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BACKGROUND: Different types of analytical methods, with different characteristics, are applied in metabolomics and lipidomics research and include untargeted, targeted and semi-targeted methods. Ultra High Performance Liquid Chromatography-Mass Spectrometry is one of the most frequently applied measurement instruments in metabolomics because of its ability to detect a large number of water-soluble and lipid metabolites over a wide range of concentrations in short analysis times. Methods applied for the detection and quantification of metabolites differ and can either report a (normalised) peak area or an absolute concentration. AIM OF REVIEW: In this tutorial we aim to (1) define similarities and differences between different analytical approaches applied in metabolomics and (2) define how amounts or absolute concentrations of endogenous metabolites can be determined together with the advantages and limitations of each approach in relation to the accuracy and precision when concentrations are reported. KEY SCIENTIFIC CONCEPTS OF REVIEW: The pre-analysis knowledge of metabolites to be targeted, the requirement for (normalised) peak responses or absolute concentrations to be reported and the number of metabolites to be reported define whether an untargeted, targeted or semi-targeted method is applied. Fully untargeted methods can only provide (normalised) peak responses and fold changes which can be reported even when the structural identity of the metabolite is not known. Targeted methods, where the analytes are known prior to the analysis, can also report fold changes. Semi-targeted methods apply a mix of characteristics of both untargeted and targeted assays. For the reporting of absolute concentrations of metabolites, the analytes are not only predefined but optimized analytical methods should be developed and validated for each analyte so that the accuracy and precision of concentration data collected for biological samples can be reported as fit for purpose and be reviewed by the scientific community.
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Espectrometría de Masas , Metabolómica , Metabolómica/métodos , Cromatografía Líquida de Alta Presión/métodos , Espectrometría de Masas/métodos , HumanosRESUMEN
INTRODUCTION: The metabolomics quality assurance and quality control consortium (mQACC) is enabling the identification, development, prioritization, and promotion of suitable reference materials (RMs) to be used in quality assurance (QA) and quality control (QC) for untargeted metabolomics research. OBJECTIVES: This review aims to highlight current RMs, and methodologies used within untargeted metabolomics and lipidomics communities to ensure standardization of results obtained from data analysis, interpretation and cross-study, and cross-laboratory comparisons. The essence of the aims is also applicable to other 'omics areas that generate high dimensional data. RESULTS: The potential for game-changing biochemical discoveries through mass spectrometry-based (MS) untargeted metabolomics and lipidomics are predicated on the evolution of more confident qualitative (and eventually quantitative) results from research laboratories. RMs are thus critical QC tools to be able to assure standardization, comparability, repeatability and reproducibility for untargeted data analysis, interpretation, to compare data within and across studies and across multiple laboratories. Standard operating procedures (SOPs) that promote, describe and exemplify the use of RMs will also improve QC for the metabolomics and lipidomics communities. CONCLUSIONS: The application of RMs described in this review may significantly improve data quality to support metabolomics and lipidomics research. The continued development and deployment of new RMs, together with interlaboratory studies and educational outreach and training, will further promote sound QA practices in the community.
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Lipidómica , Metabolómica , Espectrometría de Masas/métodos , Metabolómica/métodos , Control de Calidad , Reproducibilidad de los ResultadosRESUMEN
Mutations of the KRAS gene are found in human cancers with high frequency and result in the constitutive activation of its protein products. This leads to aberrant regulation of downstream pathways, promoting cell survival, proliferation, and tumorigenesis that drive cancer progression and negatively affect treatment outcomes. Here, we describe a workflow that can detect and quantify mutation-specific consequences of KRAS biochemistry, namely linked changes in posttranslational modifications (PTMs). We combined immunoaffinity enrichment with detection by top-down mass spectrometry to discover and quantify proteoforms with or without the Gly13Asp mutation (G13D) specifically in the KRAS4b isoform. The workflow was applied first to isogenic KRAS colorectal cancer (CRC) cell lines and then to patient CRC tumors with matching KRAS genotypes. In two cellular models, a direct link between the knockout of the mutant G13D allele and the complete nitrosylation of cysteine 118 of the remaining WT KRAS4b was observed. Analysis of tumor samples quantified the percentage of mutant KRAS4b actually present in cancer tissue and identified major differences in the levels of C-terminal carboxymethylation, a modification critical for membrane association. These data from CRC cells and human tumors suggest mechanisms of posttranslational regulation that are highly context-dependent and which lead to preferential production of specific KRAS4b proteoforms.
