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1.
Mol Cell Proteomics ; 20: 100139, 2021 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-34418567

RESUMO

Proteomics methodology has expanded to include protein structural analysis, primarily through cross-linking mass spectrometry (XL-MS) and hydrogen-deuterium exchange mass spectrometry (HX-MS). However, while the structural proteomics community has effective tools for primary data analysis, there is a need for structure modeling pipelines that are accessible to the proteomics specialist. Integrative structural biology requires the aggregation of multiple distinct types of data to generate models that satisfy all inputs. Here, we describe IMProv, an app in the Mass Spec Studio that combines XL-MS data with other structural data, such as cryo-EM densities and crystallographic structures, for integrative structure modeling on high-performance computing platforms. The resource provides an easily deployed bundle that includes the open-source Integrative Modeling Platform program (IMP) and its dependencies. IMProv also provides functionality to adjust cross-link distance restraints according to the underlying dynamics of cross-linked sites, as characterized by HX-MS. A dynamics-driven conditioning of restraint values can improve structure modeling precision, as illustrated by an integrative structure of the five-membered Polycomb Repressive Complex 2. IMProv is extensible to additional types of data.

2.
Anal Chem ; 93(9): 4246-4254, 2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33592142

RESUMO

The data analysis practices associated with hydrogen-deuterium exchange mass spectrometry (HX-MS) lag far behind that of most other MS-based protein analysis tools. A reliance on external tools from other fields and a persistent need for manual data validation restrict this powerful technology to the expert user. Here, we provide an extensive upgrade to the HX data analysis suite available in the Mass Spec Studio in the form of two new apps (HX-PIPE and HX-DEAL), completing a workflow that provides an HX-tailored peptide identification capability, accelerated validation routines, automated spectral deconvolution strategies, and a rich set of exportable graphics and statistical reports. With these new tools, we demonstrate that the peptide identifications obtained from undeuterated samples generated at the start of a project contain information that helps predict and control the extent of manual validation required. We also uncover a large fraction of HX-usable peptides that remains unidentified in most experiments. We show that automated spectral deconvolution routines can identify exchange regimes in a project-wide manner, although they remain difficult to accurately assign in all scenarios. Taken together, these new tools provide a robust and complete solution suitable for the analysis of high-complexity HX-MS data.

3.
Structure ; 29(5): 467-478.e6, 2021 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-33412091

RESUMO

In the non-homologous end-joining (NHEJ) of a DNA double-strand break, DNA ends are bound and protected by DNA-PK, which synapses across the break to tether the broken ends and initiate repair. There is little clarity surrounding the nature of the synaptic complex and the mechanism governing the transition to repair. We report an integrative structure of the synaptic complex at a precision of 13.5 Å, revealing a symmetric head-to-head arrangement with a large offset in the DNA ends and an extensive end-protection mechanism involving a previously uncharacterized plug domain. Hydrogen/deuterium exchange mass spectrometry identifies an allosteric pathway connecting DNA end-binding with the kinase domain that places DNA-PK under tension in the kinase-active state. We present a model for the transition from end-protection to repair, where the synaptic complex supports hierarchical processing of the ends and scaffold assembly, requiring displacement of the catalytic subunit and tension release through kinase activity.

4.
Nat Commun ; 11(1): 6233, 2020 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-33277478

RESUMO

The KEOPS complex, which is conserved across archaea and eukaryotes, is composed of four core subunits; Pcc1, Kae1, Bud32 and Cgi121. KEOPS is crucial for the fitness of all organisms examined. In humans, pathogenic mutations in KEOPS genes lead to Galloway-Mowat syndrome, an autosomal-recessive disease causing childhood lethality. Kae1 catalyzes the universal and essential tRNA modification N6-threonylcarbamoyl adenosine, but the precise roles of all other KEOPS subunits remain an enigma. Here we show using structure-guided studies that Cgi121 recruits tRNA to KEOPS by binding to its 3' CCA tail. A composite model of KEOPS bound to tRNA reveals that all KEOPS subunits form an extended tRNA-binding surface that we have validated in vitro and in vivo to mediate the interaction with the tRNA substrate and its modification. These findings provide a framework for understanding the inner workings of KEOPS and delineate why all KEOPS subunits are essential.


