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1.
Biochemistry ; 50(38): 8251-60, 2011 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-21859100

RESUMO

The technique of hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) has been applied to a mesophilic (E. coli) dihydrofolate reductase under conditions that allow direct comparison to a thermophilic (B. stearothermophilus) ortholog, Ec-DHFR and Bs-DHFR, respectively. The analysis of hydrogen-deuterium exchange patterns within proteolytically derived peptides allows spatial resolution, while requiring a series of controls to compare orthologous proteins with only ca. 40% sequence identity. These controls include the determination of primary structure effects on intrinsic rate constants for HDX as well as the use of existing 3-dimensional structures to evaluate the distance of each backbone amide hydrogen to the protein surface. Only a single peptide from the Ec-DHFR is found to be substantially more flexible than the Bs-DHFR at 25 °C in a region located within the protein interior at the intersection of the cofactor and substrate-binding sites. The surrounding regions of the enzyme are either unchanged or more flexible in the thermophilic DHFR from B. stearothermophilus. The region with increased flexibility in Ec-DHFR corresponds to one of two regions previously proposed to control the enthalpic barrier for hydride transfer in Bs-DHFR [Oyeyemi et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 10074].


Assuntos
Tetra-Hidrofolato Desidrogenase/química , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Medição da Troca de Deutério , Estabilidade Enzimática , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Geobacillus stearothermophilus/enzimologia , Geobacillus stearothermophilus/genética , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Homologia Estrutural de Proteína , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismo , Termodinâmica
2.
J Proteome Res ; 10(7): 3060-75, 2011 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-21599010

RESUMO

When analyzing proteins in complex samples using tandem mass spectrometry of peptides generated by proteolysis, the inference of proteins can be ambiguous, even with well-validated peptides. Unresolved questions include whether to show all possible proteins vs a minimal list, what to do when proteins are inferred ambiguously, and how to quantify peptides that bridge multiple proteins, each with distinguishing evidence. Here we describe IsoformResolver, a peptide-centric protein inference algorithm that clusters proteins in two ways, one based on peptides experimentally identified from MS/MS spectra, and the other based on peptides derived from an in silico digest of the protein database. MS/MS-derived protein groups report minimal list proteins in the context of all possible proteins, without redundantly listing peptides. In silico-derived protein groups pull together functionally related proteins, providing stable identifiers. The peptide-centric grouping strategy used by IsoformResolver allows proteins to be displayed together when they share peptides in common, providing a comprehensive yet concise way to organize protein profiles. It also summarizes information on spectral counts and is especially useful for comparing results from multiple LC-MS/MS experiments. Finally, we examine the relatedness of proteins within IsoformResolver groups and compare its performance to other protein inference software.


Assuntos
Mineração de Dados/métodos , Fragmentos de Peptídeos/análise , Isoformas de Proteínas , Proteômica/métodos , Algoritmos , Cromatografia Líquida , Bases de Dados de Proteínas , Humanos , Fragmentos de Peptídeos/química , Isoformas de Proteínas/análise , Isoformas de Proteínas/química , Software , Espectrometria de Massas em Tandem , Tripsina/metabolismo
3.
J Dermatol Sci ; 61(1): 51-9, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21146962

