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1.
Nat Methods ; 18(3): 303-308, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33589837

RESUMEN

Current proteomic approaches disassemble and digest nucleosome particles, blurring readouts of the 'histone code'. To preserve nucleosome-level information, we developed Nuc-MS, which displays the landscape of histone variants and their post-translational modifications (PTMs) in a single mass spectrum. Combined with immunoprecipitation, Nuc-MS quantified nucleosome co-occupancy of histone H3.3 with variant H2A.Z (sixfold over bulk) and the co-occurrence of oncogenic H3.3K27M with euchromatic marks (for example, a >15-fold enrichment of dimethylated H3K79me2). Nuc-MS is highly concordant with chromatin immunoprecipitation-sequencing (ChIP-seq) and offers a new readout of nucleosome-level biology.


Asunto(s)
Histonas/metabolismo , Nucleosomas/metabolismo , Proteómica/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Línea Celular , Inmunoprecipitación de Cromatina/métodos , Células HEK293 , Código de Histonas , Humanos , Metilación
2.
Anal Chem ; 94(42): 14593-14602, 2022 10 25.
Artículo en Inglés | MEDLINE | ID: mdl-36179215

RESUMEN

Immune monitoring in cancer immunotherapy involves screening CD8+ T-cell responses against neoantigens, the tumor-specific peptides presented by Major histocompatibility complex Class I (MHCI) on the cell surface. High-throughput immune monitoring requires methods to produce and characterize small quantities of thousands of MHCI-peptide complexes that may be tested for a patient's T-cell response. MHCI synthesis has been achieved using a photocleavable peptide that is exchanged by the neoantigen; however, assays that measure peptide exchange currently disassemble the complex prior to analysis─precluding direct molecular characterization. Here, we use native mass spectrometry (MS) to profile intact recombinant MHCI complexes and directly measure peptide exchange. Coupled with size-exclusion chromatography or capillary-zone electrophoresis, the assay identified all tested human leukocyte antigen (HLA)/peptide combinations in the nanomole to picomole range with minimal run time, reconciling the synthetic and analytical requirements of MHCI-peptide screening with the downstream T-cell assays. We further show that the assay can be "multiplexed" by measuring exchange of multiple peptides simultaneously and also enables calculation of Vc50, a measure of gas-phase stability. Additionally, MHCI complexes were fragmented by top-down sequencing, demonstrating that the intact complex, peptide sequence, and their binding affinity can be determined in a single analysis. This screening tool for MHCI-neoantigen complexes represents a step toward the application of state-of-the-art MS technology in translational settings. Not only is this assay already informing on the viability of immunotherapy in practice, the platform also holds promise to inspire novel MS readouts for increasingly complex biomolecules used in the diagnosis and treatment of disease.


Asunto(s)
Antígenos de Histocompatibilidad Clase I , Péptidos , Humanos , Antígenos de Histocompatibilidad Clase I/metabolismo , Péptidos/química , Espectrometría de Masas , Antígenos HLA , Antígenos de Neoplasias
3.
Nat Methods ; 16(7): 587-594, 2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-31249407

RESUMEN

One gene can give rise to many functionally distinct proteoforms, each of which has a characteristic molecular mass. Top-down mass spectrometry enables the analysis of intact proteins and proteoforms. Here members of the Consortium for Top-Down Proteomics provide a decision tree that guides researchers to robust protocols for mass analysis of intact proteins (antibodies, membrane proteins and others) from mixtures of varying complexity. We also present cross-platform analytical benchmarks using a protein standard sample, to allow users to gauge their proficiency.


Asunto(s)
Benchmarking , Espectrometría de Masas/métodos , Proteínas/química , Desnaturalización Proteica , Procesamiento Proteico-Postraduccional , Proteómica
4.
Anal Chem ; 93(12): 5151-5160, 2021 03 30.
Artículo en Inglés | MEDLINE | ID: mdl-33749242