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Neoplasias Colorrectales/enzimología , Mutación Missense , Proteínas de Neoplasias/análisis , Mutación Puntual , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas p21(ras)/análisis , Secuencia de Aminoácidos , Línea Celular Tumoral , Membrana Celular/metabolismo , Cromatografía Liquida , Neoplasias Colorrectales/genética , Cisteína/química , Humanos , Metilación , Modelos Moleculares , Proteínas de Neoplasias/química , Proteínas de Neoplasias/aislamiento & purificación , Nitrosación , Prenilación , Conformación Proteica , Proteómica/métodos , Proteínas Proto-Oncogénicas p21(ras)/química , Proteínas Proto-Oncogénicas p21(ras)/aislamiento & purificación , Proteínas Recombinantes/química , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Espectrometría de Masas en TándemRESUMEN
We describe here the agreed upon first development steps and priority objectives of a community engagement effort to address current challenges in quality assurance (QA) and quality control (QC) in untargeted metabolomic studies. This has included (1) a QA and QC questionnaire responded to by the metabolomics community in 2015 which recommended education of the metabolomics community, development of appropriate standard reference materials and providing incentives for laboratories to apply QA and QC; (2) a 2-day 'Think Tank on Quality Assurance and Quality Control for Untargeted Metabolomic Studies' held at the National Cancer Institute's Shady Grove Campus and (3) establishment of the Metabolomics Quality Assurance and Quality Control Consortium (mQACC) to drive forward developments in a coordinated manner.
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Metabolómica/métodos , Metabolómica/normas , Humanos , Laboratorios , Control de Calidad , Mejoramiento de la CalidadRESUMEN
Bottom-up proteomics relies on the use of proteases and is the method of choice for identifying thousands of protein groups in complex samples. Top-down proteomics has been shown to be robust for direct analysis of small proteins and offers a solution to the "peptide-to-protein" inference problem inherent with bottom-up approaches. Here, we describe the first large-scale integration of genomic, bottom-up and top-down proteomic data for the comparative analysis of patient-derived mouse xenograft models of basal and luminal B human breast cancer, WHIM2 and WHIM16, respectively. Using these well-characterized xenograft models established by the National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium, we compared and contrasted the performance of bottom-up and top-down proteomics to detect cancer-specific aberrations at the peptide and proteoform levels and to measure differential expression of proteins and proteoforms. Bottom-up proteomic analysis of the tumor xenografts detected almost 10 times as many coding nucleotide polymorphisms and peptides resulting from novel splice junctions than top-down. For proteins in the range of 0-30 kDa, where quantitation was performed using both approaches, bottom-up proteomics quantified 3,519 protein groups from 49,185 peptides, while top-down proteomics quantified 982 proteoforms mapping to 358 proteins. Examples of both concordant and discordant quantitation were found in a â¼60:40 ratio, providing a unique opportunity for top-down to fill in missing information. The two techniques showed complementary performance, with bottom-up yielding eight times more identifications of 0-30 kDa proteins in xenograft proteomes, but failing to detect differences in certain posttranslational modifications (PTMs), such as phosphorylation pattern changes of alpha-endosulfine. This work illustrates the potency of a combined bottom-up and top-down proteomics approach to deepen our knowledge of cancer biology, especially when genomic data are available.
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Neoplasias de la Mama/metabolismo , Xenoinjertos/metabolismo , Proteoma/metabolismo , Proteómica/métodos , Animales , Neoplasias de la Mama/genética , Cromatografía Líquida de Alta Presión , Femenino , Genotipo , Humanos , Ratones , Peso Molecular , Péptidos/genética , Péptidos/metabolismo , Polimorfismo de Nucleótido Simple , Proteoma/química , Proteoma/genética , Espectrometría de Masas en Tándem , Trasplante HeterólogoRESUMEN
The normal cellular role of α-synuclein is of potential importance in understanding diseases in which an aggregated form of the protein has been implicated. A potential loss or change in the normal function of α-synuclein could play a role in the aetiology of diseases such as Parkinson's disease. Recently, it has been suggested that α-synuclein could cause the enzymatic reduction of iron and a cellular increase in Fe(II) levels. Experiments were carried out to determine if such activity could be measured in vivo. Experiments with rats overexpressing human α-synuclein in nigral dopaminergic neurons demonstrated a correlation between α-synuclein expression and ferrireductase activity. Furthermore, studies on tissue from Parkinson's disease patient brains showed a significant decrease in ferrireductase activity, possibly due to deposition of large amounts of inactive protein. Cellular studies suggest that increase ferrireductase activity results in increased levels of dopamine metabolites and increased sensitivity to the toxicity of DOPAL. These findings demonstrate that α-synuclein ferrireductase activity is present in vivo and its alteration may play a role in neuron loss in disease.