RESUMO

BACKGROUND: In normal mammalian epidermis, cell division occurs primarily in the basal layer where cells are attached to the basement membrane. Upon release from the basement membrane, these basal cells stop dividing and begin to differentiate and stratify producing cornified cells expressing differentiation markers, including the keratin bundling protein filaggrin, and cornified envelope proteins. Little is understood about the regulatory mechanisms of these processes. A rat epidermal keratinocyte cell line synthesizing and processing profilaggrin at confluence in a synchronous manner for 4-5 days provides a useful culture model for epidermal differentiation. Profilaggrin expression in this cell line however decreases with passaging, and its processing involves extensive nonspecific proteolysis. OBJECTIVE: Our objective was to identify culture conditions that effect the decrease in profilaggrin expression with passaging and nonspecific proteolysis of profilaggrin in order to study epidermal differentiation more closely. METHOD: The large amount of nonspecific proteolysis suggested autophagocytosis. To test this, cells were cultured in the presence of 3-methyladenine (3-MA). Two known gradients in epidermis are decreasing serum components and increasing calcium concentrations in the upper cell layers. To determine whether these gradients effected processing, cells were cultured in serum/DMEM or in serum-free KGM and under varying external calcium concentrations. Cells were also cultured in presence of aminoguanidine in an attempt to maintain profilaggrin expression with passaging. RESULTS: Profilaggrin expression was enhanced in the presence of 3-MA, with optimum around 6mM. In the absence of aminoguanidine, profilaggrin expression decreased as a function of increasing passage number; in its presence, profilaggrin expression remained high in some, but not in all of the independently maintained cell lines. Thus, culturing in aminoguanidine was necessary, but not sufficient, for sustained ability to express profilaggrin at confluence. Production of filaggrin from profilaggrin was maximized in a serum-free medium with [Ca(2+)] at 5mM. Filaggrin associates with phospholipid vesicles in vitro forming aggregates similar to those seen in vivo, suggesting that filaggrin release induces vesicular aggregation and autophagocytosis. CONCLUSION: We have used a keratinocyte cell line that synthesizes and processes profilaggrin after confluence as a culture model to study epidermal differentiation. In this system profilaggrin processing must be preceded by inhibition of autophagosome formation and/or modulation of vesicular trafficking, and these processes are regulated by epidermal calcium and serum factor gradients.


Assuntos
Cálcio/metabolismo , Técnicas de Cultura/métodos , Proteínas de Filamentos Intermediários/metabolismo , Queratinócitos/metabolismo , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Autofagia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Proteínas Filagrinas , Guanidinas/farmacologia , Proteínas de Filamentos Intermediários/efeitos dos fármacos , Ratos
4.
Proc Natl Acad Sci U S A ; 107(22): 10074-9, 2010 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-20534574

RESUMO

We report hydrogen deuterium exchange by mass spectrometry (HDX-MS) as a function of temperature in a thermophilic dihydrofolate reductase from Bacillus stearothermophilus (Bs-DHFR). Protein stability, probed with circular dichroism, established an accessible temperature range of 10 degrees C to 55 degrees C for the interrogation of HDX-MS. Although both the rate and extent of HDX are sensitive to temperature, the majority of peptides showed rapid kinetics of exchange, allowing us to focus on plateau values for the maximal extent of exchange at each temperature. Arrhenius plots of the ratio of hydrogens exchanged at 5 h normalized to the number of exchangeable hydrogens vs. 1/T provides an estimate for the apparent enthalpic change of local unfolding, DeltaH degrees (unf(avg)). Most regions in the enzyme show DeltaH degrees (unf(avg)) < or = 2.0 kcal/mol, close to the value of kT; by contrast, significantly elevated values for DeltaH degrees (unf(avg)) are observed in regions within the core of protein that contain the cofactor and substrate-binding sites. Our technique introduces a new strategy for probing the temperature dependence of local protein unfolding within native proteins. These findings are discussed in the context of the demonstrated role for nuclear tunneling in hydride transfer from NADPH to dihydrofolate, and relate the observed enthalpic changes to two classes of motion, preorganization and reorganization, that have been proposed to control the efficiency of hydrogenic wave function overlap. Our findings suggest that the enthalpic contribution to the heavy atom environmental reorganizations controlling the hydrogenic wave function overlap will be dominated by regions of the protein proximal to the bound cofactor and substrate.


Assuntos
Tetra-Hidrofolato Desidrogenase/química , Tetra-Hidrofolato Desidrogenase/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Deutério , Estabilidade Enzimática , Geobacillus stearothermophilus/enzimologia , Geobacillus stearothermophilus/genética , Hidrogênio , Cinética , Modelos Moleculares , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Movimento (Física) , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Temperatura , Tetra-Hidrofolato Desidrogenase/genética , Termodinâmica
5.
Mol Cell Proteomics ; 8(10): 2308-20, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19620624