RESUMEN

We report a novel platform [native capillary zone electrophoresis-top-down mass spectrometry (nCZE-TDMS)] for the separation and characterization of whole nucleosomes, their histone subunits, and post-translational modifications (PTMs). As the repeating unit of chromatin, mononucleosomes (Nucs) are an ∼200 kDa complex of DNA and histone proteins involved in the regulation of key cellular processes central to human health and disease. Unraveling the covalent modification landscape of histones and their defined stoichiometries within Nucs helps to explain epigenetic regulatory mechanisms. In nCZE-TDMS, online Nuc separation is followed by a three-tier tandem MS approach that measures the intact mass of Nucs, ejects and detects the constituent histones, and fragments to sequence the histone. The new platform was optimized with synthetic Nucs to significantly reduce both sample requirements and cost compared to direct infusion. Limits of detection were in the low-attomole range, with linearity of over ∼3 orders of magnitude. The nCZE-TDMS platform was applied to endogenous Nucs from two cell lines distinguished by overexpression or knockout of histone methyltransferase NSD2/MMSET, where analysis of constituent histones revealed changes in histone abundances over the course of the CZE separation. We are confident the nCZE-TDMS platform will help advance nucleosome-level research in the fields of chromatin and epigenetics.


Asunto(s)
Electroforesis Capilar , Nucleosomas , Histonas/metabolismo , Humanos , Espectrometría de Masas , Procesamiento Proteico-Postraduccional
5.
Clin Chem Lab Med ; 59(4): 653-661, 2021 03 26.
Artículo en Inglés | MEDLINE | ID: mdl-33079696

RESUMEN

OBJECTIVES: Multiple myeloma (MM) is a malignant plasma cell neoplasm, requiring the integration of clinical examination, laboratory and radiological investigations for diagnosis. Detection and isotypic identification of the monoclonal protein(s) and measurement of other relevant biomarkers in serum and urine are pivotal analyses. However, occasionally this approach fails to characterize complex protein signatures. Here we describe the development and application of next generation mass spectrometry (MS) techniques, and a novel adaptation of immunofixation, to interrogate non-canonical monoclonal immunoproteins. METHODS: Immunoprecipitation immunofixation (IP-IFE) was performed on a Sebia Hydrasys Scan2. Middle-down de novo sequencing and native MS were performed with multiple instruments (21T FT-ICR, Q Exactive HF, Orbitrap Fusion Lumos, and Orbitrap Eclipse). Post-acquisition data analysis was performed using Xcalibur Qual Browser, ProSight Lite, and TDValidator. RESULTS: We adapted a novel variation of immunofixation electrophoresis (IFE) with an antibody-specific immunosubtraction step, providing insight into the clonal signature of gamma-zone monoclonal immunoglobulin (M-protein) species. We developed and applied advanced mass spectrometric techniques such as middle-down de novo sequencing to attain in-depth characterization of the primary sequence of an M-protein. Quaternary structures of M-proteins were elucidated by native MS, revealing a previously unprecedented non-covalently associated hetero-tetrameric immunoglobulin. CONCLUSIONS: Next generation proteomic solutions offer great potential for characterizing complex protein structures and may eventually replace current electrophoretic approaches for the identification and quantification of M-proteins. They can also contribute to greater understanding of MM pathogenesis, enabling classification of patients into new subtypes, improved risk stratification and the potential to inform decisions on future personalized treatment modalities.


Asunto(s)
Mieloma Múltiple , Proteínas de Mieloma , Proteómica/métodos , Anticuerpos Monoclonales , Humanos , Inmunoelectroforesis , Espectrometría de Masas , Mieloma Múltiple/diagnóstico
6.
Int J Mass Spectrom ; 4652021 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-34539228

RESUMEN

The combined use of electrospray ionization run in so-called "native mode" with top-down mass spectrometry (nTDMS) is enhancing both structural biology and discovery proteomics by providing three levels of information in a single experiment: the intact mass of a protein or complex, the masses of its subunits and non-covalent cofactors, and fragment ion masses from direct dissociation of subunits that capture the primary sequence and combinations of diverse post-translational modifications (PTMs). While intact mass data are readily deconvoluted using well-known software options, the analysis of fragmentation data that result from a tandem MS experiment - essential for proteoform characterization - is not yet standardized. In this tutorial, we offer a decision-tree for the analysis of nTDMS experiments on protein complexes and diverse bioassemblies. We include an overview of strategies to navigate this type of analysis, provide example data sets, and highlight software for the hypothesis-driven interrogation of fragment ions for localization of PTMs, metals, and cofactors on native proteoforms. Throughout we have emphasized the key features (deconvolution, search mode, validation, other) that the reader can consider when deciding upon their specific experimental and data processing design using both open-access and commercial software.