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Encéfalo/metabolismo , Enfermedad de Parkinson/metabolismo , alfa-Sinucleína/metabolismo , Animales , FMN Reductasa/metabolismo , Femenino , Humanos , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Lymphocytes are immune cells that are critical for the maintenance of adaptive immunity. Differentiation of lymphoid progenitors yields B-, T-, and NK-cell subtypes that individually correlate with specific forms of leukemia or lymphoma. Therefore, it is imperative a precise method of cell categorization is utilized to detect differences in distinct disease states present in patients. One viable means of classification involves evaluation of the cell surface proteome of lymphoid malignancies. Specifically, this manuscript details the use of an antibody independent approach known as Cell Surface Capture Technology, to assess the N-glycoproteome of four human lymphocyte cell lines. Altogether, 404 cell surface N-glycoproteins were identified as markers for specific cell types involved in lymphocytic malignancies, including 82 N-glycoproteins that had not been previously been described for B or T cells within the Cell Surface Protein Atlas. Comparative analysis, hierarchical clustering techniques, and label-free quantitation were used to reveal proteins most informative for each cell type. Undoubtedly, the characterization of the cell surface proteome of lymphoid malignancies is a first step toward improving personalized diagnosis and treatment of leukemia and lymphoma.
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Biomarcadores de Tumor/metabolismo , Membrana Celular/metabolismo , Glicoproteínas/metabolismo , Leucemia/metabolismo , Linfocitos/metabolismo , Linfoma/metabolismo , Proteoma/análisis , Células Cultivadas , Humanos , Leucemia/patología , Linfocitos/citología , Linfoma/patología , Proteómica/métodosRESUMEN
α-Synuclein (α-syn) is a cytosolic protein known for its association with neurodegenerative diseases, including Parkinson's disease and other synucleinopathies. The potential cellular function of α-synuclein may be of consequence for understanding the pathogenesis of such diseases. Previous work has suggested that α-synuclein can catalyze the reduction of iron as a ferrireductase. We performed a detailed analysis of the steady-state kinetics of recombinant α-syn ferrireductase activity and for disease-associated variants. Our study illustrates that the ferrireductase activity we observed is clearly commensurate with bona fide enzyme activity and suggests a mechanistic rationale for the activity and the relationship to cellular regulation of the pool of Fe(III) and Fe(II). Using cell-based studies, we examined the functionally active conformation and found that the major catalytically active form is a putative membrane-associated tetramer. Using an artificial membrane environment with recombinant protein, we demonstrate that secondary structure folding of α-synuclein is insufficient to allow enzyme activity and the absolute specificity of the tertiary/quaternary structure is the primary requirement. Finally, we explored the steady-state kinetics of a range of disease α-synuclein variants and found that variants involved in neurodegenerative disease exhibited major changes in their enzymatic activity. We discuss these data in the context of a potential disease-associated mechanism for aberrant α-synuclein ferrireductase activity.