RESUMO

RhoA controls changes in cell morphology and invasion associated with cancer phenotypes. Cell lines derived from melanoma tumors at varying stages revealed that RhoA is selectively activated in cells of metastatic origin. We describe a functional proteomics strategy to identify proteins regulated by RhoA and report a previously uncharacterized human protein, named "mediator of RhoA-dependent invasion (MRDI)," that is induced in metastatic cells by constitutive RhoA activation and promotes cell invasion. In human melanomas, MRDI localization correlated with stage, showing nuclear localization in nevi and early stage tumors and cytoplasmic localization with plasma membrane accentuation in late stage tumors. Consistent with its role in promoting cell invasion, MRDI localized to cell protrusions and leading edge membranes in cultured cells and was required for cell motility, tyrosine phosphorylation of focal adhesion kinase, and modulation of actin stress fibers. Unexpectedly MRDI had enzymatic function as an isomerase that converts the S-adenosylmethionine catabolite 5-methylribose 1-phosphate into 5-methylribulose 1-phosphate. The enzymatic function of MRDI was required for methionine salvage from S-adenosylmethionine but distinct from its function in cell invasion. Thus, mechanisms used by signal transduction pathways to control cell movement have evolved from proteins with ancient function in amino acid metabolism.


Assuntos
Aldose-Cetose Isomerases/metabolismo , Melanoma , Metionina/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Aldose-Cetose Isomerases/genética , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Ativação Enzimática , Feminino , Humanos , Melanoma/enzimologia , Melanoma/patologia , Metionina/química , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Estrutura Molecular , Invasividade Neoplásica , Metástase Neoplásica , Proteômica/métodos , Interferência de RNA , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , Transdução de Sinais/fisiologia , Transplante Heterólogo , Proteína rhoA de Ligação ao GTP/genética
6.
Mol Cell ; 34(1): 115-31, 2009 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-19362540

RESUMO

Melanoma and other cancers harbor oncogenic mutations in the protein kinase B-Raf, which leads to constitutive activation and dysregulation of MAP kinase signaling. In order to elucidate molecular determinants responsible for B-Raf control of cancer phenotypes, we present a method for phosphoprotein profiling, using negative ionization mass spectrometry to detect phosphopeptides based on their fragment ion signature caused by release of PO(3)(-). The method provides an alternative strategy for phosphoproteomics, circumventing affinity enrichment of phosphopeptides and isotopic labeling of samples. Ninety phosphorylation events were regulated by oncogenic B-Raf signaling, based on their responses to treating melanoma cells with MKK1/2 inhibitor. Regulated phosphoproteins included known signaling effectors and cytoskeletal regulators. We investigated MINERVA/FAM129B, a target belonging to a protein family with unknown category and function, and established the importance of this protein and its MAP kinase-dependent phosphorylation in controlling melanoma cell invasion into three-dimensional collagen matrix.


Assuntos
Melanoma/metabolismo , Proteômica , Proteínas Proto-Oncogênicas B-raf/metabolismo , Linhagem Celular Tumoral , Humanos , Sistema de Sinalização das MAP Quinases , Espectrometria de Massas , Mutação , Fosfoproteínas/análise , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosfoproteínas/fisiologia , Fosforilação , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/fisiologia , Especificidade por Substrato
7.
Mol Cell Proteomics ; 8(4): 857-69, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19106086

RESUMO

Identifying peptides from mass spectrometric fragmentation data (MS/MS spectra) using search strategies that map protein sequences to spectra is computationally expensive. An alternative strategy uses direct spectrum-to-spectrum matching against a reference library of previously observed MS/MS that has the advantage of evaluating matches using fragment ion intensities and other ion types than the simple set normally used. However, this approach is limited by the small sizes of the available peptide MS/MS libraries and the inability to evaluate the rate of false assignments. In this study, we observed good performance of simulated spectra generated by the kinetic model implemented in MassAnalyzer (Zhang, Z. (2004) Prediction of low-energy collision-induced dissociation spectra of peptides. Anal. Chem. 76, 3908-3922; Zhang, Z. (2005) Prediction of low-energy collision-induced dissociation spectra of peptides with three or more charges. Anal. Chem. 77, 6364-6373) as a substitute for the reference libraries used by the spectrum-to-spectrum search programs X!Hunter and BiblioSpec and similar results in comparison with the spectrum-to-sequence program Mascot. We also demonstrate the use of simulated spectra for searching against decoy sequences to estimate false discovery rates. Although we found lower score discrimination with spectrum-to-spectrum searches than with Mascot, particularly for higher charge forms, comparable peptide assignments with low false discovery rate were achieved by examining consensus between X!Hunter and Mascot, filtering results by mass accuracy, and ignoring score thresholds. Protein identification results are comparable to those achieved when evaluating consensus between Sequest and Mascot. Run times with large scale data sets using X!Hunter with the simulated spectral library are 7 times faster than Mascot and 80 times faster than Sequest with the human International Protein Index (IPI) database. We conclude that simulated spectral libraries greatly expand the search space available for spectrum-to-spectrum searching while enabling principled analyses and that the approach can be used in consensus strategies for large scale studies while reducing search times.