7.
J Proteome Res ; 19(3): 1346-1350, 2020 03 06.
Artículo en Inglés | MEDLINE | ID: mdl-32032494

RESUMEN

Charge detection mass spectrometry (CDMS) is mainly utilized to determine the mass of intact molecules. Previous applications of CDMS have determined the mass-to-charge ratio and the charge of large polymers, DNA molecules, and native protein complexes, from which corresponding mass values could be assigned. Recent advances have demonstrated that CDMS using an Orbitrap mass analyzer yields the reliable assignment of integer charge states that enables individual ion mass spectrometry (I2MS) and spectral output directly into the mass domain. Here I2MS analysis was extended to isotopically resolved fragment ions from intact proteoforms for the first time. With a radically different bias for ion readout, I2MS identified low-abundance fragment ions containing many hundreds of residues that were undetectable by standard Orbitrap measurements, leading to a doubling in the sequence coverage of triosephosphate isomerase. Thus MS/MS with the detection of individual ions (MS/I2MS) provides a far greater ability to detect high mass fragment ions and exhibits strong complementarity to traditional spectral readout in this, its first application to top-down mass spectrometry.


Asunto(s)
Proteómica , Espectrometría de Masas en Tándem , Iones
8.
Nat Chem Biol ; 14(1): 36-41, 2018 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-29131144

RESUMEN

Protein complexes exhibit great diversity in protein membership, post-translational modifications and noncovalent cofactors, enabling them to function as the actuators of many important biological processes. The exposition of these molecular features using current methods lacks either throughput or molecular specificity, ultimately limiting the use of protein complexes as direct analytical targets in a wide range of applications. Here, we apply native proteomics, enabled by a multistage tandem MS approach, to characterize 125 intact endogenous complexes and 217 distinct proteoforms derived from mouse heart and human cancer cell lines in discovery mode. The native conditions preserved soluble protein-protein interactions, high-stoichiometry noncovalent cofactors, covalent modifications to cysteines, and, remarkably, superoxide ligands bound to the metal cofactor of superoxide dismutase 2. These data enable precise compositional analysis of protein complexes as they exist in the cell and demonstrate a new approach that uses MS as a bridge to structural biology.


Asunto(s)
Complejos Multiproteicos/química , Multimerización de Proteína , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Animales , Línea Celular Tumoral , Humanos , Ratones , Complejos Multiproteicos/genética , Conformación Proteica , Procesamiento Proteico-Postraduccional , Subunidades de Proteína/química , Subunidades de Proteína/genética
9.
J Neurochem ; 148(6): 822-836, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30565253

RESUMEN

Amyloid ß oligomers (AßOs) accumulate early in Alzheimer's disease (AD) and experimentally cause memory dysfunction and the major pathologies associated with AD, for example, tau abnormalities, synapse loss, oxidative damage, and cognitive dysfunction. In order to develop the most effective AßO-targeting diagnostics and therapeutics, the AßO structures contributing to AD-associated toxicity must be elucidated. Here, we investigate the structural properties and pathogenic relevance of AßOs stabilized by the bifunctional crosslinker 1,5-difluoro-2,4-dinitrobenzene (DFDNB). We find that DFDNB stabilizes synthetic Aß in a soluble oligomeric conformation. With DFDNB, solutions of Aß that would otherwise convert to large aggregates instead yield solutions of stable AßOs, predominantly in the 50-300 kDa range, that are maintained for at least 12 days at 37°C. Structures were determined by biochemical and native top-down mass spectrometry analyses. Assayed in neuronal cultures and i.c.v.-injected mice, the DFDNB-stabilized AßOs were found to induce tau hyperphosphorylation, inhibit choline acetyltransferase, and provoke neuroinflammation. Most interestingly, DFDNB crosslinking was found to stabilize an AßO conformation particularly potent in inducing memory dysfunction in mice. Taken together, these data support the utility of DFDNB crosslinking as a tool for stabilizing pathogenic AßOs in structure-function studies.


Asunto(s)
Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/química , Reactivos de Enlaces Cruzados/farmacología , Neuronas/patología , Animales , Humanos , Ratones , Ratas
10.
Biochemistry ; 57(25): 3515-3523, 2018 06 26.
Artículo en Inglés | MEDLINE | ID: mdl-29694778

RESUMEN

Methanobactins (Mbns) are ribosomally produced, post-translationally modified bacterial natural products with a high affinity for copper. MbnN, a pyridoxal 5'-phosphate-dependent aminotransferase, performs a transamination reaction that is the last step in the biosynthesis of Mbns produced by several Methylosinus species. Our bioinformatic analyses indicate that MbnNs likely derive from histidinol-phosphate aminotransferases (HisCs), which play a key role in histidine biosynthesis. A comparison of the HisC active site with the predicted MbnN structure suggests that MbnN's active site is altered to accommodate the larger and more hydrophobic substrates necessary for Mbn biosynthesis. Moreover, we have confirmed that MbnN is capable of catalyzing the final transamination step in Mbn biosynthesis in vitro and in vivo. We also demonstrate that without this final modification, Mbn exhibits significantly decreased stability under physiological conditions. An examination of other Mbns and Mbn operons suggests that N-terminal protection of this family of natural products is of critical importance and that several different means of N-terminal stabilization have evolved independently in Mbn subfamilies.


