/efectos de los fármacos , Femenino , Humanos , Canales Iónicos/deficiencia , Canales Iónicos/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Proteínas Mitocondriales/deficiencia , Proteínas Mitocondriales/genética , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Oxidación-Reducción , Compuestos de Sulfhidrilo/metabolismo , Termogénesis/efectos de los fármacos , Proteína Desacopladora 1RESUMEN
Thermogenic brown and beige adipose tissues dissipate chemical energy as heat, and their thermogenic activities can combat obesity and diabetes. Herein the functional adaptations to cold of brown and beige adipose depots are examined using quantitative mitochondrial proteomics. We identify arginine/creatine metabolism as a beige adipose signature and demonstrate that creatine enhances respiration in beige-fat mitochondria when ADP is limiting. In murine beige fat, cold exposure stimulates mitochondrial creatine kinase activity and induces coordinated expression of genes associated with creatine metabolism. Pharmacological reduction of creatine levels decreases whole-body energy expenditure after administration of a ß3-agonist and reduces beige and brown adipose metabolic rate. Genes of creatine metabolism are compensatorily induced when UCP1-dependent thermogenesis is ablated, and creatine reduction in Ucp1-deficient mice reduces core body temperature. These findings link a futile cycle of creatine metabolism to adipose tissue energy expenditure and thermal homeostasis. PAPERCLIP.
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Tejido Adiposo Pardo/metabolismo , Creatina/metabolismo , Termogénesis , Adenosina Difosfato/metabolismo , Tejido Adiposo/metabolismo , Animales , Metabolismo Energético , Homeostasis , Humanos , Canales Iónicos/metabolismo , Ratones , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Obesidad/metabolismo , Proteína Desacopladora 1RESUMEN
The composition of the nucleoplasm determines the behavior of key processes such as transcription, yet there is still no reliable and quantitative resource of nuclear proteins. Furthermore, it is still unclear how the distinct nuclear and cytoplasmic compositions are maintained. To describe the nuclear proteome quantitatively, we isolated the large nuclei of frog oocytes via microdissection and measured the nucleocytoplasmic partitioning of â¼9,000 proteins by mass spectrometry. Most proteins localize entirely to either nucleus or cytoplasm; only â¼17% partition equally. A protein's native size in a complex, but not polypeptide molecular weight, is predictive of localization: partitioned proteins exhibit native sizes larger than â¼100 kDa, whereas natively smaller proteins are equidistributed. To evaluate the role of nuclear export in maintaining localization, we inhibited Exportin 1. This resulted in the expected re-localization of proteins toward the nucleus, but only 3% of the proteome was affected. Thus, complex assembly and passive retention, rather than continuous active transport, is the dominant mechanism for the maintenance of nuclear and cytoplasmic proteomes.
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Proteínas Anfibias/genética , Proteínas Nucleares/genética , Proteoma/genética , Xenopus/genética , Proteínas Anfibias/metabolismo , Animales , Núcleo Celular/genética , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas Nucleares/metabolismo , Oocitos/metabolismo , Proteoma/metabolismo , Xenopus/metabolismoRESUMEN
Isobaric labeling strategies for mass spectrometry-based proteomics enable multiplexed simultaneous quantification of samples and therefore substantially increase the sample throughput in proteomics. However, despite these benefits, current limits to multiplexing capacity are prohibitive for large sample sizes and impose limitations on experimental design. Here, we introduce a novel mechanism for increasing the multiplexing density of isobaric reagents. We present Combinatorial Isobaric Mass Tags (CMTs), an isobaric labeling architecture with the unique ability to generate multiple series of reporter ions simultaneously. We demonstrate that utilization of multiple reporter ion series improves multiplexing capacity of CMT with respect to a commercially available isobaric labeling reagent with preserved quantitative accuracy and depth of coverage in complex mixtures. We provide a blueprint for the realization of 16-plex reagents with 1 Da spacing between reporter ions and up to 28-plex at 6 mDa spacing using only 5 heavy isotopes per reagent. We anticipate that this improvement in multiplexing capacity will further advance the application of quantitative proteomics, particularly in high-throughput screening assays.
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Espectrometría de Masas/métodos , Péptidos/análisis , Proteómica/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Indicadores y Reactivos/química , Iones/químicaRESUMEN
Protein interactions form a network whose structure drives cellular function and whose organization informs biological inquiry. Using high-throughput affinity-purification mass spectrometry, we identify interacting partners for 2,594 human proteins in HEK293T cells. The resulting network (BioPlex) contains 23,744 interactions among 7,668 proteins with 86% previously undocumented. BioPlex accurately depicts known complexes, attaining 80%-100% coverage for most CORUM complexes. The network readily subdivides into communities that correspond to complexes or clusters of functionally related proteins. More generally, network architecture reflects cellular localization, biological process, and molecular function, enabling functional characterization of thousands of proteins. Network structure also reveals associations among thousands of protein domains, suggesting a basis for examining structurally related proteins. Finally, BioPlex, in combination with other approaches, can be used to reveal interactions of biological or clinical significance. For example, mutations in the membrane protein VAPB implicated in familial amyotrophic lateral sclerosis perturb a defined community of interactors.