Assuntos
Proteínas Arqueais/química , Methanocaldococcus/metabolismo , Complexos Multiproteicos/química , RNA de Transferência/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Cristalografia por Raios X , Methanocaldococcus/genética , Modelos Moleculares , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Conformação de Ácido Nucleico , Ligação Proteica , Domínios Proteicos , RNA de Transferência/genética , RNA de Transferência/metabolismo , RNA de Transferência de Lisina/química , RNA de Transferência de Lisina/genética , RNA de Transferência de Lisina/metabolismo
5.
Commun Biol ; 3(1): 735, 2020 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-33277614

RESUMO

The TRAnsport Protein Particle (TRAPP) complexes act as Guanine nucleotide exchange factors (GEFs) for Rab GTPases, which are master regulators of membrane trafficking in eukaryotic cells. In metazoans, there are two large multi-protein TRAPP complexes: TRAPPII and TRAPPIII, with the TRAPPII complex able to activate both Rab1 and Rab11. Here we present detailed biochemical characterisation of Rab-GEF specificity of the human TRAPPII complex, and molecular insight into Rab binding. GEF assays of the TRAPPII complex against a panel of 20 different Rab GTPases revealed GEF activity on Rab43 and Rab19. Electron microscopy and chemical cross-linking revealed the architecture of mammalian TRAPPII. Hydrogen deuterium exchange MS showed that Rab1, Rab11 and Rab43 share a conserved binding interface. Clinical mutations in Rab11, and phosphomimics of Rab43, showed decreased TRAPPII GEF mediated exchange. Finally, we designed a Rab11 mutation that maintained TRAPPII-mediated GEF activity while decreasing activity of the Rab11-GEF SH3BP5, providing a tool to dissect Rab11 signalling. Overall, our results provide insight into the GTPase specificity of TRAPPII, and how clinical mutations disrupt this regulation.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Linhagem Celular , Cromatografia Líquida , Humanos , Insetos , Modelos Moleculares , Conformação Proteica , Isoformas de Proteínas , Especificidade por Substrato , Espectrometria de Massas em Tandem , Proteínas rab de Ligação ao GTP/genética
6.
J Proteomics ; 225: 103844, 2020 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-32480078

RESUMO

Structural Mass Spectrometry (SMS) provides a comprehensive toolbox for the analysis of protein structure and function. It offers multiple sources of structural information that are increasingly useful for integrative structural modeling of complex protein systems. As MS-based structural workflows scale to larger systems, consistent and coherent data interpretation resources are needed to better support modeling. Unlike the proteomics community, practitioners of SMS lack adequate computational tools. Here, we review new developments in the Mass Spec Studio: an expandable ecosystem of workflows for the analysis of complementary SMS techniques with linkages to modeling. Current functionality in the Studio (version 2) supports three major SMS workflows (crosslinking, hydrogen/deuterium exchange and covalent labelling) and two pipelines for structural modeling, with a special focus on data integration. The Mass Spec Studio is an architecture focused on rapid and robust extension of functionality by a community of developers. SIGNIFICANCE: This review surveys the new data analysis capabilities within the Mass Spec Studio, a rich framework for rapid software development specifically targeting the community of structural proteomics and structural mass spectrometry. Updates to crosslinking, hydrogen/deuterium-exchange and covalent labeling apps are provided as well as a utility for translating such analyses into restraints that support integrative structural modeling. These new capabilities, together with the underlying design tools and content, provide the community with a wealth of resources to tackle complex structural problem and design new approaches to data analysis.


Assuntos
Ecossistema , Proteínas , Espectrometria de Massas , Proteômica , Software
7.
J Am Soc Mass Spectrom ; 31(2): 207-216, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-32031402

RESUMO

The functional properties of a protein are strongly influenced by its topography, or the solvent-facing contour map of its surface. Together with crosslinking, covalent labeling mass spectrometry (CL-MS) has the potential to contribute topographical data through the measurement of surface accessibility. However, recent efforts to correlate measures of surface accessibility with labeling yield have been met with mixed success. Most applications of CL-MS involve differential analysis of protein interactions (i.e., footprinting experiments) where such inconsistencies have limited effect. Extending CL-MS into structural analysis requires an improved evaluation of the relationship between labeling and surface exposure. In this study, we applied recently developed diazirine reagents to obtain deep coverage of the large motor domain of Eg5 (a mitotic kinesin), and together with computational methods we correlated labeling yields with accessibility data in a number of ways. We observe that correlations can indeed be seen at a local structural level, but these correlations do not extend across the structure. The lack of correlation arises from the influence of protein dynamics and chemical composition on reagent partitioning and, thus, also on labeling yield. We conclude that our use of CL-MS data should be considered in light of "chemical accessibility" rather than "solvent accessibility" and suggest that CL-MS data would be a useful tool in the fundamental study of protein-solute interactions.