Asunto(s)
Vías Biosintéticas , Imidazoles/metabolismo , Methylosinus/enzimología , Oligopéptidos/metabolismo , Transaminasas/metabolismo , Dominio Catalítico , Imidazoles/química , Methylosinus/química , Methylosinus/metabolismo , Modelos Moleculares , Oligopéptidos/química , Especificidad por Sustrato , Transaminasas/química
11.
Nat Commun ; 15(1): 3259, 2024 Apr 16.
Artículo en Inglés | MEDLINE | ID: mdl-38627419

RESUMEN

The heterogeneity inherent in today's biotherapeutics, especially as a result of heavy glycosylation, can affect a molecule's safety and efficacy. Characterizing this heterogeneity is crucial for drug development and quality assessment, but existing methods are limited in their ability to analyze intact glycoproteins or other heterogeneous biotherapeutics. Here, we present an approach to the molecular assessment of biotherapeutics that uses proton-transfer charge-reduction with gas-phase fractionation to analyze intact heterogeneous and/or glycosylated proteins by mass spectrometry. The method provides a detailed landscape of the intact molecular weights present in biotherapeutic protein preparations in a single experiment. For glycoproteins in particular, the method may offer insights into glycan composition when coupled with a suitable bioinformatic strategy. We tested the approach on various biotherapeutic molecules, including Fc-fusion, VHH-fusion, and peptide-bound MHC class II complexes to demonstrate efficacy in measuring the proteoform-level diversity of biotherapeutics. Notably, we inferred the glycoform distribution for hundreds of molecular weights for the eight-times glycosylated fusion drug IL22-Fc, enabling correlations between glycoform sub-populations and the drug's pharmacological properties. Our method is broadly applicable and provides a powerful tool to assess the molecular heterogeneity of emerging biotherapeutics.


Asunto(s)
Glicoproteínas , Polisacáridos , Glicosilación , Glicoproteínas/metabolismo , Espectrometría de Masas/métodos , Polisacáridos/metabolismo
12.
ACS Chem Biol ; 17(10): 2769-2780, 2022 10 21.
Artículo en Inglés | MEDLINE | ID: mdl-35951581

RESUMEN

Triosephosphate isomerase (TPI) performs the 5th step in glycolysis, operates near the limit of diffusion, and is involved in "moonlighting" functions. Its dimer was found singly phosphorylated at Ser20 (pSer20) in human cells, with this post-translational modification (PTM) showing context-dependent stoichiometry and loss under oxidative stress. We generated synthetic pSer20 proteoforms using cell-free protein synthesis that showed enhanced TPI activity by 4-fold relative to unmodified TPI. Molecular dynamics simulations show that the phosphorylation enables a channel to form that shuttles substrate into the active site. Refolding, kinetic, and crystallographic analyses of point mutants including S20E/G/Q indicate that hetero-dimerization and subunit asymmetry are key features of TPI. Moreover, characterization of an endogenous human TPI tetramer also implicates tetramerization in enzymatic regulation. S20 is highly conserved across eukaryotic TPI, yet most prokaryotes contain E/D at this site, suggesting that phosphorylation of human TPI evolved a new switch to optionally boost an already fast enzyme. Overall, complete characterization of TPI shows how endogenous proteoform discovery can prioritize functional versus bystander PTMs.


