RESUMO
Mitochondria are epicentres of eukaryotic metabolism and bioenergetics. Pioneering efforts in recent decades have established the core protein componentry of these organelles1 and have linked their dysfunction to more than 150 distinct disorders2,3. Still, hundreds of mitochondrial proteins lack clear functions4, and the underlying genetic basis for approximately 40% of mitochondrial disorders remains unresolved5. Here, to establish a more complete functional compendium of human mitochondrial proteins, we profiled more than 200 CRISPR-mediated HAP1 cell knockout lines using mass spectrometry-based multiomics analyses. This effort generated approximately 8.3 million distinct biomolecule measurements, providing a deep survey of the cellular responses to mitochondrial perturbations and laying a foundation for mechanistic investigations into protein function. Guided by these data, we discovered that PIGY upstream open reading frame (PYURF) is an S-adenosylmethionine-dependent methyltransferase chaperone that supports both complex I assembly and coenzyme Q biosynthesis and is disrupted in a previously unresolved multisystemic mitochondrial disorder. We further linked the putative zinc transporter SLC30A9 to mitochondrial ribosomes and OxPhos integrity and established RAB5IF as the second gene harbouring pathogenic variants that cause cerebrofaciothoracic dysplasia. Our data, which can be explored through the interactive online MITOMICS.app resource, suggest biological roles for many other orphan mitochondrial proteins that still lack robust functional characterization and define a rich cell signature of mitochondrial dysfunction that can support the genetic diagnosis of mitochondrial diseases.
Assuntos
Mitocôndrias , Proteínas Mitocondriais , Proteínas de Transporte de Cátions , Proteínas de Ciclo Celular , Metabolismo Energético , Humanos , Espectrometria de Massas , Mitocôndrias/genética , Mitocôndrias/metabolismo , Doenças Mitocondriais/genética , Doenças Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Fatores de Transcrição , Proteínas rab5 de Ligação ao GTPRESUMO
Therapeutic RNAs are routinely modified during their synthesis to ensure proper drug uptake, stability, and efficacy. Phosphorothioate (PS) RNA, molecules in which one or more backbone phosphates are modified with a sulfur atom in place of standard nonbridging oxygen, is one of the most common modifications because of ease of synthesis and pharmacokinetic benefits. Quality assessment of RNA synthesis, including modification incorporation, is essential for drug selectivity and performance, and the synthetic nature of the PS linkage incorporation often reveals impurities. Here, we present a comprehensive analysis of PS RNA via tandem mass spectrometry (MS). We show that activated ion-negative electron transfer dissociation MS/MS is especially useful in diagnosing PS incorporation, producing diagnostic a- and z-type ions at PS linkage sites, beyond the standard d- and w-type ions. Analysis using resonant and beam-type collision-based activation reveals that, overall, more intense sequence ions and base-loss ions result when a PS modification is present. Furthermore, we report increased detection of b- and x-type product ions at sites of PS incorporation, in addition to the standard c- and y-type ions. This work reveals that the gas-phase chemical stability afforded by sulfur alters RNA dissociation and necessitates inclusion of additional product ions for MS/MS of PS RNA.
Assuntos
RNA , Espectrometria de Massas em Tandem , Espectrometria de Massas em Tandem/métodos , RNA/metabolismo , Oligonucleotídeos Fosforotioatos/químicaRESUMO
Single-cell proteomics is a powerful approach to precisely profile protein landscapes within individual cells toward a comprehensive understanding of proteomic functions and tissue and cellular states. The inherent challenges associated with limited starting material demand heightened analytical sensitivity. Just as advances in sample preparation maximize the amount of material that makes it from the cell to the mass spectrometer, we strive to maximize the number of ions that make it from ion source to the detector. In isobaric tagging experiments, limited reporter ion generation limits quantitative accuracy and precision. The combination of infrared photoactivation and ion parking circumvents the m/z dependence inherent in HCD, maximizing reporter generation and avoiding unintended degradation of TMT reporter molecules in infrared-tandem mass tags (IR-TMT). The method was applied to single-cell human proteomes using 18-plex TMTpro, resulting in 4-5-fold increases in reporter signal compared to conventional SPS-MS3 approaches. IR-TMT enables faster duty cycles, higher throughput, and increased peptide identification and quantification. Comparative experiments showcase 4-5-fold lower injection times for IR-TMT, providing superior sensitivity without compromising accuracy. In all, IR-TMT enhances the dynamic range of proteomic experiments and is compatible with gas-phase fractionation and real-time searching, promising increased gains in the study of cellular heterogeneity.