Assuntos
Diazometano/química , Cinesina/química , Espectrometria de Massas/métodos , Humanos , Indicadores e Reagentes , Modelos Moleculares , Conformação Proteica
8.
Structure ; 27(12): 1745-1759, 2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31780431

RESUMO

Structures of biomolecular systems are increasingly computed by integrative modeling. In this approach, a structural model is constructed by combining information from multiple sources, including varied experimental methods and prior models. In 2019, a Workshop was held as a Biophysical Society Satellite Meeting to assess progress and discuss further requirements for archiving integrative structures. The primary goal of the Workshop was to build consensus for addressing the challenges involved in creating common data standards, building methods for federated data exchange, and developing mechanisms for validating integrative structures. The summary of the Workshop and the recommendations that emerged are presented here.


Assuntos
Biologia Computacional/métodos , Bases de Dados de Proteínas , Modelos Moleculares , Conformação Proteica , Proteínas/química , Cristalografia por Raios X , Espectroscopia de Ressonância Magnética
9.
Prog Biophys Mol Biol ; 147: 92-102, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31570166

RESUMO

X-ray crystallography and electron microscopy maps resolved to 3-8 Šare generally sufficient for tracing the path of the polypeptide chain in space, while often insufficient for unambiguously registering the sequence on the path (i.e., threading). Frequently, however, additional information is available from other biophysical experiments, physical principles, statistical analyses, and other prior models. Here, we formulate an integrative approach for sequence assignment to a partial backbone model as an optimization problem, which requires three main components: the representation of the system, the scoring function, and the optimization method. The method is implemented in the open source Integrative Modeling Platform (IMP) (https://integrativemodeling.org), allowing a number of different terms in the scoring function. We apply this method to localizing the sequence assignment within a 199-residue disordered region of three structured and sequence unassigned helices in the DNA-PKcs crystallographic structure, using chemical crosslinks, hydrogen deuterium exchange, and sequence connectivity. The resulting ensemble of threading models provides two major solutions, one of which suggests that the crucial ABCDE cluster of phosphorylation sites cannot undergo intra-molecular autophosphorylation without a conformational rearrangement. The ensemble of solutions embodies the most accurate and precise sequence threading given the available information.


Assuntos
Proteína Quinase Ativada por DNA/química , Proteína Quinase Ativada por DNA/metabolismo , Medição da Troca de Deutério , Cristalografia por Raios X , Fosforilação , Conformação Proteica em alfa-Hélice
10.
Anal Biochem ; 586: 113416, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31499019

RESUMO

Microtubules (MTs) are key components in the cytoskeleton of the eukaryotic cell, and play roles in processes such as intracellular transport and cell division. An improved understanding MT regulation requires structural analysis of the extensive interactions between the MT lattice and its regulatory proteins, but MT interactions are challenging for even the most advanced structural methods to characterize. Integrative methods involving crosslinking mass spectrometry (XL-MS) can extend structural analysis to many interaction classes, but the representation of MTs in crosslinking data-sets has been surprisingly low. Here, we explore the basis for the underrepresentation of the MT lattice and present an enhanced method for mapping MT structural features using an optimized set of reagents, together with fluorescence detection to ensure MT structural integrity. Through the application of stringent identification criteria, 91 unique crosslinks were identified, 78 of which were uniquely matched to 7 distinct structural features of the MT lattice. Of note, 4 crosslinks were detected for the lattice-A protofilament organization. The lattice-A structure defines a "seam" or discontinuity in MTs and is an emerging site of interest for MT regulation. Our methodology should be broadly applicable to integrative structural studies involving any MT-protein interaction.