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FMN Reductasa/metabolismo , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Proteínas del Tejido Nervioso/metabolismo , Neuronas/enzimología , alfa-Sinucleína/metabolismo , Sustitución de Aminoácidos , Sitios de Unión , Biocatálisis , Línea Celular Tumoral , FMN Reductasa/química , FMN Reductasa/genética , Humanos , Liposomas , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Peso Molecular , Mutación , Nanoestructuras/química , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Solubilidad , Especificidad por Sustrato , alfa-Sinucleína/química , alfa-Sinucleína/genéticaRESUMEN
The ability to site-specifically incorporate non-canonical amino acids (ncAAs) into proteins has made possible the study of protein structure and function in fundamentally new ways, as well as the bio synthesis of unnatural polymers. However, the task of site-specifically incorporating multiple ncAAs into proteins with high purity and yield continues to present a challenge. At the heart of this challenge lies the lower efficiency of engineered orthogonal translation system components compared to their natural counterparts (e.g., translation elements that specifically use a ncAA and do not interact with the cell's natural translation apparatus). Here, we show that evolving and tuning expression levels of multiple components of an engineered translation system together as a whole enhances ncAA incorporation efficiency. Specifically, we increase protein yield when incorporating multiple p-azido-phenylalanine(pAzF) residues into proteins by (i) evolving the Methanocaldococcus jannaschii p-azido-phenylalanyl-tRNA synthetase anti-codon binding domain, (ii) evolving the elongation factor Tu amino acid-binding pocket, and (iii) tuning the expression of evolved translation machinery components in a single vector. Use of the evolved translation machinery in a genomically recoded organism lacking release factor one enabled enhanced multi-site ncAA incorporation into proteins. We anticipate that our approach to orthogonal translation system development will accelerate and expand our ability to site-specifically incorporate multiple ncAAs into proteins and biopolymers, advancing new horizons for synthetic and chemical biotechnology. Biotechnol. Bioeng. 2017;114: 1074-1086. © 2016 Wiley Periodicals, Inc.
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Aminoácidos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Biosíntesis de Proteínas , Ingeniería de Proteínas/métodos , Aminoacil-ARNt Sintetasas/metabolismo , Modelos Moleculares , Factor Tu de Elongación Peptídica/metabolismo , Biosíntesis de Proteínas/genética , Biosíntesis de Proteínas/fisiología , Proteínas/genética , Proteínas/metabolismoRESUMEN
Echinococcus granulosus is the causative agent of cystic hydatid disease, a neglected zoonosis responsible for high morbidity and mortality. Several molecular mechanisms underlying parasite biology remain poorly understood. Here, E. granulosus subcellular fractions were analyzed by top down and bottom up proteomics for protein identification and characterization of co-translational and post-translational modifications (CTMs and PTMs, respectively). Nuclear and cytosolic extracts of E. granulosus protoscoleces were fractionated by 10% GELFrEE and proteins under 30 kDa were analyzed by LC-MS/MS. By top down analysis, 186 proteins and 207 proteoforms were identified, of which 122 and 52 proteoforms were exclusively detected in nuclear and cytosolic fractions, respectively. CTMs were evident as 71% of the proteoforms had methionine excised and 47% were N-terminal acetylated. In addition, in silico internal acetylation prediction coupled with top down MS allowed the characterization of 9 proteins differentially acetylated, including histones. Bottom up analysis increased the overall number of identified proteins in nuclear and cytosolic fractions to 154 and 112, respectively. Overall, our results provided the first description of the low mass proteome of E. granulosus subcellular fractions and highlighted proteoforms with CTMs and PTMS whose characterization may lead to another level of understanding about molecular mechanisms controlling parasitic flatworm biology.
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Echinococcus granulosus/metabolismo , Proteínas del Helminto/aislamiento & purificación , Histonas/aislamiento & purificación , Procesamiento Proteico-Postraduccional , Proteoma/aislamiento & purificación , Proteómica/métodos , Acetilación , Secuencia de Aminoácidos , Animales , Bovinos , Núcleo Celular/química , Núcleo Celular/parasitología , Cromatografía Liquida , Citosol/química , Citosol/parasitología , Equinococosis/parasitología , Equinococosis/patología , Echinococcus granulosus/genética , Echinococcus granulosus/crecimiento & desarrollo , Células Epiteliales/química , Células Epiteliales/parasitología , Proteínas del Helminto/genética , Proteínas del Helminto/metabolismo , Histonas/genética , Histonas/metabolismo , Estadios del Ciclo de Vida/genética , Pulmón/química , Pulmón/parasitología , Metionina/química , Metionina/metabolismo , Anotación de Secuencia Molecular , Datos de Secuencia Molecular , Proteoma/genética , Proteoma/metabolismo , Proteómica/instrumentación , Espectrometría de Masas en TándemRESUMEN
Site-specific incorporation of non-standard amino acids (NSAAs) into proteins opens the way to novel biological insights and applications in biotechnology. Here, we describe the development of a high yielding cell-free protein synthesis (CFPS) platform for NSAA incorporation from crude extracts of genomically recoded Escherichia coli lacking release factor 1. We used genome engineering to construct synthetic organisms that, upon cell lysis, lead to improved extract performance. We targeted five potential negative effectors to be disabled: the nuclease genes rna, rnb, csdA, mazF, and endA. Using our most productive extract from strain MCJ.559 (csdA(-) endA(-)), we synthesized 550±40 µg mL(-1) of modified superfolder green fluorescent protein containing p-acetyl-L-phenylalanine. This yield was increased to â¼1300 µg mL(-1) when using a semicontinuous method. Our work has implications for using whole genome editing for CFPS strain development, expanding the chemistry of biological systems, and cell-free synthetic biology.