RESUMO
Mitochondria are essential for numerous cellular processes, yet hundreds of their proteins lack robust functional annotation. To reveal functions for these proteins (termed MXPs), we assessed condition-specific protein-protein interactions for 50 select MXPs using affinity enrichment mass spectrometry. Our data connect MXPs to diverse mitochondrial processes, including multiple aspects of respiratory chain function. Building upon these observations, we validated C17orf89 as a complex I (CI) assembly factor. Disruption of C17orf89 markedly reduced CI activity, and its depletion is found in an unresolved case of CI deficiency. We likewise discovered that LYRM5 interacts with and deflavinates the electron-transferring flavoprotein that shuttles electrons to coenzyme Q (CoQ). Finally, we identified a dynamic human CoQ biosynthetic complex involving multiple MXPs whose topology we map using purified components. Collectively, our data lend mechanistic insight into respiratory chain-related activities and prioritize hundreds of additional interactions for further exploration of mitochondrial protein function.
Assuntos
Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas , Proteômica/métodos , Bases de Dados de Proteínas , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Células HEK293 , Células Hep G2 , Humanos , Proteínas Mitocondriais/genética , Interferência de RNA , Transdução de Sinais , Transfecção , Ubiquinona/metabolismoRESUMO
The UbiB protein kinase-like (PKL) family is widespread, comprising one-quarter of microbial PKLs and five human homologs, yet its biochemical activities remain obscure. COQ8A (ADCK3) is a mammalian UbiB protein associated with ubiquinone (CoQ) biosynthesis and an ataxia (ARCA2) through unclear means. We show that mice lacking COQ8A develop a slowly progressive cerebellar ataxia linked to Purkinje cell dysfunction and mild exercise intolerance, recapitulating ARCA2. Interspecies biochemical analyses show that COQ8A and yeast Coq8p specifically stabilize a CoQ biosynthesis complex through unorthodox PKL functions. Although COQ8 was predicted to be a protein kinase, we demonstrate that it lacks canonical protein kinase activity in trans. Instead, COQ8 has ATPase activity and interacts with lipid CoQ intermediates, functions that are likely conserved across all domains of life. Collectively, our results lend insight into the molecular activities of the ancient UbiB family and elucidate the biochemical underpinnings of a human disease.
Assuntos
Comportamento Animal , Ataxia Cerebelar/enzimologia , Cerebelo/enzimologia , Proteínas Mitocondriais/deficiência , Músculo Esquelético/enzimologia , Ubiquinona/deficiência , Animais , Células COS , Ataxia Cerebelar/genética , Ataxia Cerebelar/fisiopatologia , Ataxia Cerebelar/psicologia , Cerebelo/fisiopatologia , Cerebelo/ultraestrutura , Chlorocebus aethiops , Modelos Animais de Doenças , Tolerância ao Exercício , Feminino , Predisposição Genética para Doença , Células HEK293 , Humanos , Metabolismo dos Lipídeos , Masculino , Aprendizagem em Labirinto , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Modelos Moleculares , Atividade Motora , Força Muscular , Músculo Esquelético/fisiopatologia , Fenótipo , Ligação Proteica , Conformação Proteica , Proteômica/métodos , Reconhecimento Psicológico , Teste de Desempenho do Rota-Rod , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Convulsões/enzimologia , Convulsões/genética , Convulsões/fisiopatologia , Relação Estrutura-Atividade , Fatores de Tempo , Transfecção , Ubiquinona/química , Ubiquinona/genéticaRESUMO
Addressing mixtures and heterogeneity in structural biology requires approaches that can differentiate and separate structures based on mass and conformation. Mass spectrometry (MS) provides tools for measuring and isolating gas-phase ions. The development of native MS including electrospray ionization allowed for manipulation and analysis of intact noncovalent biomolecules as ions in the gas phase, leading to detailed measurements of structural heterogeneity. Conversely, transmission electron microscopy (TEM) generates detailed images of biomolecular complexes that show an overall structure. Our matrix-landing approach uses native MS to probe and select biomolecular ions of interest for subsequent TEM imaging, thus unifying information on mass, stoichiometry, heterogeneity, etc., available via native MS with TEM images. Here, we prepare TEM grids of protein complexes purified via quadrupolar isolation and matrix-landing and generate 3D reconstructions of the isolated complexes. Our results show that these complexes maintain their structure through gas-phase isolation.