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Mapas de Interacción de Proteínas , Proteómica/métodos , Esclerosis Amiotrófica Lateral/genética , Humanos , Espectrometría de Masas , Mapeo de Interacción de Proteínas , Proteínas/química , Proteínas/aislamiento & purificación , Proteínas/metabolismoRESUMEN
As a driver for many biological processes, phosphorylation remains an area of intense research interest. Advances in multiplexed quantitation utilizing isobaric tags (e.g., TMT and iTRAQ) have the potential to create a new paradigm in quantitative proteomics. New instrumentation and software are propelling these multiplexed workflows forward, which results in more accurate, sensitive, and reproducible quantitation across tens of thousands of phosphopeptides. This study assesses the performance of multiplexed quantitative phosphoproteomics on the Orbitrap Fusion mass spectrometer. Utilizing a two-phosphoproteome model of precursor ion interference, we assessed the accuracy of phosphopeptide quantitation across a variety of experimental approaches. These methods included the use of synchronous precursor selection (SPS) to enhance TMT reporter ion intensity and accuracy. We found that (i) ratio distortion remained a problem for phosphopeptide analysis in multiplexed quantitative workflows, (ii) ratio distortion can be overcome by the use of an SPS-MS3 scan, (iii) interfering ions generally possessed a different charge state than the target precursor, and (iv) selecting only the phosphate neutral loss peak (single notch) for the MS3 scan still provided accurate ratio measurements. Remarkably, these data suggest that the underlying cause of interference may not be due to coeluting and cofragmented peptides but instead from consistent, low level background fragmentation. Finally, as a proof-of-concept 10-plex experiment, we compared phosphopeptide levels from five murine brains to five livers. In total, the SPS-MS3 method quantified 38â¯247 phosphopeptides, corresponding to 11â¯000 phosphorylation sites. With 10 measurements recorded for each phosphopeptide, this equates to more than 628â¯000 binary comparisons collected in less than 48 h.
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Química Encefálica , Cromatografía Líquida de Alta Presión/métodos , Hígado/química , Espectrometría de Masas/métodos , Fosfopéptidos/análisis , Proteómica/métodos , Animales , Cromatografía de Fase Inversa/métodos , Masculino , Ratones , FosforilaciónRESUMEN
Multiplexed quantitation via isobaric chemical tags (e.g., tandem mass tags (TMT) and isobaric tags for relative and absolute quantitation (iTRAQ)) has the potential to revolutionize quantitative proteomics. However, until recently the utility of these tags was questionable due to reporter ion ratio distortion resulting from fragmentation of coisolated interfering species. These interfering signals can be negated through additional gas-phase manipulations (e.g., MS/MS/MS (MS3) and proton-transfer reactions (PTR)). These methods, however, have a significant sensitivity penalty. Using isolation waveforms with multiple frequency notches (i.e., synchronous precursor selection, SPS), we coisolated and cofragmented multiple MS2 fragment ions, thereby increasing the number of reporter ions in the MS3 spectrum 10-fold over the standard MS3 method (i.e., MultiNotch MS3). By increasing the reporter ion signals, this method improves the dynamic range of reporter ion quantitation, reduces reporter ion signal variance, and ultimately produces more high-quality quantitative measurements. To demonstrate utility, we analyzed biological triplicates of eight colon cancer cell lines using the MultiNotch MS3 method. Across all the replicates we quantified 8,378 proteins in union and 6,168 proteins in common. Taking into account that each of these quantified proteins contains eight distinct cell-line measurements, this data set encompasses 174,704 quantitative ratios each measured in triplicate across the biological replicates. Herein, we demonstrate that the MultiNotch MS3 method uniquely combines multiplexing capacity with quantitative sensitivity and accuracy, drastically increasing the informational value obtainable from proteomic experiments.
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Neoplasias del Colon/metabolismo , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Algoritmos , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión/métodos , Células HeLa , Humanos , Iones , Isocitrato Deshidrogenasa/análisis , Isocitrato Deshidrogenasa/metabolismo , Análisis de Componente Principal , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Proteína Smad4/análisis , Proteína Smad4/metabolismo , Espectrometría de Masas en Tándem/instrumentaciónRESUMEN
The human gut microbiota is an important metabolic organ, yet little is known about how its individual species interact, establish dominant positions, and respond to changes in environmental factors such as diet. In this study, gnotobiotic mice were colonized with an artificial microbiota comprising 12 sequenced human gut bacterial species and fed oscillating diets of disparate composition. Rapid, reproducible, and reversible changes in the structure of this assemblage were observed. Time-series microbial RNA-Seq analyses revealed staggered functional responses to diet shifts throughout the assemblage that were heavily focused on carbohydrate and amino acid metabolism. High-resolution shotgun metaproteomics confirmed many of these responses at a protein level. One member, Bacteroides cellulosilyticus WH2, proved exceptionally fit regardless of diet. Its genome encoded more carbohydrate active enzymes than any previously sequenced member of the Bacteroidetes. Transcriptional profiling indicated that B. cellulosilyticus WH2 is an adaptive forager that tailors its versatile carbohydrate utilization strategy to available dietary polysaccharides, with a strong emphasis on plant-derived xylans abundant in dietary staples like cereal grains. Two highly expressed, diet-specific polysaccharide utilization loci (PULs) in B. cellulosilyticus WH2 were identified, one with characteristics of xylan utilization systems. Introduction of a B. cellulosilyticus WH2 library comprising >90,000 isogenic transposon mutants into gnotobiotic mice, along with the other artificial community members, confirmed that these loci represent critical diet-specific fitness determinants. Carbohydrates that trigger dramatic increases in expression of these two loci and many of the organism's 111 other predicted PULs were identified by RNA-Seq during in vitro growth on 31 distinct carbohydrate substrates, allowing us to better interpret in vivo RNA-Seq and proteomics data. These results offer insight into how gut microbes adapt to dietary perturbations at both a community level and from the perspective of a well-adapted symbiont with exceptional saccharolytic capabilities, and illustrate the value of artificial communities.