RESUMEN
Many growth factors and cytokines signal by binding to the extracellular domains of their receptors and driving association and transphosphorylation of the receptor intracellular tyrosine kinase domains, initiating downstream signaling cascades. To enable systematic exploration of how receptor valency and geometry affect signaling outcomes, we designed cyclic homo-oligomers with up to 8 subunits using repeat protein building blocks that can be modularly extended. By incorporating a de novo-designed fibroblast growth factor receptor (FGFR)-binding module into these scaffolds, we generated a series of synthetic signaling ligands that exhibit potent valency- and geometry-dependent Ca2+ release and mitogen-activated protein kinase (MAPK) pathway activation. The high specificity of the designed agonists reveals distinct roles for two FGFR splice variants in driving arterial endothelium and perivascular cell fates during early vascular development. Our designed modular assemblies should be broadly useful for unraveling the complexities of signaling in key developmental transitions and for developing future therapeutic applications.
Asunto(s)
Diferenciación Celular , Factores de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento de Fibroblastos , Transducción de Señal , Animales , Humanos , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Ratones , Ligandos , Calcio/metabolismo , Sistema de Señalización de MAP QuinasasRESUMEN
Thousands of interactions assemble proteins into modules that impart spatial and functional organization to the cellular proteome. Through affinity-purification mass spectrometry, we have created two proteome-scale, cell-line-specific interaction networks. The first, BioPlex 3.0, results from affinity purification of 10,128 human proteins-half the proteome-in 293T cells and includes 118,162 interactions among 14,586 proteins. The second results from 5,522 immunoprecipitations in HCT116 cells. These networks model the interactome whose structure encodes protein function, localization, and complex membership. Comparison across cell lines validates thousands of interactions and reveals extensive customization. Whereas shared interactions reside in core complexes and involve essential proteins, cell-specific interactions link these complexes, "rewiring" subnetworks within each cell's interactome. Interactions covary among proteins of shared function as the proteome remodels to produce each cell's phenotype. Viewable interactively online through BioPlexExplorer, these networks define principles of proteome organization and enable unknown protein characterization.
Asunto(s)
Mapeo de Interacción de Proteínas/métodos , Mapas de Interacción de Proteínas/genética , Proteoma/genética , Biología Computacional/métodos , Células HCT116/metabolismo , Células HEK293/metabolismo , Humanos , Espectrometría de Masas/métodos , Mapas de Interacción de Proteínas/fisiología , Proteoma/metabolismo , Proteómica/métodosRESUMEN
Mammalian tissues engage in specialized physiology that is regulated through reversible modification of protein cysteine residues by reactive oxygen species (ROS). ROS regulate a myriad of biological processes, but the protein targets of ROS modification that drive tissue-specific physiology in vivo are largely unknown. Here, we develop Oximouse, a comprehensive and quantitative mapping of the mouse cysteine redox proteome in vivo. We use Oximouse to establish several paradigms of physiological redox signaling. We define and validate cysteine redox networks within each tissue that are tissue selective and underlie tissue-specific biology. We describe a common mechanism for encoding cysteine redox sensitivity by electrostatic gating. Moreover, we comprehensively identify redox-modified disease networks that remodel in aged mice, establishing a systemic molecular basis for the long-standing proposed links between redox dysregulation and tissue aging. We provide the Oximouse compendium as a framework for understanding mechanisms of redox regulation in physiology and aging.
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Envejecimiento/genética , Cisteína/genética , Proteínas/genética , Proteoma/genética , Envejecimiento/metabolismo , Envejecimiento/patología , Animales , Cisteína/metabolismo , Humanos , Ratones , Especificidad de Órganos/genética , Oxidación-Reducción , Estrés Oxidativo/genética , Proteómica/métodos , Especies Reactivas de Oxígeno , Transducción de Señal/genéticaRESUMEN
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.