Assuntos
Reagentes para Ligações Cruzadas/química , Microtúbulos/química , Reagentes para Ligações Cruzadas/síntese química , Modelos Moleculares , Estrutura Molecular , Polimerização
11.
Cell ; 178(5): 1205-1221.e17, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31442408

RESUMO

A hallmark feature of inflammation is the orchestrated recruitment of neutrophils from the bloodstream into inflamed tissue. Although selectins and integrins mediate recruitment in many tissues, they have a minimal role in the lungs and liver. Exploiting an unbiased in vivo functional screen, we identified a lung and liver homing peptide that functionally abrogates neutrophil recruitment to these organs. Using biochemical, genetic, and confocal intravital imaging approaches, we identified dipeptidase-1 (DPEP1) as the target and established its role as a physical adhesion receptor for neutrophil sequestration independent of its enzymatic activity. Importantly, genetic ablation or functional peptide blocking of DPEP1 significantly reduced neutrophil recruitment to the lungs and liver and provided improved survival in models of endotoxemia. Our data establish DPEP1 as a major adhesion receptor on the lung and liver endothelium and identify a therapeutic target for neutrophil-driven inflammatory diseases of the lungs.


Assuntos
Dipeptidases/metabolismo , Neutrófilos/fisiologia , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Animais , Cilastatina/farmacologia , Cilastatina/uso terapêutico , Dipeptidases/antagonistas & inibidores , Dipeptidases/genética , Modelos Animais de Doenças , Endotoxemia/mortalidade , Endotoxemia/patologia , Endotoxemia/prevenção & controle , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Humanos , Lipopolissacarídeos/farmacologia , Fígado/efeitos dos fármacos , Fígado/imunologia , Fígado/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos SCID , Infiltração de Neutrófilos/efeitos dos fármacos , Peptídeos/síntese química , Peptídeos/química , Peptídeos/farmacologia , Taxa de Sobrevida
12.
Nat Methods ; 16(7): 595-602, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31249422

RESUMO

Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful biophysical technique being increasingly applied to a wide variety of problems. As the HDX-MS community continues to grow, adoption of best practices in data collection, analysis, presentation and interpretation will greatly enhance the accessibility of this technique to nonspecialists. Here we provide recommendations arising from community discussions emerging out of the first International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX; 2017). It is meant to represent both a consensus viewpoint and an opportunity to stimulate further additions and refinements as the field advances.


Assuntos
Medição da Troca de Deutério/métodos , Espectrometria de Massas/métodos , Análise de Dados , Concentração de Íons de Hidrogênio
13.
Anal Chem ; 91(13): 8492-8499, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31198032

RESUMO

Covalent labeling with mass spectrometry (CL-MS) provides a direct measure of the chemical and structural features of proteins with the potential for resolution at the amino-acid level. Unfortunately, most applications of CL-MS are limited to narrowly defined differential analyses, where small numbers of residues are compared between two or more protein states. Extending the utility of high-resolution CL-MS for structure-based applications requires more robust computational routines and the development of methodology capable of reporting of labeling yield accurately. Here, we provide a substantial improvement in the analysis of CL-MS data with the development of an extended plug-in built within the Mass Spec Studio development framework (MSS-CLEAN). All elements of data analysis-from database search to site-resolved and normalized labeling output-are accommodated, as illustrated through the nonselective labeling of the human kinesin Eg5 with photoconverted 3,3'-azibutan-1-ol. In developing the new features within the CL-MS plug-in, we identified additional complexities associated with the application of CL reagents, arising primarily from digestion-induced bias in yield measurements and ambiguities in site localization. A strategy is presented involving the use of redundant site labeling data from overlapping peptides, the imputation of missing data, and a normalization routine to determine relative protection factors. These elements together provide for a robust structural interpretation of CL-MS/MS data while minimizing the over-reporting of labeling site resolution. Finally, to minimize bias, we recommend that digestion strategies for the generation of useful overlapping peptides involve the application of complementary enzymes that drive digestion to completion.