Asunto(s)
Simulación de Dinámica Molecular , Triosa-Fosfato Isomerasa , Humanos , Triosa-Fosfato Isomerasa/química , Triosa-Fosfato Isomerasa/metabolismo , Fosforilación , Dominio Catalítico , Cinética
13.
J Am Soc Mass Spectrom ; 31(3): 574-581, 2020 Mar 04.
Artículo en Inglés | MEDLINE | ID: mdl-31971796

RESUMEN

New tools and techniques have dramatically accelerated the field of structural biology over the past several decades. One potent and relatively new technique that is now being utilized by an increasing number of laboratories is the combination of so-called "native" electrospray ionization (ESI) with mass spectrometry (MS) for the characterization of proteins and their noncovalent complexes. However, native ESI-MS produces species at increasingly higher m/z with increasing molecular weight, leading to substantial differences when compared to traditional mass spectrometric approaches using denaturing ESI solutions. Herein, these differences are explored both theoretically and experimentally to understand the role that charge state and isotopic distributions have on signal-to-noise (S/N) as a function of complex molecular weight and how the reduced collisional cross sections of proteins electrosprayed under native solution conditions can lead to improved data quality in image current mass analyzers, such as Orbitrap and FT-ICR. Quantifying ion signal differences under native and denatured conditions revealed enhanced S/N and a more gradual decay in S/N with increasing mass under native conditions. Charge state and isotopic S/N models, supported by experimental results, indicate that analysis of proteins under native conditions at 100 kDa will be 17 times more sensitive than analysis under denatured conditions at the same mass. Higher masses produce even larger sensitivity gains. Furthermore, reduced cross sections under native conditions lead to lower levels of ion decay within an Orbitrap scan event over long transient acquisition times, enabling isotopic resolution of species with molecular weights well in excess of those typically resolved under denatured conditions.


Asunto(s)
Proteínas/química , Animales , Humanos , Iones/química , Desnaturalización Proteica , Espectrometría de Masa por Ionización de Electrospray , Electricidad Estática
14.
J Am Soc Mass Spectrom ; 31(7): 1398-1409, 2020 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-32436704

RESUMEN

Protein fragmentation is a critical component of top-down proteomics, enabling gene-specific protein identification and full proteoform characterization. The factors that influence protein fragmentation include precursor charge, structure, and primary sequence, which have been explored extensively for collision-induced dissociation (CID). Recently, noticeable differences in CID-based fragmentation were reported for native versus denatured proteins, motivating the need for scoring metrics that are tailored specifically to native top-down mass spectrometry (nTDMS). To this end, position and intensity were tracked for 10,252 fragment ions produced by higher-energy collisional dissociation (HCD) of 159 native monomers and 70 complexes. We used published structural data to explore the relationship between fragmentation and protein topology and revealed that fragmentation events occur at a large range of relative residue solvent accessibility. Additionally, our analysis found that fragment ions at sites with an N-terminal aspartic acid or a C-terminal proline make up on average 40 and 27%, respectively, of the total matched fragment ion intensity in nTDMS. Percent intensity contributed by each amino acid was determined and converted into weights to (1) update the previously published C-score and (2) construct a native Fragmentation Propensity Score. Both scoring systems showed an improvement in protein identification or characterization in comparison to traditional methods and overall increased confidence in results with fewer matched fragment ions but with high probability nTDMS fragmentation patterns. Given the rise of nTDMS as a tool for structural mass spectrometry, we forward these scoring metrics as new methods to enhance analysis of nTDMS data.


Asunto(s)
Iones , Proteoma , Proteómica/métodos , Animales , Línea Celular , Bases de Datos de Proteínas , Humanos , Iones/análisis , Iones/química , Espectrometría de Masas , Ratones , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/química , Proteoma/análisis , Proteoma/química
15.
J Am Soc Mass Spectrom ; 30(7): 1190-1198, 2019 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-30963455

RESUMEN

Native mass spectrometry (nMS) is a technique growing at the interface of analytical chemistry, structural biology, and proteomics that enables the detection and partial characterization of non-covalent protein assemblies. Currently, the standardization and dissemination of nMS is hampered by technical challenges associated with instrument operation, benchmarking, and optimization over time. Here, we provide a standard operating procedure for acquiring high-quality native mass spectra of 30-300 kDa proteins using an Orbitrap mass spectrometer. By describing reproducible sample preparation, loading, ionization, and nMS analysis, we forward two proteoforms and three complexes as possible standards to advance training and longitudinal assessment of instrument performance. Spectral data for five standards can guide assessment of instrument parameters, data production, and data analysis. By introducing this set of standards and protocols, we aim to help normalize native mass spectrometry practices across labs and provide benchmarks for reproducibility and high-quality data production in the years ahead. Graphical abstract.