Asunto(s)
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
BAX is a pro-apoptotic protein that transforms from a cytosolic monomer into a toxic oligomer that permeabilizes the mitochondrial outer membrane. How BAX monomers assemble into a higher-order conformation, and the structural determinants essential to membrane permeabilization, remain a mechanistic mystery. A key hurdle has been the inability to generate a homogeneous BAX oligomer (BAXO) for analysis. Here, we report the production and characterization of a full-length BAXO that recapitulates physiologic BAX activation. Multidisciplinary studies revealed striking conformational consequences of oligomerization and insight into the macromolecular structure of oligomeric BAX. Importantly, BAXO enabled the assignment of specific roles to particular residues and α helices that mediate individual steps of the BAX activation pathway, including unexpected functionalities of BAX α6 and α9 in driving membrane disruption. Our results provide the first glimpse of a full-length and functional BAXO, revealing structural requirements for the elusive execution phase of mitochondrial apoptosis.
Asunto(s)
Apoptosis , Mitocondrias/patología , Membranas Mitocondriales/metabolismo , Multimerización de Proteína , Proteína X Asociada a bcl-2/química , Proteína X Asociada a bcl-2/metabolismo , Animales , Transporte Biológico , Permeabilidad de la Membrana Celular , Citosol/metabolismo , Humanos , Ratones , Mitocondrias/metabolismo , Modelos Moleculares , Conformación Proteica , Proteínas Proto-Oncogénicas c-fosRESUMEN
Stochastic, intensity-based precursor isolation can result in isotopically enriched fragment ions. This problem is exacerbated for large peptides and stable isotope labeling experiments using deuterium or 15N. For stable isotope labeling experiments, incomplete and ubiquitous labeling strategies result in the isolation of peptide ions composed of many distinct structural isomers. Unfortunately, existing proteomics search algorithms do not account for this variability in isotopic incorporation, and thus often yield poor peptide and protein identification rates. We sought to resolve this shortcoming by deriving the expected isotopic distributions of each fragment ion and incorporating them into the theoretical mass spectra used for peptide-spectrum-matching. We adapted the Comet search platform to integrate a modified spectral prediction algorithm we term Conditional fragment Ion Distribution Search (CIDS). Comet-CIDS uses a traditional database searching strategy, but for each candidate peptide we compute the isotopic distribution of each fragment to better match the observed m/z distributions. Evaluating previously generated D2O and 15N labeled data sets, we found that Comet-CIDS identified more confident peptide spectral matches and higher protein sequence coverage compared to traditional theoretical spectra generation, with the magnitude of improvement largely determined by the amount of labeling in the sample.
Asunto(s)
Péptidos , Proteínas , Péptidos/química , Proteínas/metabolismo , Secuencia de Aminoácidos , Probabilidad , IonesRESUMEN
Sample multiplexed quantitative proteomics assays have proved to be a highly versatile means to assay molecular phenotypes. Yet, stochastic precursor selection and precursor coisolation can dramatically reduce the efficiency of data acquisition and quantitative accuracy. To address this, intelligent data acquisition (IDA) strategies have recently been developed to improve instrument efficiency and quantitative accuracy for both discovery and targeted methods. Toward this end, we sought to develop and implement a new real-time spectral library searching (RTLS) workflow that could enable intelligent scan triggering and peak selection within milliseconds of scan acquisition. To ensure ease of use and general applicability, we built an application to read in diverse spectral libraries and file types from both empirical and predicted spectral libraries. We demonstrate that RTLS methods enable improved quantitation of multiplexed samples, particularly with consideration for quantitation from chimeric fragment spectra. We used RTLS to profile proteome responses to small molecule perturbations and were able to quantify up to 15% more significantly regulated proteins in half the gradient time compared to traditional methods. Taken together, the development of RTLS expands the IDA toolbox to improve instrument efficiency and quantitative accuracy for sample multiplexed analyses.