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Biotecnología , Escherichia coli/genética , Escherichia coli/metabolismo , Ingeniería Genética , Factores de Terminación de Péptidos/deficiencia , Biosíntesis de Proteínas , Aminoácidos/química , Aminoácidos/metabolismo , Sistema Libre de Células , Proteínas de Escherichia coli/genética , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/química , Factores de Terminación de Péptidos/genéticaRESUMEN
We report a simple temperature-responsive bioconjugate system comprising superfolder green fluorescent protein (sfGFP) decorated with poly[(oligo ethylene glycol) methyl ether methacrylate] (PEGMA) polymers. We used amber suppression to site-specifically incorporate the non-canonical azide-functional amino acid p-azidophenylalanine (pAzF) into sfGFP at different positions. The azide moiety on modified sfGFP was then coupled using copper-catalyzed "click" chemistry with the alkyne terminus of a PEGMA synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization. The protein in the resulting bioconjugate was found to remain functionally active (i.e., fluorescent) after conjugation. Turbidity measurements revealed that the point of attachment of the polymer onto the protein scaffold has an impact on the thermoresponsive behavior of the resultant bioconjugate. Furthermore, small-angle X-ray scattering analysis showed the wrapping of the polymer around the protein in a temperature-dependent fashion. Our work demonstrates that standard genetic manipulation combined with an expanded genetic code provides an easy way to construct functional hybrid biomaterials where the location of the conjugation site on the protein plays an important role in determining material properties. We anticipate that our approach could be generalized for the synthesis of complex functional materials with precisely defined domain orientation, connectivity, and composition.
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Proteínas Fluorescentes Verdes/química , Metacrilatos/química , Polietilenglicoles/química , Temperatura , Azidas/química , Hidrodinámica , Modelos Moleculares , Fenilalanina/análogos & derivados , Fenilalanina/química , Conformación ProteicaRESUMEN
The direct analysis of intact proteins via MS offers compelling advantages in comparison to alternative methods due to the direct and unambiguous identification and characterization of protein sequences it provides. The inability to efficiently analyze proteins in the "middle mass range," defined here as proteins from 30 to 80 kDa, in a robust fashion has limited the adoption of these "top-down" methods. Largely, a result of poor liquid chromatographic performance, the limitations in this mass range may be addressed by alternative separations that replace chromatography. Herein, the short migration times of CZE-ESI-MS/MS have been extended to size-sorted whole proteins in complex mixtures from Pseudomonas aeruginosa PA01. An electrokinetically pumped nanospray interface, a coated capillary, and a stacking method for on-column sample concentration were developed to achieve high-loading capacity and separation resolution. We achieved full width at half maximum of 8-16 s for model proteins up to 29 kDa and identified 30 proteins in the mass range of 30-80 kDa from P. aeruginosa PA01 whole cell lysate. These results suggest that CZE-ESI-MS/MS is capable of identifying proteins in the middle mass range in top-down proteomics.
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Electroforesis Capilar/métodos , Proteínas/análisis , Proteínas/química , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Reproducibilidad de los Resultados , Espectrometría de Masa por Ionización de Electrospray/métodosRESUMEN
Pilot Project #1--the identification and characterization of human histone H4 proteoforms by top-down MS--is the first project launched by the Consortium for Top-Down Proteomics (CTDP) to refine and validate top-down MS. Within the initial results from seven participating laboratories, all reported the probability-based identification of human histone H4 (UniProt accession P62805) with expectation values ranging from 10(-13) to 10(-105). Regarding characterization, a total of 74 proteoforms were reported, with 21 done so unambiguously; one new PTM, K79ac, was identified. Inter-laboratory comparison reveals aspects of the results that are consistent, such as the localization of individual PTMs and binary combinations, while other aspects are more variable, such as the accurate characterization of low-abundance proteoforms harboring >2 PTMs. An open-access tool and discussion of proteoform scoring are included, along with a description of general challenges that lie ahead including improved proteoform separations prior to mass spectrometric analysis, better instrumentation performance, and software development.