Assuntos
Imageamento Tridimensional , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas/métodos , Íons/química , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
We describe an apparatus for the cryogenic landing of particles from the ion beam of a mass spectrometer onto transmission electron microscope grids for cryo-electron microscopy. This system also allows for the controlled formation of thin films of amorphous ice on the grid surface. We demonstrate that as compared to room temperature landings, the use of this cryogenic landing device greatly improves the structural preservation of deposited protein-protein complexes. Furthermore, landing under cryogenic conditions can increase the diversity of particle orientations, allowing for improved 3D structural interpretation. We conclude that this approach allows for the direct coupling of mass spectrometry with cryo-electron microscopy.
Assuntos
Microscopia Crioeletrônica , Microscopia Crioeletrônica/métodos , Espectrometria de MassasRESUMO
Mass spectrometry (MS)-based proteomics is a powerful technology to globally profile protein abundances, activities, interactions, and modifications. The extreme complexity of proteomics samples, which often contain hundreds of thousands of analytes, necessitates continuous development of MS techniques and instrumentation to improve speed, sensitivity, precision, and accuracy, among other analytical characteristics. Here, we systematically evaluated the Orbitrap Ascend Tribrid mass spectrometer in the context of shotgun proteomics, and we compared its performance to that of the previous generation of Tribrid instrumentsâthe Orbitrap Eclipse. The updated architecture of the Orbitrap Ascend includes a second ion-routing multipole (IRM) in front of the redesigned C-trap/Orbitrap and a new ion funnel that allows gentler ion introduction, among other changes. These modifications in Ascend hardware configuration enabled an increase in parallelizable ion injection time during higher-energy collisional dissociation (HCD) Orbitrap tandem MS (FTMS2) analysis of â¼5 ms. This enhancement was particularly valuable in the analyses of limited sample amounts, where improvements in sensitivity resulted in up to 140% increase in the number of identified tryptic peptides. Further, analysis of phosphorylated peptides enriched from the K562 human cell line yielded up to â¼50% increase in the number of unique phosphopeptides and localized phosphosites. Strikingly, we also observed a â¼2-fold boost in the number of detected N-glycopeptides, likely owing to the improvements in ion transmission and sensitivity. In addition, we performed the multiplexed quantitative proteomics analyses of TMT11-plex labeled HEK293T tryptic peptides and observed 9-14% increase in the number of quantified peptides. In conclusion, the Orbitrap Ascend consistently outperformed its predecessor the Orbitrap Eclipse in various bottom-up proteomic analyses, and we anticipate that it will generate reproducible and in-depth datasets for numerous proteomic applications.