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Péptidos , Proteómica , Proteómica/métodos , Péptidos/análisis , Proteoma/análisis , Biblioteca de Genes , Flujo de Trabajo , Biblioteca de PéptidosRESUMEN
Isobaric labeling empowers proteome-wide expression measurements simultaneously across multiple samples. Here an expanded set of 16 isobaric reagents based on an isobutyl-proline immonium ion reporter structure (TMTpro) is presented. These reagents have similar characteristics to existing tandem mass tag reagents but with increased fragmentation efficiency and signal. In a proteome-scale example dataset, we compared eight common cell lines with and without Torin1 treatment with three replicates, quantifying more than 8,800 proteins (mean of 7.5 peptides per protein) per replicate with an analysis time of only 1.1 h per proteome. Finally, we modified the thermal stability assay to examine proteome-wide melting shifts after treatment with DMSO, 1 or 20 µM staurosporine with five replicates. This assay identified and dose-stratified staurosporine binding to 228 cellular kinases in just one, 18-h experiment. TMTpro reagents allow complex experimental designs-all with essentially no missing values across the 16 samples and no loss in quantitative integrity.
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Péptidos/química , Proteoma/química , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Línea Celular , Humanos , Marcaje IsotópicoRESUMEN
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.
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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
Pathway proteomics strategies measure protein expression changes in specific cellular processes that carry out related functions. Using targeted tandem mass tags-based sample multiplexing, hundreds of proteins can be quantified across 10 or more samples simultaneously. To facilitate these highly complex experiments, we introduce a strategy that provides complete control over targeted sample multiplexing experiments, termed Tomahto, and present its implementation on the Orbitrap Tribrid mass spectrometer platform. Importantly, this software monitors via the external desktop computer to the data stream and inserts optimized MS2 and MS3 scans in real time based on an application programming interface with the mass spectrometer. Hundreds of proteins of interest from diverse biological samples can be targeted and accurately quantified in a sensitive and high-throughput fashion. It achieves sensitivity comparable to, if not better than, deep fractionation and requires minimal total sample input (â¼10 µg). As a proof-of-principle experiment, we selected four pathways important in metabolism- and inflammation-related processes (260 proteins/520 peptides) and measured their abundance across 90 samples (nine tissues from five old and five young mice) to explore effects of aging. Tissue-specific aging is presented here and we highlight the role of inflammation- and metabolism-related processes in white adipose tissue. We validated our approach through comparison with a global proteome survey across the tissues, work that we also provide as a general resource for the community.
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Envejecimiento/genética , Proteoma/genética , Proteómica/métodos , Programas Informáticos , Animales , Ensayos Analíticos de Alto Rendimiento/métodos , Inflamación/genética , Espectrometría de Masas/métodos , Redes y Vías Metabólicas/genética , Ratones , Especificidad de Órganos/genética , Péptidos/genéticaRESUMEN
Oxidation of cysteine thiols by physiological reactive oxygen species (ROS) initiates thermogenesis in brown and beige adipose tissues. Cellular selenocysteines, where sulfur is replaced with selenium, exhibit enhanced reactivity with ROS. Despite their critical roles in physiology, methods for broad and direct detection of proteogenic selenocysteines are limited. Here we developed a mass spectrometric method to interrogate incorporation of selenium into proteins. Unexpectedly, this approach revealed facultative incorporation of selenium as selenocysteine or selenomethionine into proteins that lack canonical encoding for selenocysteine. Selenium was selectively incorporated into regulatory sites on key metabolic proteins, including as selenocysteine-replacing cysteine at position 253 in uncoupling protein 1 (UCP1). This facultative utilization of selenium was initiated by increasing cellular levels of organic, but not inorganic, forms of selenium. Remarkably, dietary selenium supplementation elevated facultative incorporation into UCP1, elevated energy expenditure through thermogenic adipose tissue, and protected against obesity. Together, these findings reveal the existence of facultative protein selenation, which correlates with impacts on thermogenic adipocyte function and presumably other biological processes as well.