Asunto(s)
Espectrometría de Masas/métodos , Multimerización de Proteína , Proteínas/química , Alcohol Deshidrogenasa/química , Animales , Anhidrasas Carbónicas/química , Bovinos , Modelos Moleculares , Piruvato Quinasa/química , Conejos , Reproducibilidad de los Resultados , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/química
16.
Nat Commun ; 10(1): 2675, 2019 06 17.
Artículo en Inglés | MEDLINE | ID: mdl-31209220

RESUMEN

Aerobic methane oxidation is catalyzed by particulate methane monooxygenase (pMMO), a copper-dependent, membrane metalloenzyme composed of subunits PmoA, PmoB, and PmoC. Characterization of the copper active site has been limited by challenges in spectroscopic analysis stemming from the presence of multiple copper binding sites, effects of detergent solubilization on activity and crystal structures, and the lack of a heterologous expression system. Here we utilize nanodiscs coupled with native top-down mass spectrometry (nTDMS) to determine the copper stoichiometry in each pMMO subunit and to detect post-translational modifications (PTMs). These results indicate the presence of a mononuclear copper center in both PmoB and PmoC. pMMO-nanodisc complexes with a higher stoichiometry of copper-bound PmoC exhibit increased activity, suggesting that the PmoC copper site plays a role in methane oxidation activity. These results provide key insights into the pMMO copper centers and demonstrate the ability of nTDMS to characterize complex membrane-bound metalloenzymes.


Asunto(s)
Proteínas Bacterianas/metabolismo , Espectrometría de Masas/métodos , Methylococcaceae/metabolismo , Modelos Moleculares , Oxigenasas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/ultraestructura , Sitios de Unión , Biocatálisis , Dominio Catalítico , Cobre/química , Cobre/metabolismo , Microscopía por Crioelectrón , Metano/metabolismo , Metanol/metabolismo , Methylococcaceae/química , Methylococcaceae/ultraestructura , Oxidación-Reducción , Oxigenasas/química , Oxigenasas/ultraestructura , Procesamiento Proteico-Postraduccional
17.
Science ; 359(6382): 1411-1416, 2018 Mar 23.
Artículo en Inglés | MEDLINE | ID: mdl-29567715

RESUMEN

Metal homeostasis poses a major challenge to microbes, which must acquire scarce elements for core metabolic processes. Methanobactin, an extensively modified copper-chelating peptide, was one of the earliest natural products shown to enable microbial acquisition of a metal other than iron. We describe the core biosynthetic machinery responsible for the characteristic posttranslational modifications that grant methanobactin its specificity and affinity for copper. A heterodimer comprising MbnB, a DUF692 family iron enzyme, and MbnC, a protein from a previously unknown family, performs a dioxygen-dependent four-electron oxidation of the precursor peptide (MbnA) to install an oxazolone and an adjacent thioamide, the characteristic methanobactin bidentate copper ligands. MbnB and MbnC homologs are encoded together and separately in many bacterial genomes, suggesting functions beyond their roles in methanobactin biosynthesis.


Asunto(s)
Cobre/metabolismo , Methylosinus trichosporium/metabolismo , Oligopéptidos/biosíntesis , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Genoma Bacteriano , Imidazoles/química , Imidazoles/metabolismo , Ligandos , Methylosinus trichosporium/genética , Oligopéptidos/química , Oligopéptidos/genética , Oligopéptidos/metabolismo , Oxidación-Reducción , Oxígeno/metabolismo , Conformación Proteica en Hélice alfa , Multimerización de Proteína
18.
Curr Protoc Bioinformatics ; 56: 13.30.1-13.30.11, 2016 12 08.
Artículo en Inglés | MEDLINE | ID: mdl-27930806

RESUMEN

Recent advances in top-down mass spectrometry using native electrospray now enable the analysis of intact protein complexes with relatively small sample amounts in an untargeted mode. Here, we describe how to characterize both homo- and heteropolymeric complexes with high molecular specificity using input data produced by tandem mass spectrometry of whole protein assemblies. The tool described is a "search engine for multi-proteoform complexes," (SEMPC) and is available for free online. The output is a list of candidate multi-proteoform complexes and scoring metrics, which are used to define a distinct set of one or more unique protein subunits, their overall stoichiometry in the intact complex, and their pre- and post-translational modifications. Thus, we present an approach for the identification and characterization of intact protein complexes from native mass spectrometry data. © 2016 by John Wiley & Sons, Inc.


Asunto(s)
Internet , Proteínas/química , Proteómica/métodos , Motor de Búsqueda , Procesamiento Proteico-Postraduccional , Proteómica/instrumentación , Espectrometría de Masas en Tándem
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