Assuntos
Proteínas , Proteômica , Humanos , Proteômica/métodos , Células HEK293 , Proteínas/química , Espectrometria de Massas em Tandem/métodos , FosfopeptídeosRESUMO
Oral microbiome influences human health, specifically prediabetes and type 2 diabetes (Pre-DM/DM) and periodontal diseases (PDs), through complex microbial interactions. To explore these relations, we performed 16S rDNA sequencing, metabolomics, lipidomics, and proteomics analyses on supragingival dental plaque collected from individuals with Pre-DM/DM (n = 39), Pre-DM/DM and PD (n = 37), PD alone (n = 11), or neither (n = 10). We identified on average 2790 operational taxonomic units and 2025 microbial and host proteins per sample and quantified 110 metabolites and 415 lipids. Plaque samples from Pre-DM/DM patients contained higher abundance of Fusobacterium and Tannerella than plaques from metabolically healthy patients. Phosphatidylcholines, plasmenyl phosphatidylcholines, ceramides containing non-OH fatty acids, and host proteins related to actin filament rearrangement were elevated in plaques from PD versus non-PD samples. Cross-omic correlation analysis enabled the detection of a strong association between Lautropia and monomethyl phosphatidylethanolamine (PE-NMe), which is striking because synthesis of PE-NMe is uncommon in oral bacteria. Lipidomics analysis of in vitro cultures of Lautropia mirabilis confirmed the synthesis of PE-NMe by the bacteria. This comprehensive analysis revealed a novel microbial metabolic pathway and significant associations of host-derived proteins with PD.
Assuntos
Proteínas de Bactérias/metabolismo , Burkholderiaceae/metabolismo , Placa Dentária/química , Placa Dentária/microbiologia , Diabetes Mellitus Tipo 2/microbiologia , Doenças Periodontais/microbiologia , Adulto , Idoso , Burkholderiaceae/genética , Feminino , Humanos , Masculino , Metabolômica , Pessoa de Meia-Idade , Proteômica , RNA Ribossômico 16S , Adulto JovemRESUMO
Recently, we described the use of a chemical matrix for landing and preserving the cations of protein-protein complexes within a mass spectrometer (MS) instrument. By use of a glycerol-landing matrix, we used negative stain transmission electron microscopy (TEM) to obtain a three-dimensional (3D) reconstruction of landed GroEL complexes. Here, we investigate the utilities of other chemical matrices for their abilities to land, preserve, and allow for direct imaging of these cationic particles using TEM. We report here that poly(propylene) glycol (PPG) offers superior performance over glycerol for matrix landing. We demonstrated the utility of the PPG matrix landing using three protein-protein complexesâGroEL, the 20S proteasome core particle, and ß-galactosidaseâand obtained a 3D reconstruction of each complex from matrix-landed particles. These structures have no detectable differences from the structures obtained using conventional preparation methods, suggesting the structures are well preserved at least to the resolution limit of the reconstructions (â¼20 Å). We conclude that matrix landing offers a direct approach to couple native MS with TEM for protein structure determination.
Assuntos
Glicerol , Proteínas , Microscopia Eletrônica , Espectrometria de Massas , Proteínas/análiseRESUMO
Isobaric tagging facilitates multiplexed experiments that can determine sequences and relative amounts of peptides in biological samples using tandem mass spectrometry (MSn). Limited reporter ion generation limits quantitative accuracy and precision. As reporter ions are susceptible to unintended fragmentation and scattering by high-energy collisions, we activated peptides with IR photons and prevented successive dissociation of generated reporter ions with ion parking, which altogether boosted reporter ion yield by up to 55%. Even so, unintended co-isolation of contaminating peaks in MS2 experiments distorts reporter ion intensities and can distort quantitative information. MS3 experiments address contamination by generating reporter ions via collisional activation (HCD) of one or more peptide product ions rather than the isolated peptide precursor ion. Because HCD performance is related to m/z, activation of multiple synchronously isolated product ions generates less than optimal reporter ion intensities. In this work, we show that using infrared multiphoton dissociation, which is not dependent on m/z, to generate reporter ions from 10 synchronously isolated peptide product ions results in a 2.4-fold increase in reporter ion intensities, significantly enhancing the sensitivity and dynamic range of quantitation via isobaric tagging.