Assuntos
Simulação por Computador , Espectrometria de Massas , Biblioteca de Peptídeos , Proteínas/análise , Sequência de Aminoácidos , Linhagem Celular Tumoral , Bases de Dados de Proteínas , Humanos , Peptídeos/química , Proteínas/química , Curva ROC , Padrões de Referência , Software
8.
J Mol Biol ; 379(5): 1075-93, 2008 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-18501927

RESUMO

Hydrogen-deuterium exchange measurements represent a powerful approach to investigating changes in conformation and conformational mobility in proteins. Here, we examine p38alpha MAP kinase (MAPK) by hydrogen-exchange (HX) mass spectrometry to determine whether changes in conformational mobility may be induced by kinase phosphorylation and activation. Factors influencing sequence coverage in the HX mass spectrometry experiment, which show that varying sampling depths, instruments, and peptide search strategies yield the highest coverage of exchangeable amides, are examined. Patterns of regional deuteration in p38alpha are consistent with tertiary structure and similar to deuteration patterns previously determined for extracellular-signal-regulated kinase (ERK) 2, indicating that MAPKs are conserved with respect to the extent of local amide HX. Activation of p38alpha alters HX in five regions, which are interpreted by comparing X-ray structures of unphosphorylated p38alpha and X-ray structures of phosphorylated p38gamma. Conformational differences account for altered HX within the activation lip, the P+1 site, and the active site. In contrast, HX alterations are ascribed to activation-induced effects on conformational mobility, within substrate-docking sites (alphaF-alphaG, beta7-beta8), the C-terminal core (alphaE), and the N-terminal core region (beta4-beta5, alphaL16, alphaC). Activation also decreases HX in a 3-10 helix at the C-terminal extension of p38alpha. Although this helix in ERK2 forms a dimerization interface that becomes protected from HX upon activation, analytical ultracentrifugation shows that this does not occur in p38alpha because both unphosphorylated and diphosphorylated forms are monomeric. Finally, HX patterns in monophosphorylated p38alpha are similar to those in unphosphorylated kinase, indicating that the major activation lip remodeling events occur only after diphosphorylation. Importantly, patterns of activation-induced HX show differences between p38alpha and ERK2 despite their similarities in overall deuteration, suggesting that although MAPKs are closely related with respect to primary sequence and tertiary structure, they have distinct mechanisms for dynamic control of enzyme function.


Assuntos
Proteína Quinase 14 Ativada por Mitógeno/química , Amidas/química , Sequência de Aminoácidos , Animais , Cromatografia Líquida , Cristalografia por Raios X , Medição da Troca de Deutério , Ativação Enzimática , Hidrogênio/química , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/química , Proteína Quinase 14 Ativada por Mitógeno/genética , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Dados de Sequência Molecular , Fosforilação , Conformação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometria de Massas em Tandem
9.
Nat Methods ; 4(10): 798-806, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17901869

RESUMO

Post-translational modifications of proteins control many biological processes, and examining their diversity is critical for understanding mechanisms of cell regulation. Mass spectrometry is a fundamental tool for detecting and mapping covalent modifications and quantifying their changes. Modern approaches have made large-scale experiments possible, screening complex mixtures of proteins for alterations in chemical modifications. By profiling protein chemistries, biologists can gain deeper insight into biological control. The aim of this review is introduce biologists to current strategies in mass spectrometry-based proteomics that are used to characterize protein post-translational modifications, noting strengths and shortcomings of various approaches.