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Proteómica/métodos , Cromatografía Liquida/métodos , Análisis por Conglomerados , Células HeLa , Histonas/análisis , Histonas/química , Humanos , Espectrometría de Masas/métodos , Proyectos Piloto , Procesamiento Proteico-Postraduccional , Programas InformáticosRESUMEN
The ability to study organisms by direct analysis of their proteomes without digestion via mass spectrometry has benefited greatly from recent advances in separation techniques, instrumentation, and bioinformatics. However, improvements to data acquisition logic have lagged in comparison. Past workflows for Top Down Proteomics (TDPs) have focused on high throughput at the expense of maximal protein coverage and characterization. This mode of data acquisition has led to enormous overlap in the identification of highly abundant proteins in subsequent LC-MS injections. Furthermore, a wealth of data is left underutilized by analyzing each newly targeted species as unique, rather than as part of a collection of fragmentation events on a distinct proteoform. Here, we present a major advance in software for acquisition of TDP data that incorporates a fully automated workflow able to detect intact masses, guide fragmentation to achieve maximal identification and characterization of intact protein species, and perform database search online to yield real-time protein identifications. On Pseudomonas aeruginosa, the software combines fragmentation events of the same precursor with previously obtained fragments to achieve improved characterization of the target form by an average of 42 orders of magnitude in confidence. When HCD fragmentation optimization was applied to intact proteins ions, there was an 18.5 order of magnitude gain in confidence. These improved metrics set the stage for increased proteome coverage and characterization of higher order organisms in the future for sharply improved control over MS instruments in a project- and lab-wide context.
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Ensayos Analíticos de Alto Rendimiento/métodos , Sistemas en Línea , Proteómica/métodos , Estadística como Asunto/métodos , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Datos de Secuencia Molecular , Pseudomonas aeruginosaRESUMEN
With the prospect of resolving whole protein molecules into their myriad proteoforms on a proteomic scale, the question of their quantitative analysis in discovery mode comes to the fore. Here, we demonstrate a robust pipeline for the identification and stringent scoring of abundance changes of whole protein forms <30 kDa in a complex system. The input is ~100-400 µg of total protein for each biological replicate, and the outputs are graphical displays depicting statistical confidence metrics for each proteoform (i.e., a volcano plot and representations of the technical and biological variation). A key part of the pipeline is the hierarchical linear model that is tailored to the original design of the study. Here, we apply this new pipeline to measure the proteoform-level effects of deleting a histone deacetylase (rpd3) in S. cerevisiae. Over 100 proteoform changes were detected above a 5% false positive threshold in WT vs the Δrpd3 mutant, including the validating observation of hyperacetylation of histone H4 and both H2B isoforms. Ultimately, this approach to label-free top down proteomics in discovery mode is a critical technical advance for testing the hypothesis that whole proteoforms can link more tightly to complex phenotypes in cell and disease biology than do peptides created in shotgun proteomics.
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Proteínas/química , Proteómica/métodos , Histona Desacetilasas/análisis , Histona Desacetilasas/genética , Mutación/genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genéticaRESUMEN
It has long been understood that it is proteins, expressed and post-translationally modified, that are the primary regulators of both the fate and the function of cells. The ability to measure differences in the expression of the constellation of unique protein forms (proteoforms) with complete molecular specificity has the potential to sharply improve the return on investment for mass spectrometry-based proteomics in translational research and clinical diagnostics.