Assuntos
Peptídeos , Espectrometria de Massas em Tandem , Indicadores e Reagentes , Íons , Peptídeos/química , Espectrometria de Massas em Tandem/métodosRESUMO
Mass spectrometry (MS) serves as the centerpiece technology for proteome, lipidome, and metabolome analysis. To gain a better understanding of the multifaceted networks of myriad regulatory layers in complex organisms, integration of different multiomic layers is increasingly performed, including joint extraction methods of diverse biomolecular classes and comprehensive data analyses of different omics. Despite the versatility of MS systems, fractured methodology drives nearly all MS laboratories to specialize in analysis of a single ome at the exclusion of the others. Although liquid chromatography-mass spectrometry (LC-MS) analysis is similar for different biomolecular classes, the integration on the instrument level is lagging behind. The recent advancements in high flow proteomics enable us to take a first step towards integration of protein and lipid analysis. Here, we describe a technology to achieve broad and deep coverage of multiple molecular classes simultaneously through multi-omic single-shot technology (MOST), requiring only one column, one LC-MS instrument, and a simplified workflow. MOST achieved great robustness and reproducibility. Its application to a Saccharomyces cerevisiae study consisting of 20 conditions revealed 2842 protein groups and 325 lipids and potential molecular relationships.
Assuntos
Lipidômica , Proteoma , Cromatografia Líquida , Reprodutibilidade dos Testes , TecnologiaRESUMO
Calorie restriction (CR) extends life span in diverse species. Mitochondria play a key role in CR adaptation; however, the molecular details remain elusive. We developed and applied a quantitative mass spectrometry method to probe the liver mitochondrial acetyl-proteome during CR versus control diet in mice that were wild-type or lacked the protein deacetylase SIRT3. Quantification of 3,285 acetylation sites-2,193 from mitochondrial proteins-rendered a comprehensive atlas of the acetyl-proteome and enabled global site-specific, relative acetyl occupancy measurements between all four experimental conditions. Bioinformatic and biochemical analyses provided additional support for the effects of specific acetylation on mitochondrial protein function. Our results (1) reveal widespread reprogramming of mitochondrial protein acetylation in response to CR and SIRT3, (2) identify three biochemically distinct classes of acetylation sites, and (3) provide evidence that SIRT3 is a prominent regulator in CR adaptation by coordinately deacetylating proteins involved in diverse pathways of metabolism and mitochondrial maintenance.
Assuntos
Restrição Calórica , Proteínas Mitocondriais/metabolismo , Proteoma/metabolismo , Sirtuína 3/fisiologia , Acetilcoenzima A/metabolismo , Acetilação , Adaptação Fisiológica , Motivos de Aminoácidos , Sequência de Aminoácidos , Aminoácidos/metabolismo , Animais , Metabolismo dos Carboidratos , Células Cultivadas , Cromatografia por Troca Iônica , Análise por Conglomerados , Sequência Consenso , Expressão Gênica , Genes Mitocondriais , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Hepáticas/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/isolamento & purificação , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Processamento de Proteína Pós-Traducional , Proteoma/química , Proteoma/isolamento & purificação , Sirtuína 3/química , Sirtuína 3/isolamento & purificação , Sirtuína 3/metabolismo , Coloração e Rotulagem , Espectrometria de Massas em TandemRESUMO
Mass spectrometry is the premier tool for identifying and quantifying protein phosphorylation on a global scale. Analysis of phosphopeptides requires enrichment, and even after the samples remain highly complex and exhibit broad dynamic range of abundance. Achieving maximal depth of coverage for phosphoproteomics therefore typically necessitates offline liquid chromatography prefractionation, a time-consuming and laborious approach. Here, we incorporate a recently commercialized aerodynamic high-field asymmetric waveform ion mobility spectrometry (FAIMS) device into the phosphoproteomic workflow. We characterize the effects of phosphorylation on the FAIMS separation, describe optimized compensation voltage settings for unlabeled phosphopeptides, and demonstrate the advantages of FAIMS-enabled gas-phase fractionation. Standard FAIMS single-shot analyses identified around 15-20% additional phosphorylation sites than control experiments without FAIMS. In comparison to liquid chromatography prefractionation, FAIMS experiments yielded similar or superior results when analyzing up to four discrete gas-phase fractions. Although using FAIMS led to a modest reduction in the precision of quantitative measurements when using label-free approaches, the data collected with FAIMS yielded a 26% increase in total reproducible measurements. Overall, we conclude that the new FAIMS technology is a valuable addition to any phosphoproteomic workflow, with greater benefits emerging from longer analyses and higher amounts of material.
