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1.
Cells ; 10(11)2021 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-34831095

RESUMO

Human fetal progenitor tenocytes (hFPT) produced in defined cell bank systems have recently been characterized and qualified as potential therapeutic cell sources in tendon regenerative medicine. In view of further developing the manufacture processes of such cell-based active pharmaceutical ingredients (API), the effects of hypoxic in vitro culture expansion on key cellular characteristics or process parameters were evaluated. To this end, multiple aspects were comparatively assessed in normoxic incubation (i.e., 5% CO2 and 21% O2, standard conditions) or in hypoxic incubation (i.e., 5% CO2 and 2% O2, optimized conditions). Experimentally investigated parameters and endpoints included cellular proliferation, cellular morphology and size distribution, cell surface marker panels, cell susceptibility toward adipogenic and osteogenic induction, while relative protein expression levels were analyzed by quantitative mass spectrometry. The results outlined conserved critical cellular characteristics (i.e., cell surface marker panels, cellular phenotype under chemical induction) and modified key cellular parameters (i.e., cell size distribution, endpoint cell yields, matrix protein contents) potentially procuring tangible benefits for next-generation cell manufacturing workflows. Specific proteomic analyses further shed some light on the cellular effects of hypoxia, potentially orienting further hFPT processing for cell-based, cell-free API manufacture. Overall, this study indicated that hypoxic incubation impacts specific hFPT key properties while preserving critical quality attributes (i.e., as compared to normoxic incubation), enabling efficient manufacture of tenocyte-based APIs for homologous standardized transplant products.


Assuntos
Preparações Farmacêuticas/síntese química , Medicina Regenerativa , Tendões/transplante , Tenócitos/patologia , Adipogenia , Biomarcadores/metabolismo , Hipóxia Celular/efeitos dos fármacos , Proliferação de Células , Forma Celular , Tamanho Celular , Células Cultivadas , Regulação para Baixo , Proteínas da Matriz Extracelular/metabolismo , Feto/citologia , Ontologia Genética , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Modelos Biológicos , Osteogênese , Fenótipo , Padrões de Referência , Tenócitos/efeitos dos fármacos , Fatores de Tempo , Regulação para Cima
3.
Cell Rep ; 26(4): 1044-1058.e5, 2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30673600

RESUMO

Meiotic progression in S. pombe is regulated by stage-specific gene expression and translation, changes in RNA stability, expression of anti-sense transcripts, and targeted proteolysis of regulatory proteins. We have used SILAC labeling to examine the relative levels of proteins in diploid S. pombe cells during meiosis. Among the 3,268 proteins quantified at all time points, the levels of 880 proteins changed at least 2-fold; the majority of proteins showed stepwise increases or decreases during the meiotic divisions, while some changed transiently. Overall, we observed reductions in proteins involved in anabolism and increases in proteins involved in catabolism. We also observed increases in the levels of proteins of the ESCRT-III complex and revealed a role for ESCRT-III components in chromosome segregation and spore formation. Correlation with studies of meiotic gene expression and ribosome occupancy reveals that many of the changes in steady-state protein levels are post-transcriptional.


Assuntos
Segregação de Cromossomos/fisiologia , Cromossomos Fúngicos/metabolismo , Regulação Fúngica da Expressão Gênica/fisiologia , Meiose/fisiologia , Proteoma/biossíntese , Proteínas de Schizosaccharomyces pombe/biossíntese , Schizosaccharomyces/metabolismo , Cromossomos Fúngicos/genética , Proteoma/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética
4.
Bioconjug Chem ; 29(10): 3385-3393, 2018 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-30141619

RESUMO

While it is well established that the surface of a nanoparticle plays a pivotal role for the protein corona, the vast number of proteins present in biological media render general conclusions about affinities between nanoparticle surfaces and proteins nontrivial. Recently published articles increasingly reveal differences between systems and an ever increasing number of influencing factors for the protein corona. In contrast, the present study posits that the reported differences may, at least in part, be due to poor experimental design, which leads to biased results. The present study investigates protein adsorption onto silica nanoparticles with different chemical groups on the surface by the statistical analysis of triplicate measurements as well as control measurements. We demonstrate that 60% of the identified protein types did not have any significant affinities for the nanoparticles. Of the remaining 40%, 60% were driven by surface charges and only 40% preferentially adsorbed onto specific surface groups. Furthermore, we found that of the 20 most abundant proteins in the serum, only five bound to the nanoparticles studied here. We illustrate the importance of control replicate experiments to avoid exaggerated differences between systems and to properly quantify the differences and similarities between comparable systems.