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Tejido Adiposo/metabolismo , Cisteína/metabolismo , Obesidad/metabolismo , Selenio/metabolismo , Termogénesis , Proteína Desacopladora 1/metabolismo , Tejido Adiposo/fisiología , Animales , Células Cultivadas , Masculino , Espectrometría de Masas/métodos , Ratones , Ratones Endogámicos C57BL , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Quantitative mass spectrometry measurements of peptides necessarily incorporate sequence-specific biases that reflect the behavior of the peptide during enzymatic digestion and liquid chromatography and in a mass spectrometer. These sequence-specific effects impair quantification accuracy, yielding peptide quantities that are systematically under- or overestimated. We provide empirical evidence for the existence of such biases, and we use a deep neural network, called Pepper, to automatically identify and reduce these biases. The model generalizes to new proteins and new runs within a related set of tandem mass spectrometry experiments, and the learned coefficients themselves reflect expected physicochemical properties of the corresponding peptide sequences. The resulting adjusted abundance measurements are more correlated with mRNA-based gene expression measurements than the unadjusted measurements. Pepper is suitable for data generated on a variety of mass spectrometry instruments and can be used with labeled or label-free approaches and with data-independent or data-dependent acquisition.
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Péptidos , Espectrometría de Masas en Tándem , Secuencia de Aminoácidos , Sesgo , Aprendizaje Automático , Péptidos/análisis , Espectrometría de Masas en Tándem/métodosRESUMEN
Eukaryotic protein phosphorylation modulates nearly every major biological process. Phosphorylation regulates protein activity, mediates cellular signal transduction, and manipulates cellular structure. Consequently, the dysregulation of kinase and phosphatase pathways has been linked to a multitude of diseases. Mass spectrometry-based proteomic techniques are increasingly used for the global interrogation of perturbations in phosphorylation-based cellular signaling. Strategies for studying phosphoproteomes require high-specificity enrichment, sensitive detection, and accurate localization of phosphorylation sites with advanced LC-MS/MS techniques and downstream informatics. Sample multiplexing with isobaric tags has also been integral to recent advancements in throughput and sensitivity for phosphoproteomic studies. Each of these facets of phosphoproteomics analysis present distinct challenges and thus opportunities for improvement and innovation. Here, we review current methodologies, explore persistent challenges, and discuss the outlook for isobaric tag-based quantitative phosphoproteomic analysis.
Asunto(s)
Proteómica , Espectrometría de Masas en Tándem , Cromatografía Liquida , FosforilaciónRESUMEN
Accurate assignment of monoisotopic peaks is essential for the identification of peptides in bottom-up proteomics. Misassignment or inaccurate attribution of peptidic ions leads to lower sensitivity and fewer total peptide identifications. In the present work, we present a performant, open-source, cross-platform algorithm, Monocle, for the rapid reassignment of instrument-assigned precursor peaks to monoisotopic peptide assignments. We demonstrate that the present algorithm can be integrated into many common proteomic pipelines and provides rapid conversion from multiple data source types. Finally, we show that our monoisotopic peak assignment results in up to a twofold increase in total peptide identifications compared to analyses lacking monoisotopic correction and a 44% improvement over previous monoisotopic peak correction algorithms.
Asunto(s)
Proteoma , Proteómica , Algoritmos , Péptidos , Espectrometría de Masas en TándemRESUMEN
Inactivating mutations in the ubiquitously expressed membrane trafficking component GMAP-210 (encoded by Trip11) cause achondrogenesis type 1A (ACG1A). ACG1A is surprisingly tissue specific, mainly affecting cartilage development. Bone development is also abnormal, but as chondrogenesis and osteogenesis are closely coupled, this could be a secondary consequence of the cartilage defect. A possible explanation for the tissue specificity of ACG1A is that cartilage and bone are highly secretory tissues with a high use of the membrane trafficking machinery. The perinatal lethality of ACG1A prevents investigating this hypothesis. We therefore generated mice with conditional Trip11 knockout alleles and inactivated Trip11 in chondrocytes, osteoblasts, osteoclasts and pancreas acinar cells, all highly secretory cell types. We discovered that the ACG1A skeletal phenotype is solely due to absence of GMAP-210 in chondrocytes. Mice lacking GMAP-210 in osteoblasts, osteoclasts and acinar cells were normal. When we inactivated Trip11 in primary chondrocyte cultures, GMAP-210 deficiency affected trafficking of a subset of chondrocyte-expressed proteins rather than globally impairing membrane trafficking. Thus, GMAP-210 is essential for trafficking specific cargoes in chondrocytes but is dispensable in other highly secretory cells.