Assuntos
Espectrometria de Massas/instrumentação , Fosfoproteínas/metabolismo , Proteômica/instrumentação , Sítios de Ligação , Fosforilação , Fluxo de TrabalhoRESUMO
Modified oligonucleotides represent a promising avenue for drug development, with small interfering RNAs (siRNA) and microRNAs gaining traction in the therapeutic market. Mass spectrometry (MS)-based analysis offers many benefits for characterizing modified nucleic acids. Negative electron transfer dissociation (NETD) has proven valuable in sequencing oligonucleotide anions, particularly because it can retain modifications while generating sequence-informative fragments. We show that NETD can be successfully implemented on a widely available quadrupole-Orbitrap-linear ion trap mass spectrometer that uses a front-end glow discharge source to generate radical fluoranthene reagent cations. We characterize both unmodified and modified ribonucleic acids and present the first application of activated-ion negative electron transfer dissociation (AI-NETD) to nucleic acids. AI-NETD achieved 100% sequence coverage for both a 6-mer (5'-rGmUrArCmUrG-3') with 2'-O-methyl modifications and a 21-mer (5'-rCrArUrCrCrUrCrUrArGrArGrGrArUrArGrArArUrG-3'), the luciferase antisense siRNA. Both NETD and AI-NETD afforded complete sequence coverage of these molecules while maintaining a relatively low degree of undesired base-loss products and internal products relative to collision-based methods.
Assuntos
MicroRNAs/análise , RNA Interferente Pequeno/análise , Sequência de Aminoácidos , Transporte de Elétrons , Espectrometria de MassasRESUMO
Monoclonal antibodies (mAbs) are important therapeutic glycoproteins, but their large size and structural complexity make them difficult to rapidly characterize. Top-down mass spectrometry (MS) has the potential to overcome challenges of other common approaches by minimizing sample preparation and preserving endogenous modifications. However, comprehensive mAb characterization requires generation of many, well-resolved fragments and remains challenging. While ETD retains modifications and cleaves disulfide bonds-making it attractive for mAb characterization-it can be less effective for precursors having high m/z values. Activated ion electron transfer dissociation (AI-ETD) uses concurrent infrared photoactivation to promote product ion generation and has proven effective in increasing sequence coverage of intact proteins. Here, we present the first application of AI-ETD to mAb sequencing. For the standard NIST mAb, we observe a high degree of complementarity between fragments generated using standard ETD with a short reaction time and AI-ETD with a long reaction time. Most importantly, AI-ETD reveals disulfide-bound regions that have been intractable, thus far, for sequencing with top-down MS. We conclude AI-ETD has the potential to rapidly and comprehensively analyze intact mAbs.
Assuntos
Anticorpos Monoclonais/química , Técnicas Eletroquímicas/métodos , Sequência de Aminoácidos , Transporte de Elétrons , Íons/químicaRESUMO
Photoactivation and photodissociation have long proven to be useful tools in tandem mass spectrometry, but implementation often involves cumbersome and potentially dangerous configurations. Here, we redress this problem by using a fiber-optic cable to couple an infrared (IR) laser to a mass spectrometer for robust, efficient, and safe photoactivation experiments. Transmitting 10.6 µm IR photons through a hollow-core fiber, we show that such fiber-assisted activated ion-electron transfer dissociation (AI-ETD) and IR multiphoton dissociation (IRMPD) experiments can be carried out as effectively as traditional mirror-based implementations. We report on the transmission efficiency of the hollow-core fiber for conducting photoactivation experiments and perform various intact protein and peptide analyses to illustrate the benefits of fiber-assisted AI-ETD, namely, a simplified system for irradiating the two-dimensional linear ion trap volume concurrent with ETD reactions to limit uninformative nondissociative events and thereby amplify sequence coverage. We also describe a calibration scheme for the routine analysis of IR laser alignment and power through the fiber and into the dual cell quadrupolar linear ion trap. In all, these advances allow for a more robust, straightforward, and safe instrumentation platform, permitting implementation of AI-ETD and IRMPD on commercial mass spectrometers and broadening the accessibility of these techniques.