Assuntos
Nanopartículas/química , Coroa de Proteína/química , Adsorção , Magnetismo , Microscopia Eletrônica de Transmissão , Espectroscopia Fotoeletrônica , Reprodutibilidade dos Testes , Dióxido de Silício/química , Propriedades de Superfície
5.
J Biol Chem ; 292(44): 18129-18144, 2017 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-28893905

RESUMO

Lysine acetylation is a widespread posttranslational modification affecting many biological pathways. Recent studies indicate that acetylated lysine residues mainly exhibit low acetylation occupancy, but challenges in sample preparation and analysis make it difficult to confidently assign these numbers, limiting understanding of their biological significance. Here, we tested three common sample preparation methods to determine their suitability for assessing acetylation stoichiometry in three human cell lines, identifying the acetylation occupancy in more than 1,300 proteins from each cell line. The stoichiometric analysis in combination with quantitative proteomics also enabled us to explore their functional roles. We found that higher abundance of the deacetylase sirtuin 1 (SIRT1) correlated with lower acetylation occupancy and lower levels of ribosomal proteins, including those involved in ribosome biogenesis and rRNA processing. Treatment with the SIRT1 inhibitor EX-527 confirmed SIRT1's role in the regulation of pre-rRNA synthesis and processing. Specifically, proteins involved in pre-rRNA transcription, including subunits of the polymerase I and SL1 complexes and the RNA polymerase I-specific transcription initiation factor RRN3, were up-regulated after SIRT1 inhibition. Moreover, many protein effectors and regulators of pre-rRNA processing needed for rRNA maturation were also up-regulated after EX-527 treatment with the outcome that pre-rRNA and 28S rRNA levels also increased. More generally, we found that SIRT1 inhibition down-regulates metabolic pathways, including glycolysis and pyruvate metabolism. Together, these results provide the largest data set thus far of lysine acetylation stoichiometry (available via ProteomeXchange with identifier PXD005903) and set the stage for further biological investigations of this central posttranslational modification.


Assuntos
Regulação da Expressão Gênica , Lisina/metabolismo , Processamento de Proteína Pós-Traducional , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Ribossômico/metabolismo , Sirtuína 1/metabolismo , Acetilação/efeitos dos fármacos , Métodos Analíticos de Preparação de Amostras , Carbazóis/farmacologia , Linhagem Celular Transformada , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Humanos , Cinética , Mapeamento de Peptídeos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteômica/métodos , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , RNA Ribossômico 28S/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Sirtuína 1/antagonistas & inibidores , Sirtuína 1/genética , Espectrometria de Massas em Tandem
6.
Analyst ; 142(20): 3805-3815, 2017 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-28695931

RESUMO

Nanoparticles (NPs) in contact with a biological environment get covered by proteins and some are loosely bound and some are tightly bound. The latter form a hard protein corona (HPC) which is known to determine their biological behavior. Therefore, in order to study the biological behaviour of NPs one needs to start from the HPC. However, established methods and standards of HPC isolation are still not known. This is especially a challenge in the case of magnetic NPs which form a major branch of nanomedicine. Therefore, we developed a novel HPC isolation method, a multi-step centrifugation method (MSCM), for single-domain magnetic NPs. The MSCM was applied to iron oxide NPs in interaction with human blood and lymph serum with different dilutions in triplicate. The analysis of the composition of the obtained HPCs showed the reproducibility of the MSCM. This new method was also compared with the existing magnetic separation method (MagSep) and a study of the obtained HPC allowed us to establish the validity limits of MagSep and MSCM on only superparamagnetic NPs and on any single-domain magnetic NPs, respectively. Surprisingly, the HPCs obtained by these two isolation methods were quite different, up to 50%, suggesting that only these proteins, which are found in the HPCs of both isolation methods, are in fact real HPCs.

7.
Small ; 13(29)2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28582610

RESUMO

In biological environments, the surface of nanoparticles (NPs) are modified by protein corona (PC) that determines their biological behavior. Unfortunately, in vitro tests still give different PC than in vivo tests causing in vitro-in vivo discrepancy; hence, in vitro studies are not indicative for the NPs' behavior in vivo. Here is demonstrated that PC in vitro is strongly influenced by the type of extracellular fluid (ECF), blood or lymph, by their high and low flow conditions and transitions between ECFs, and a combination of these parameters. As a result, this in vitro study approaches fluidic and dynamic variations to which NPs are exposed in vivo: different ECF that NPs encounter first in different injection routes, different transitions in-between ECFs during circulation, and simultaneous change in the exposed flow in these transitions. The most-abundant proteins in PCs are found to be not the most abundant in ECFs, but those having high affinity for binding to the surface of NPs. Moreover, some proteins are differently abundant in PCs at different flows, which indicate force-promoted binding, catch bonds. These results suggest that future in vitro studies should consider more complex incubation conditions to improve the in vitro-in vivo consistency necessary for translational research.


Assuntos
Nanopartículas/química , Coroa de Proteína/química , Animais , Líquido Extracelular/química , Humanos
8.
Biochem J ; 467(1): 77-90, 2015 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-25588078