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Acondroplasia , Alelos , Desarrollo Óseo/genética , Cartílago , Fenotipo , Acondroplasia/genética , Acondroplasia/metabolismo , Acondroplasia/patología , Animales , Transporte Biológico Activo/genética , Cartílago/anomalías , Cartílago/metabolismo , Cartílago/patología , Condrocitos/metabolismo , Condrocitos/patología , Proteínas del Citoesqueleto , Ratones , Ratones Noqueados , Proteínas Nucleares/metabolismo , Osteoblastos/metabolismo , Osteoblastos/patología , Osteoclastos/metabolismo , Osteoclastos/patologíaRESUMEN
INTRODUCTION: Protein phosphorylation is a primary mechanism of signal transduction in cellular systems. Isobaric tagging can be used to investigate alterations in phosphorylation events in sample multiplexing experiments where quantification extends across all conditions. As such, innovations in tandem mass tag methods can facilitate the expansion of the depth and breadth of phosphoproteomic analyses. AREAS COVERED: This review discusses the current state of tandem mass tag-centric phosphoproteomics and highlights advances in reagent chemistry, instrumentation, data acquisition, and data analysis. We stress that approaches for phosphoproteomic investigations require high-specificity enrichment, sensitive detection, and accurate phosphorylation site localization. EXPERT OPINION: Tandem mass tag-centric phosphoproteomics will continue to be an important conduit for our understanding of signal transduction in living organisms. We anticipate that progress in phosphopeptide enrichment methodologies, enhancements in instrumentation and data acquisition technologies, and further refinements in analytical strategies will be key to the discovery of biologically relevant findings from phosphoproteomics studies.
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Proteómica , Transducción de Señal , Humanos , Espectrometría de Masas , Fosfopéptidos/metabolismo , Fosfoproteínas/metabolismo , FosforilaciónRESUMEN
Phosphorylation is a post-translational modification with a vital role in cellular signaling. Isobaric labeling-based strategies, such as tandem mass tags (TMT), can measure the relative phosphorylation states of peptides in a multiplexed format. However, the low stoichiometry of protein phosphorylation constrains the depth of phosphopeptide analysis by mass spectrometry. As such, robust and sensitive workflows are required. Here we evaluate and optimize high-Field Asymmetric waveform Ion Mobility Spectrometry (FAIMS) coupled to Orbitrap Tribrid mass spectrometers for the analysis of TMT-labeled phosphopeptides. We determined that using FAIMS-MS3 with three compensation voltages (CV) in a single method (e.g., CV = -40/-60/-80 V) maximizes phosphopeptide coverage while minimizing inter-CV overlap. Furthermore, consecutive analyses using MSA-CID (multistage activation collision-induced dissociation) and HCD (higher-energy collisional dissociation) fragmentation at the MS2 stage increases the depth of phosphorylation analysis. The methodology and results outlined herein provide a template for tailoring optimized FAIMS-based methods.
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Espectrometría de Movilidad Iónica/métodos , Fosfopéptidos/análisis , Proteómica/métodos , Espectrometría de Movilidad Iónica/instrumentación , Fosfopéptidos/química , Fosfopéptidos/metabolismo , Fosforilación , Proteómica/instrumentación , Flujo de TrabajoRESUMEN
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.
Asunto(s)
Proteoma , Proteómica , Fraccionamiento Químico , Iones , PéptidosRESUMEN
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
The rise of sample multiplexing in quantitative proteomics for the dissection of complex phenotypic comparisons has been advanced by the development of ever more sensitive and robust instrumentation. Here, we evaluated the utility of the Orbitrap Eclipse Tribrid mass spectrometer (advanced quadrupole filter, optimized FTMS scan overhead) and new instrument control software features (Precursor Fit filtering, TurboTMT and Real-time Peptide Search filtering). Multidimensional comparisons of these novel features increased total peptide identifications by 20% for SPS-MS3 methods and 14% for HRMS2 methods. Importantly Real-time Peptide Search filtering enabled a â¼2× throughput improvement for quantification. Across the board, these sensitivity increases were attained without sacrificing quantitative accuracy. New hardware and software features enable more efficient characterization in pursuit of comparative whole proteome insights.