Assuntos
Espectrometria de Massas/métodos , Processamento de Proteína Pós-Traducional , Acetilação , Cisteína/química , Bases de Dados de Proteínas , Marcação por Isótopo , Oxirredução , Fosfopeptídeos/isolamento & purificação , Fosforilação , Mapeamento de Interação de Proteínas , Espectrometria de Massas em Tandem/métodos , Ubiquitina/metabolismo
10.
J Biol Chem ; 282(42): 30553-61, 2007 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-17728254

RESUMO

The Mps1 protein kinase is required for proper assembly of the mitotic spindle, checkpoint signaling, and several other aspects of cell growth and differentiation. Mps1 regulation is mediated by cell cycle-dependent changes in transcription and protein level. There is also a strong correlation between hyperphosphorylated mitotic forms of Mps1 and increased kinase activity. We investigated the role that autophosphorylation plays in regulating human Mps1 (hMps1) protein kinase activity. Here we report that hyperphosphorylated hMps1 forms are not the only active forms of the kinase. However, autophosphorylation of hMps1 within the activation loop is required for full activity in vitro. Mass spectrometry analysis of de novo synthesized enzyme in Escherichia coli identified autophosphorylation sites at residues Thr(675), Thr(676), and Thr(686), but phosphatase-treated and reactivated enzyme was only phosphorylated on Thr(676). Mutation of Thr(676) in hMps1 or the corresponding Thr(591) residue within yeast Mps1 reduces kinase activity in vitro. We find that overexpression of an hMps1-T676A mutation inhibits centrosome duplication in RPE1 cells. Likewise, yeast cells harboring mps1-T591A as the sole MPS1 allele are not viable. Our data strongly support the conclusion that site-specific Mps1 autophosphorylation within the activation loop is required for full activity in vitro and function in vivo.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Centrossomo/enzimologia , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Fuso Acromático/enzimologia , Alelos , Substituição de Aminoácidos , Proteínas de Ciclo Celular/genética , Diferenciação Celular/fisiologia , Linhagem Celular , Proliferação de Células , Sobrevivência Celular/genética , Ativação Enzimática/genética , Escherichia coli/genética , Humanos , Espectrometria de Massas , Mutação de Sentido Incorreto , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fuso Acromático/genética , Treonina/genética , Treonina/metabolismo
11.
J Proteome Res ; 6(6): 2176-85, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17503796

RESUMO

The pro-inflammatory cytokines, Tumor Necrosis Factor-alpha (TNFalpha) and Interleukin-1 (IL-1) mediate the innate immune response. Dysregulation of the innate immune response contributes to the pathogenesis of cancer, arthritis, and congestive heart failure. TNFalpha- and IL-1-induced changes in gene expression are mediated by similar transcription factors; however, TNFalpha and IL-1 receptor knock-out mice differ in their sensitivities to a known initiator (lipopolysaccharide, LPS) of the innate immune response. The contrasting responses to LPS indicate that TNFalpha and IL-1 regulate different processes. A large-scale proteomic analysis of TNFalpha- and IL-1-induced responses was undertaken to identify processes uniquely regulated by TNFalpha and IL-1. When combined with genomic studies, our results indicate that TNFalpha, but not IL-1, mediates cell cycle arrest.


Assuntos
Genômica , Interleucina-1/farmacologia , Proteômica , Fator de Necrose Tumoral alfa/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Permeabilidade da Membrana Celular/efeitos dos fármacos , Citoplasma/efeitos dos fármacos , Citoplasma/genética , Citoplasma/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Membranas Mitocondriais/efeitos dos fármacos , Proteínas/análise , Proteínas/genética , RNA Mensageiro/análise , Transcrição Gênica/efeitos dos fármacos
12.
ACS Chem Biol ; 2(1): 39-52, 2007 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-17243782

RESUMO

Proteomics addresses the important goal of determining the chemistry and composition of proteins in biological samples. Mass-spectrometry-based strategies have been highly successful in identifying and profiling proteins in complex mixtures; however, although depth of sampling continues to improve, a general recognition exists that no study has yet achieved complete protein coverage in any tissue, cell type, subcellular component, or fluid. The development of new approaches for comprehensively surveying highly complex protein mixtures, distinguishing protein isoforms, quantifying changes in protein abundance between different samples, and mapping post-translational modifications are areas of active research. These will be needed to achieve the "systems-wide" protein profiling goals of defining molecular responses to cell perturbations and obtaining biomarker information for disease detection, prognosis, and responses to therapy. We review recent progress in approaching these problems and present examples of successful applications and the outlook for the future.