Assuntos
Marcação por Isótopo/métodos , Cinesina/análise , Software , Espectrometria de Massas em Tandem/métodos , Humanos , Cinesina/química , Modelos Moleculares , Conformação Proteica
14.
Nat Commun ; 10(1): 241, 2019 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-30651562

RESUMO

Cell survival after oxidative DNA damage requires signaling, repair and transcriptional events often enabled by nucleosome displacement, exchange or removal by chromatin remodeling enzymes. Here, we show that Chromodomain Helicase DNA-binding protein 6 (CHD6), distinct to other CHD enzymes, is stabilized during oxidative stress via reduced degradation. CHD6 relocates rapidly to DNA damage in a manner dependent upon oxidative lesions and a conserved N-terminal poly(ADP-ribose)-dependent recruitment motif, with later retention requiring the double chromodomain and central core. CHD6 ablation increases reactive oxygen species persistence and impairs anti-oxidant transcriptional responses, leading to elevated DNA breakage and poly(ADP-ribose) induction that cannot be rescued by catalytic or double chromodomain mutants. Despite no overt epigenetic or DNA repair abnormalities, CHD6 loss leads to impaired cell survival after chronic oxidative stress, abnormal chromatin relaxation, amplified DNA damage signaling and checkpoint hypersensitivity. We suggest that CHD6 is a key regulator of the oxidative DNA damage response.


Assuntos
Montagem e Desmontagem da Cromatina/fisiologia , Dano ao DNA/fisiologia , DNA Helicases/metabolismo , Reparo do DNA/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Estresse Oxidativo/fisiologia , Células A549 , Sobrevivência Celular/fisiologia , Dano ao DNA/efeitos da radiação , DNA Helicases/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/fisiologia , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos da radiação , Células HEK293 , Humanos , Microscopia Intravital , Lasers/efeitos adversos , Proteínas do Tecido Nervoso/genética , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo
15.
J Proteome Res ; 18(3): 934-946, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30582701

RESUMO

Structure-based approaches to the delineation of immunogens for vaccine development have a throughput requirement that is difficult to meet in practice with conventional methods of structure determination. Here we present a strategy for rapid and accurate structure generation in support of antigen engineering programs. The approach is developed around the modeling of interactions between host transferrin (Tf) and the bacterial vaccine target transferrin binding protein B (TbpB) from Gram-negative pathogens such as Neisseria meningitidis. Using an approach based solely on cross-linking mass spectrometry (XL-MS) data, monomeric structural models, and the Integrative Modeling Platform (IMP), we demonstrate that converged representations of the Tf:TbpB interactions can be returned that accurately reflect the binding interface and the relative orientation of the monomeric units, with the capacity to scale to the analysis of interactions from any number of additional strains. We show that a key element to accurate modeling involves the application of hetero-bifunctional cross-linkers incorporating fast-acting photoactivatable diazirines coupled with conventional amine-targeting N-hydroxysuccinimide esters, and we demonstrate that conventional homo-bifunctional reagents used in cross-linking kinetically trap dynamic states in the ensemble. Therefore, the application of both classes of cross-linker provides an opportunity to empirically detect protein dynamics during integrative structural modeling.


Assuntos
Proteínas de Bactérias/imunologia , Reagentes para Ligações Cruzadas/química , Espectrometria de Massas/métodos , Receptores da Transferrina/imunologia , Proteínas de Bactérias/metabolismo , Vacinas Bacterianas/imunologia , Reagentes para Ligações Cruzadas/efeitos da radiação , Bactérias Gram-Negativas , Modelos Moleculares , Neisseria meningitidis , Receptores da Transferrina/metabolismo , Proteína B de Ligação a Transferrina/imunologia , Proteína B de Ligação a Transferrina/metabolismo
16.
Anal Chem ; 90(15): 9077-9084, 2018 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-29975514

RESUMO

Quantification in proteomics largely relies on the incorporation of stable isotopes, with protocols that either introduce the label through metabolic incorporation or chemical tagging. Most methods rely on the use of trypsin and/or LysC to generate labeled peptides. Although alternative proteases can enhance proteome coverage, generic quantitative methods that port over to such enzymes are lacking. Here we describe a quantification strategy amenable to most proteases, which involves propionylation of metabolically labeled lysine, using a "silent stable isotope labeling by amino acids in cell culture (SILAC)" strategy that reveals isotopic labels on second-stage mass spectrometry (MS2) fragmentation in a tandem mass tag (TMT)-like manner. We selectively propionylated lysine residues prior to digestion to generate pure ArgC-like digestion for trypsin and novel ArgN-like digestions for LysargiNase, by restricting digestion at lysine. The modification offers highly complementary sequence coverage, and even enhanced protein identification rates in certain situations (GluC digestion). Propionylated lysine residues were present in the majority of identified peptides generated from digests of cell lysates and led to the consistent release of an intense cyclic imine reporter ion at mass-to-charge ratio ( m/ z) 140 using higher-energy collisional dissociation. We grew A549 cells in media containing either l-1-13C-lysine or l-6-13C-lysine, to generate proteins that share the same accurate mass when paired. Peptides were indistinguishable on the first-stage mass spectrometry (MS1) level and, upon fragmentation, released reporter ions at m/ z 140 and m/ z 141, without otherwise affecting sequence ion mass. The quantification approach is independent of the number of peptide lysines and offers a new strategy for quantitative proteomics.