Assuntos
Mioglobina/análise , Fibras Ópticas , Peptídeos/análise , Ubiquitina/análise , Animais , Calibragem , Bovinos , Cavalos , Lasers , Espectrometria de Massas , Processos FotoquímicosRESUMO
The dependence of capillary zone electrophoresis (CZE) separations on the charge state of the analyte is useful for the analysis of many post-translational modifications in proteins. In this work, we coupled CZE to an Orbitrap Fusion Lumos Tribrid platform with an advanced peak determination algorithm for phosphoproteomics analysis. A linear-polyacrylamide-coated capillary with very low electroosmotic flow was used for the separation. The optimal injection volume was between 100 and 150 nL of a solution of phosphopeptides in 30 mM ammonium bicarbonate (pH 8.2) buffer, which produces a dynamic pH junction sample injection. Larger injection volumes resulted in serious peak broadening and decreased numbers of phosphopeptide identifications. The optimized system identified 4405 phosphopeptides from 220 ng of enriched phosphopeptides from mouse brain, which represents the state-of-the-art result for single-shot CZE-ESI-MS/MS-based phosphoproteome analysis. We found that the migration time for phosphopeptides is much longer than that for non-phosphopeptides and increased along with the number of phosphorylation sites on the peptides, as expected for the additional negative charges associated with the phosphate groups. We also investigated the phosphorylation site motifs; a number of motifs appeared in the CZE-ESI-MS/MS data but not in LC-ESI-MS/MS data, which suggested the complementary performance of the techniques. The data are available via ProteomeXchange with identifier PXD012888.
Assuntos
Eletroforese Capilar , Fosfopeptídeos/isolamento & purificação , Proteômica/métodos , Espectrometria de Massas em Tandem , Algoritmos , Cromatografia Líquida , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/isolamento & purificação , Fosfopeptídeos/química , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Detecting the phosphorylation substrates of multiple kinases in a single experiment is a challenge, and new techniques are being developed to overcome this challenge. Here, we used a multiplexed assay for kinase specificity (MAKS) to identify the substrates directly and to map the phosphorylation site(s) of plant symbiotic receptor-like kinases. The symbiotic receptor-like kinases nodulation receptor-like kinase (NORK) and lysin motif domain-containing receptor-like kinase 3 (LYK3) are indispensable for the establishment of root nodule symbiosis. Although some interacting proteins have been identified for these symbiotic receptor-like kinases, very little is known about their phosphorylation substrates. Using this high-throughput approach, we identified several other potential phosphorylation targets for both these symbiotic receptor-like kinases. In particular, we also discovered the phosphorylation of LYK3 by NORK itself, which was also confirmed by pairwise kinase assays. Motif analysis of potential targets for these kinases revealed that the acidic motif xxxsDxxx was common to both of them. In summary, this high-throughput technique catalogs the potential phosphorylation substrates of multiple kinases in a single efficient experiment, the biological characterization of which should provide a better understanding of phosphorylation signaling cascade in symbiosis.
Assuntos
Medicago truncatula/enzimologia , Medicago truncatula/metabolismo , Medicago truncatula/genética , Fosforilação/genética , Fosforilação/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Simbiose/genética , Simbiose/fisiologia , Espectrometria de Massas em TandemRESUMO
Extreme sample complexity is an inherent challenge in shotgun proteomics that positions quality of chromatographic separations as one of the key determinants of attainable proteome coverage. In search of better separations, macroscopic physical characteristics of capillary columns, i.e., length and properties of stationary phase particles, are typically considered and optimized, while significance of packing bed morphology is frequently underappreciated. Here, we describe a technology that enables packing of capillary columns at excess of 30,000 psi and demonstrate that such columns exhibit reduced backpressure and remarkably reproducible chromatographic performance, improved on average by 23%. These enhancements afford up to 35% increase in the depth of commonplace bottom-up proteomic analyses, owning to augmented sensitivity and resolution of peptide separations and improvements in spectral quality. Our findings strongly corroborate advantages of ultra-high pressure packing of capillary columns for diverse shotgun proteomic workflows.