Assuntos
Espectrometria de Massas/métodos , Análise Serial de Proteínas/métodos , Proteômica/métodos , Animais , Humanos , Proteoma/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos
13.
Mol Cell Proteomics ; 6(1): 1-17, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17018520

RESUMO

A major limitation in identifying peptides from complex mixtures by shotgun proteomics is the ability of search programs to accurately assign peptide sequences using mass spectrometric fragmentation spectra (MS/MS spectra). Manual analysis is used to assess borderline identifications; however, it is error-prone and time-consuming, and criteria for acceptance or rejection are not well defined. Here we report a Manual Analysis Emulator (MAE) program that evaluates results from search programs by implementing two commonly used criteria: 1) consistency of fragment ion intensities with predicted gas phase chemistry and 2) whether a high proportion of the ion intensity (proportion of ion current (PIC)) in the MS/MS spectra can be derived from the peptide sequence. To evaluate chemical plausibility, MAE utilizes similarity (Sim) scoring against theoretical spectra simulated by MassAnalyzer software (Zhang, Z. (2004) Prediction of low-energy collision-induced dissociation spectra of peptides. Anal. Chem. 76, 3908-3922) using known gas phase chemical mechanisms. The results show that Sim scores provide significantly greater discrimination between correct and incorrect search results than achieved by Sequest XCorr scoring or Mascot Mowse scoring, allowing reliable automated validation of borderline cases. To evaluate PIC, MAE simplifies the DTA text files summarizing the MS/MS spectra and applies heuristic rules to classify the fragment ions. MAE output also provides data mining functions, which are illustrated by using PIC to identify spectral chimeras, where two or more peptide ions were sequenced together, as well as cases where fragmentation chemistry is not well predicted.


Assuntos
Espectrometria de Massas/métodos , Peptídeos/análise , Peptídeos/química , Sequência de Aminoácidos , Bases de Dados de Proteínas , Humanos , Células K562 , Dados de Sequência Molecular , Proteínas de Neoplasias/química , Análise Serial de Proteínas , Proteômica , Curva ROC , Reprodutibilidade dos Testes , Software
14.
Proc Natl Acad Sci U S A ; 103(48): 18101-6, 2006 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-17114285

RESUMO

Studies of protein kinases have identified a "gatekeeper" residue, which confers selectivity for binding nucleotides and small-molecule inhibitors. We report that, in the MAP kinase ERK2, mutations at the gatekeeper residue unexpectedly lead to autoactivation due to enhanced autophosphorylation of regulatory Tyr and Thr sites within the activation lip that control kinase activity. This occurs through an intramolecular mechanism, indicating that the gatekeeper residue indirectly constrains flexibility at the activation lip, precluding access of the phosphoacceptor residues to the catalytic base. Other residues that interact with the gatekeeper site to form a hydrophobic cluster in the N-terminal domain also cause autoactivation when mutated. Hydrogen-exchange studies of a mutant within this cluster reveal perturbations in the conserved DFG motif, predicting a route for side chain connectivity from the hydrophobic cluster to the activation lip. Mutations of residues along this route support this model, explaining how information about the gatekeeper residue identity can be transmitted to the activation lip. Thus, an N-terminal hydrophobic cluster that includes the gatekeeper forms a novel structural unit, which functions to maintain the "off" state of ERK2 before cell signal activation.


Assuntos
Sistema de Sinalização das MAP Quinases , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Animais , Ativação Enzimática , Glutamina/genética , Glutamina/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Proteína Quinase 1 Ativada por Mitógeno/química , Proteína Quinase 1 Ativada por Mitógeno/genética , Modelos Moleculares , Mutação/genética , Fosforilação , Ligação Proteica , Estrutura Quaternária de Proteína , Ratos
15.
Curr Opin Struct Biol ; 16(6): 686-92, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17085044

RESUMO

The allosteric regulation of protein kinases serves as an efficient strategy for molecular communication, event coupling and interconversion between catalytic states. Recent co-crystal structures have revealed novel ways in which kinases control activity and substrate specificity following phosphorylation, dimerization, or binding to regulatory proteins, substrates and scaffolds. In addition, hydrogen exchange coupled with mass spectrometry is emerging as a complementary strategy to probe the solution behavior of kinases; recent results have shown that allosteric regulation may involve transitions in protein motions as well as structural rearrangements.