Assuntos
Anidridos/análise , Lisina/análise , Fragmentos de Peptídeos/análise , Propionatos/análise , Proteoma/análise , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Células A549 , Animais , Bovinos , Técnicas de Cultura de Células , Células HeLa , Cavalos , Humanos , Marcação por Isótopo/métodos , Peptídeo Hidrolases/química , Proteínas/análise , Proteólise , Tripsina/química
17.
Anal Chem ; 90(5): 3083-3090, 2018 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-29405698

RESUMO

Dynamic post-translational modifications of histones regulate transcriptional gene expression in eukaryotes. Unique combinations of modifications, almost exclusively displayed at the flexible N-terminal tails on histones, create distributions of proteoforms that need to be characterized in order to understand the complexity of gene regulation and how aberrant modification patterns influence disease. Although mass spectrometry is a preferred method for the analysis of histone modifications, information is lost when using conventional trypsin-based histone methods. Newer "middle-down" protocols may retain a greater fraction of the full proteoform distribution. We describe a strategy for the simultaneous characterization of histones H3 and H4 with near-complete retention of proteoform distributions, using a conventional proteomics liquid chromatography-tandem mass spectrometry (LC-MS/MS) configuration. The selective prolyl endoprotease neprosin generates convenient peptide lengths for retention and dispersion of modified H3 and H4 peptides on reversed-phase chromatography, offering an alternative to the hydrophilic interaction liquid chromatography typically used in middle-down methods. No chemical derivatizations are required, presenting a significant advantage over the trypsin-based protocol. Over 200 proteoforms can be readily profiled in a single analysis of histones from HeLa S3 cells. An in-gel digestion protocol provides additional options for effective histone analysis.


Assuntos
Histonas/análise , Proteômica/métodos , Cromatografia Líquida , Endopeptidases/química , Células HeLa , Histonas/química , Humanos , Espectrometria de Massas , Processamento de Proteína Pós-Traducional
18.
J Am Soc Mass Spectrom ; 28(10): 2011-2021, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28799075

RESUMO

Mapping proteins with chemical reagents and mass spectrometry can generate a measure of accessible surface area, which in turn can be used to support the modeling and refinement of protein structures. Photolytically generated carbenes are a promising class of reagent for this purpose. Substituent effects appear to influence surface mapping properties, allowing for a useful measure of design control. However, to use carbene labeling data in a quantitative manner for modeling activities, we require a better understanding of their inherent amino acid reactivity, so that incorporation data can be normalized. The current study presents an analysis of the amino acid insertion frequency of aliphatic carbenes generated by the photolysis of three different diazirines: 3,3'-azibutyl-1-ammonium, 3,3'-azibutan-1-ol, and 4,4'-azipentan-1-oate. Leveraging an improved photolysis system for single-shot labeling of sub-microliter frozen samples, we used EThCD to localize insertion products in a large population of labeled peptides. Counting statistics were drawn from data-dependent LC-MS2 experiments and used to estimate the frequencies of insertion as a function of amino acid. We observed labeling of all 20 amino acids over a remarkably narrow range of insertion frequencies. However, the nature of the substituent could influence relative insertion frequencies, within a general preference for larger polar amino acids. We confirm a large (6-fold) increase in labeling yield when carbenes were photogenerated in the solid phase (77 K) relative to the liquid phase (293 K), and we suggest that carbene labeling should always be conducted in the frozen state to avoid information loss in surface mapping experiments. Graphical Abstract ᅟ.