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Investigación Biomédica , Proteínas/metabolismo , Proteoma/metabolismo , Biomarcadores/metabolismo , Humanos , Espectrometría de Masas/métodos , Procesamiento Proteico-Postraduccional , Proteolisis , ProteómicaRESUMEN
Pacidamycins are a family of uridyl tetra/pentapeptide antibiotics with antipseudomonal activities through inhibition of the translocase MraY in bacterial cell wall assembly. The biosynthetic gene cluster for pacidamycins has recently been identified through genome mining of the producer Streptomyces coeruleorubidus, and the highly dissociated nonribosomal peptide assembly line for the uridyl tetrapeptide scaffold of pacidamycin has been characterized. In this work a hypothetical protein PacB, conserved in known uridyl peptide antibiotics gene clusters, has been characterized by both genetic deletion and enzymatic analysis of the purified protein. PacB catalyzes the transfer of the alanyl residue from alanyl-tRNA to the N terminus of the tetrapeptide intermediate yielding a pentapeptide on the thio-templated nonribosomal peptide synthetase (NRPS) assembly line protein PacH. PacB thus represents a new group of tRNA-dependent peptide bond-forming enzymes in secondary metabolite biosynthesis in addition to the recently identified cyclodipeptide synthases. The characterization of PacB completes the assembly line reconstitution of pacidamycin pentapeptide antibiotic scaffolds, bridging the primary and secondary metabolic pathways by hijacking an aminoacyl-tRNA to the antibiotic biosynthetic pathway.
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Antibacterianos/biosíntesis , Antibacterianos/química , Biosíntesis de Péptidos , Péptidos/química , Peptidil Transferasas/metabolismo , Secuencia de Aminoácidos , Aminoaciltransferasas/química , Aminoaciltransferasas/genética , Aminoaciltransferasas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Biología Computacional , ADN Bacteriano/genética , Genes Bacterianos , Modelos Moleculares , Datos de Secuencia Molecular , Familia de Multigenes , Peptidil Transferasas/química , Peptidil Transferasas/genética , Nucleósidos de Pirimidina/biosíntesis , Nucleósidos de Pirimidina/química , Aminoacil-ARN de Transferencia/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Streptomyces/genética , Streptomyces/metabolismo , Especificidad por SustratoRESUMEN
Adeno-associated virus (AAV) vectors are used to deliver therapeutic transgenes, but host immune responses may interfere with transduction and transgene expression. We evaluated prophylactic corticosteroid treatment on AAV5-mediated expression in liver tissue. Wild-type C57BL/6 mice received 6 × 1013 vg/kg AAV5-HLP-hA1AT, an AAV5 vector carrying a human α1-antitrypsin (hA1AT) gene with a hepatocyte-specific promoter. Mice received 4 weeks of daily 2 mg/kg prednisolone or water starting day -1 or 0 before vector dosing. Mice that received prophylactic corticosteroids had significantly higher serum hA1AT protein than mice that did not, starting at 6 weeks and persisting to the study end at 12 weeks, potentially through a decrease in the number of low responders. RNAseq and proteomic analyses investigating mechanisms mediating the improvement of transgene expression found that prophylactic corticosteroid treatment upregulated the AAV5 coreceptor platelet-derived growth factor receptor alpha (PDGFRα) on hepatocytes and downregulated its competitive ligand PDGFα, thus increasing the uptake of AAV5 vectors. Evidently, prophylactic corticosteroid treatment also suppressed acute immune responses to AAV. Together, these mechanisms resulted in increased uptake and preservation of the transgene, allowing more vector genomes to be available to assemble into stable, full-length structures mediating long-term transgene expression. Prophylactic corticosteroids represent a potential actionable strategy to improve AAV5-mediated transgene expression and decrease intersubject variability.
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Prednisolona , Proteómica , Humanos , Ratones , Animales , Regulación hacia Arriba , Ratones Endogámicos C57BL , Hepatocitos , Transgenes , Corticoesteroides , Receptores del Factor de Crecimiento Derivado de Plaquetas/genética , Inmunidad Innata , Dependovirus/genética , Vectores Genéticos/genéticaRESUMEN
Pantetheine and its corresponding disulfide pantethine play a key role in metabolism as building blocks of coenzyme A (CoA), an essential cofactor utilized in ~4% of primary metabolism and central to fatty acid, polyketide, and nonribosomal peptide synthases. Using a combination of recombinant engineering and chemical synthesis, we show that the disulfide of N-pantoylglycyl-2-aminoethanethiol (GlyPan), with one fewer carbon than pantetheine, can rescue a mutant E. coli strain MG1655ΔpanC lacking a functional pantothenate synthetase. Using mass spectrometry, we show that the GlyPan variant is accepted by the downstream CoA biosynthetic machinery, ultimately being incorporated into essential acyl carrier proteins. These findings point to further flexibility in CoA-dependent pathways and offer the opportunity to incorporate orthogonal analogues.