Assuntos
Proteínas Quinases/química , Proteínas Quinases/metabolismo , Regulação Alostérica , Sítio Alostérico , Deutério , Dimerização , MAP Quinases Reguladas por Sinal Extracelular/química , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Hidrogênio , Espectrometria de Massas , Modelos Biológicos , Modelos Moleculares , Estrutura Quaternária de Proteína , Transdução de Sinais
16.
Proteomics ; 6(16): 4541-53, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16858730

RESUMO

Although the importance of the extracellular signal-regulated kinase (ERK) pathway in regulating the transition from G1 to S has been extensively studied, its role during the G2/M transition is less well understood. Previous reports have shown that inhibition of the ERK pathway in mammalian cells delays entry as well as progression through mitosis, suggesting the existence of molecular targets of this pathway in M phase. In this report we employed 2-DE and MS to survey proteins and PTMs in the presence versus absence of MKK1/2 inhibitor. Targets of the ERK pathway in G2/M were identified as elongation factor 2 (EF2) and nuclear matrix protein, 55 kDa (Nmt55). Phosphorylation of each protein increased under conditions of ERK pathway inhibition, suggesting indirect control of these targets; regulation of EF2 was ascribed to phosphorylation and inactivation of upstream EF2 kinase, whereas regulation of Nmt55 was ascribed to a delay in normal mitotic phosphorylation and dephosphorylation. 2-DE Western blots probed using anti-phospho-Thr-Pro antibody demonstrated that the effect of ERK inhibition is not to delay the onset of phosphorylation controlled by cdc2 and other mitotic kinases, but rather to regulate a small subset of targets in M phase in a nonoverlapping manner with cdc2.


Assuntos
Ciclo Celular/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Proteínas de Ligação a DNA , Eletroforese em Gel Bidimensional , Ativação Enzimática , Células HeLa , Humanos , Dados de Sequência Molecular , Proteínas Associadas à Matriz Nuclear/metabolismo , Fatores de Transcrição de Octâmero/metabolismo , Fator 2 de Elongação de Peptídeos/metabolismo , Fosforilação , Proteômica , Proteínas de Ligação a RNA/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
17.
Mol Cell Proteomics ; 5(8): 1359-67, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16644720

RESUMO

Rho GTPases are signal transduction effectors that control cell motility, cell attachment, and cell shape by the control of actin polymerization and tyrosine phosphorylation. To identify cellular targets regulated by Rho GTPases, we screened global protein responses to Rac1, Cdc42, and RhoA activation by two-dimensional gel electrophoresis and mass spectrometry. A total of 22 targets were identified of which 19 had never been previously linked to Rho GTPase pathways, providing novel insight into pathway function. One novel target of RhoA was protein-tyrosine phosphatase 1B (PTP1B), which catalyzes dephosphorylation of key signaling molecules in response to activation of diverse pathways. Subsequent analysis demonstrated that RhoA enhances post-translational modification of PTP1B, inactivates phosphotyrosine phosphatase activity, and up-regulates tyrosine phosphorylation of p130Cas, a key mediator of focal adhesion turnover and cell migration. Thus, protein profiling reveals a novel role for PTP1B as a mediator of RhoA-dependent phosphorylation of p130Cas.


Assuntos
Proteína Substrato Associada a Crk/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Transdução de Sinais/fisiologia , Proteína rhoA de Ligação ao GTP/metabolismo , Linhagem Celular Tumoral , Eletroforese em Gel Bidimensional , Humanos , Fosforilação , Fosfotirosina/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Proteína Tirosina Fosfatase não Receptora Tipo 1 , Proteômica , Regulação para Cima/fisiologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo
18.
J Proteome Res ; 5(3): 709-19, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16512687