Assuntos
Aminoácidos/química , Diazometano/química , Metano/análogos & derivados , Proteínas/química , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Cromatografia Líquida , Indicadores e Reagentes/química , Isomerismo , Metano/química , Fotólise
19.
Mol Cell Proteomics ; 16(6): 1162-1171, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28404794

RESUMO

Trypsin dominates bottom-up proteomics, but there are reasons to consider alternative enzymes. Improving sequence coverage, exposing proteomic "dark matter," and clustering post-translational modifications in different ways and with higher-order drive the pursuit of reagents complementary to trypsin. Additionally, enzymes that are easy to use and generate larger peptides that capitalize upon newer fragmentation technologies should have a place in proteomics. We expressed and characterized recombinant neprosin, a novel prolyl endoprotease of the DUF239 family, which preferentially cleaves C-terminal to proline residues under highly acidic conditions. Cleavage also occurs C-terminal to alanine with some frequency, but with an intriguingly high "skipping rate." Digestion proceeds to a stable end point, resulting in an average peptide mass of 2521 units and a higher dependence upon electron-transfer dissociation for peptide-spectrum matches. In contrast to most proline-cleaving enzymes, neprosin effectively degrades proteins of any size. For 1251 HeLa cell proteins identified in common using trypsin, Lys-C, and neprosin, almost 50% of the neprosin sequence contribution is unique. The high average peptide mass coupled with cleavage at residues not usually modified provide new opportunities for profiling clusters of post-translational modifications. We show that neprosin is a useful reagent for reading epigenetic marks on histones. It generates peptide 1-38 of histone H3 and peptide 1-32 of histone H4 in a single digest, permitting the analysis of co-occurring post-translational modifications in these important N-terminal tails.


Assuntos
Histonas/metabolismo , Proteômica/métodos , Células HeLa , Histonas/química , Humanos , Peptídeo Hidrolases/metabolismo , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/metabolismo
20.
Nucleic Acids Res ; 45(10): 6238-6251, 2017 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-28453785

RESUMO

Non-homologous end joining (NHEJ) repairs DNA double strand breaks in non-cycling eukaryotic cells. NHEJ relies on polynucleotide kinase/phosphatase (PNKP), which generates 5΄-phosphate/3΄-hydroxyl DNA termini that are critical for ligation by the NHEJ DNA ligase, LigIV. PNKP and LigIV require the NHEJ scaffolding protein, XRCC4. The PNKP FHA domain binds to the CK2-phosphorylated XRCC4 C-terminal tail, while LigIV uses its tandem BRCT repeats to bind the XRCC4 coiled-coil. Yet, the assembled PNKP-XRCC4-LigIV complex remains uncharacterized. Here, we report purification and characterization of a recombinant PNKP-XRCC4-LigIV complex. We show that the stable binding of PNKP in this complex requires XRCC4 phosphorylation and that only one PNKP protomer binds per XRCC4 dimer. Small angle X-ray scattering (SAXS) reveals a flexible multi-state complex that suggests that both the PNKP FHA and catalytic domains contact the XRCC4 coiled-coil and LigIV BRCT repeats. Hydrogen-deuterium exchange indicates protection of a surface on the PNKP phosphatase domain that may contact XRCC4-LigIV. A mutation on this surface (E326K) causes the hereditary neuro-developmental disorder, MCSZ. This mutation impairs PNKP recruitment to damaged DNA in human cells and provides a possible disease mechanism. Together, this work unveils multipoint contacts between PNKP and XRCC4-LigIV that regulate PNKP recruitment and activity within NHEJ.


Assuntos
Reparo do DNA por Junção de Extremidades/fisiologia , DNA Ligase Dependente de ATP/fisiologia , Enzimas Reparadoras do DNA/fisiologia , Proteínas de Ligação a DNA/fisiologia , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologia , Domínio Catalítico , Dano ao DNA , DNA Ligase Dependente de ATP/química , Enzimas Reparadoras do DNA/química , Enzimas Reparadoras do DNA/deficiência , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/química , Deutério/metabolismo , Deficiências do Desenvolvimento/genética , Humanos , Espectrometria de Massas , Microcefalia/genética , Modelos Moleculares , Complexos Multiproteicos , Mutação de Sentido Incorreto , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/deficiência , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Mutação Puntual , Ligação Proteica , Conformação Proteica , Mapeamento de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espalhamento a Baixo Ângulo , Convulsões/genética , Síndrome , Difração de Raios X
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