RESUMO

An important strategy for "shotgun proteomics" profiling involves solution proteolysis of proteins, followed by peptide separation using multidimensional liquid chromatography and automated sequencing by mass spectrometry (LC-MS/MS). Several protocols for extracting and handling membrane proteins for shotgun proteomics experiments have been reported, but few direct comparisons of different protocols have been reported. We compare four methods for preparing membrane proteins from human cells, using acid labile surfactants (ALS), urea, and mixed organic-aqueous solvents. These methods were compared with respect to their efficiency of protein solubilization and proteolysis, peptide and protein recovery, membrane protein enrichment, and peptide coverage of transmembrane proteins. Overall, approximately 50-60% of proteins recovered were membrane-associated, identified from Gene Ontology annotations and transmembrane prediction software. Samples extracted with ALS, extracted with urea followed by dilution, or extracted with urea followed by desalting yielded comparable peptide recoveries and sequence coverage of transmembrane proteins. In contrast, suboptimal proteolysis was observed with organic solvent. Urea extraction followed by desalting may be a particularly useful approach, as it is less costly than ALS and yields satisfactory protein denaturation and proteolysis under conditions that minimize reactivity with urea-derived cyanate. Spectral counting was used to compare datasets of proteins from membrane samples with those of soluble proteins from K562 cells, and to estimate fold differences in protein abundances. Proteins most highly abundant in the membrane samples showed enrichment of integral membrane protein identifications, consistent with their isolation by differential centrifugation.


Assuntos
Extratos Celulares/análise , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Proteínas de Membrana/análise , Proteínas de Neoplasias/química , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Humanos , Células K562 , Proteínas de Neoplasias/análise , Espectrometria de Massas em Tandem
19.
Anal Chem ; 78(4): 1071-84, 2006 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-16478097

RESUMO

Correct identification of a peptide sequence from MS/MS data is still a challenging research problem, particularly in proteomic analyses of higher eukaryotes where protein databases are large. The scoring methods of search programs often generate cases where incorrect peptide sequences score higher than correct peptide sequences (referred to as distraction). Because smaller databases yield less distraction and better discrimination between correct and incorrect assignments, we developed a method for editing a peptide-centric database (PC-DB) to remove unlikely sequences and strategies for enabling search programs to utilize this peptide database. Rules for unlikely missed cleavage and nontryptic proteolysis products were identified by data mining 11 849 high-confidence peptide assignments. We also evaluated ion exchange chromatographic behavior as an editing criterion to generate subset databases. When used to search a well-annotated test data set of MS/MS spectra, we found no loss of critical information using PC-DBs, validating the methods for generating and searching against the databases. On the other hand, improved confidence in peptide assignments was achieved for tryptic peptides, measured by changes in DeltaCN and RSP. Decreased distraction was also achieved, consistent with the 3-9-fold decrease in database size. Data mining identified a major class of common nonspecific proteolytic products corresponding to leucine aminopeptidase (LAP) cleavages. Large improvements in identifying LAP products were achieved using the PC-DB approach when compared with conventional searches against protein databases. These results demonstrate that peptide properties can be used to reduce database size, yielding improved accuracy and information capture due to reduced distraction, but with little loss of information compared to conventional protein database searches.


Assuntos
Bases de Dados de Proteínas , Armazenamento e Recuperação da Informação , Peptídeo Hidrolases/metabolismo , Peptídeos/química , Proteômica , Sequência de Aminoácidos , Humanos , Hidrólise , Espectrometria de Massas , Dados de Sequência Molecular , Sensibilidade e Especificidade
20.
J Theor Biol ; 240(3): 464-74, 2006 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-16325864

RESUMO

Integrating biological information from different sources to understand cellular processes is an important problem in systems biology. We use data from mRNA expression arrays and chemical kinetics to formulate a metabolic model relevant to K562 erythroleukemia cells. MAP kinase pathway activation alters the expression of metabolic enzymes in K562 cells. Our array data show changes in expression of lactate dehydrogenase (LDH) isoforms after treatment with phorbol 12-myristate 13-acetate (PMA), which activates MAP kinase signaling. We model the change in lactate production which occurs when the MAP kinase pathway is activated, using a non-equilibrium, chemical-kinetic model of homolactic fermentation. In particular, we examine the role of LDH isoforms, which catalyse the conversion of pyruvate to lactate. Changes in the isoform ratio are not the primary determinant of the production of lactate. Rather, the total concentration of LDH controls the lactate concentration.


Assuntos
Isoenzimas/metabolismo , Lactato Desidrogenases/metabolismo , Ácido Láctico/biossíntese , Sistema de Sinalização das MAP Quinases/fisiologia , Modelos Químicos , RNA Mensageiro/metabolismo , Linhagem Celular Tumoral , Ativação Enzimática , Eritrócitos/metabolismo , Fermentação , Perfilação da Expressão Gênica , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Ácido Pirúvico/metabolismo , Acetato de Tetradecanoilforbol/farmacologia
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