RESUMO

Mitogen-activated protein kinase (MAPK) pathways constitute key regulatory elements linking extracellular stimuli to nuclear gene expression. Immediate-early responsive genes (IEGs) of the activator protein 1 (AP-1) family, such as fos, achieve peak expression levels shortly after cells are stimulated with growth factors and sharply decrease thereafter. Several AU-rich binding proteins (AUBPs), including HuR (Hu-antigen R, Elav-like protein 1, ELAVL1) and KSRP (far upstream element-binding protein 2, KHSRP) bind to a fos AU-rich element (ARE) present in the 3'-UTR (untranslated region) of fos mRNA regulating its stability by a still poorly defined mechanism. We show in the present study that, whereas HuR binds and stabilizes transcribed reporter mRNAs bearing the fos 3'-UTR, KSRP counteracts this effect. Furthermore, we found that fos mRNA stability and HuR phosphorylation status are dependent on the activity of p38 MAPK in both epithelial cells and fibroblasts upon proliferative stimulation. Analysing PPI (protein-protein interaction) networks, we performed a thorough query of interacting proteins for p38 MAPKs, HuR and other AUBPs upon growth factor stimulation. This revealed novel HuR interactors including inhibitors of protein phosphatase 2 (PP2A) activity. Over-expression of two of these interactors, pp32 and APRIL (acidic leucine-rich nuclear phosphoprotein 32 family member B, ANP32B) and pharmacological inhibition of PP2A stabilized a fos reporter mRNA. Our results indicate that p38 MAPK regulates fos mRNA decay by affecting the state of phosphorylation of HuR while controlling yet to be fully elucidated PP regulatory networks.


Assuntos
Proteínas ELAV/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Mitógenos/farmacologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Regiões 3' não Traduzidas/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Proteínas ELAV/genética , Proteína Semelhante a ELAV 1 , Células HEK293 , Células HeLa , Humanos , Camundongos , Mutação , Células NIH 3T3 , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/química , Proteínas Proto-Oncogênicas c-fos/genética , RNA Mensageiro/química , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transativadores/química , Transativadores/genética , Transativadores/metabolismo
9.
Front Microbiol ; 5: 432, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25191310

RESUMO

Desulfotomaculum reducens strain MI-1 is a Gram-positive, sulfate-reducing bacterium also capable of reducing Fe(III). Metal reduction in Gram-positive bacteria is poorly understood. Here, we investigated Fe(III) reduction with lactate, a non-fermentable substrate, as the electron donor. Lactate consumption is concomitant to Fe(III) reduction, but does not support significant growth, suggesting that little energy can be conserved from this process and that it may occur fortuitously. D. reducens can reduce both soluble [Fe(III)-citrate] and insoluble (hydrous ferric oxide, HFO) Fe(III). Because physically inaccessible HFO was not reduced, we concluded that reduction requires direct contact under these experimental conditions. This implies the presence of a surface exposed reductase capable of transferring electrons from the cell to the extracellular electron acceptor. With the goal of characterizing the role of surface proteins in D. reducens and of identifying candidate Fe(III) reductases, we carried out an investigation of the surface proteome (surfaceome) of D. reducens. Cell surface exposed proteins were extracted by trypsin cell shaving or by lysozyme treatment, and analyzed by liquid chromatography-tandem mass spectrometry. This investigation revealed that the surfaceome fulfills many functions, including solute transport, protein export, maturation and hydrolysis, peptidoglycan synthesis and modification, and chemotaxis. Furthermore, a few redox-active proteins were identified. Among these, three are putatively involved in Fe(III) reduction, i.e., a membrane-bound hydrogenase 4Fe-4S cluster subunit (Dred_0462), a heterodisulfide reductase subunit A (Dred_0143) and a protein annotated as alkyl hydroperoxide reductase but likely functioning as a thiol-disulfide oxidoreductase (Dred_1533).

10.
Mol Cell Proteomics ; 13(11): 3014-28, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24997995

RESUMO

Mycobacterium tuberculosis has a remarkable ability to persist within the human host as a clinically inapparent or chronically active infection. Fatty acids are thought to be an important carbon source used by the bacteria during long term infection. Catabolism of fatty acids requires reprogramming of metabolic networks, and enzymes central to this reprogramming have been targeted for drug discovery. Mycobacterium smegmatis, a nonpathogenic relative of M. tuberculosis, is often used as a model system because of the similarity of basic cellular processes in these two species. Here, we take a quantitative proteomics-based approach to achieve a global view of how the M. smegmatis metabolic network adjusts to utilization of fatty acids as a carbon source. Two-dimensional liquid chromatography and mass spectrometry of isotopically labeled proteins identified a total of 3,067 proteins with high confidence. This number corresponds to 44% of the predicted M. smegmatis proteome and includes most of the predicted metabolic enzymes. Compared with glucose-grown cells, 162 proteins showed differential abundance in acetate- or propionate-grown cells. Among these, acetate-grown cells showed a higher abundance of proteins that could constitute a functional glycerate pathway. Gene inactivation experiments confirmed that both the glyoxylate shunt and the glycerate pathway are operational in M. smegmatis. In addition to proteins with annotated functions, we demonstrate carbon source-dependent differential abundance of proteins that have not been functionally characterized. These proteins might play as-yet-unidentified roles in mycobacterial carbon metabolism. This study reveals several novel features of carbon assimilation in M. smegmatis, which suggests significant functional plasticity of metabolic networks in this organism.


Assuntos
Acetatos/metabolismo , Metabolismo Energético/fisiologia , Glucose/metabolismo , Mycobacterium smegmatis/enzimologia , Propionatos/metabolismo , Proteínas de Bactérias/metabolismo , Carbono/metabolismo , Ácidos Graxos/metabolismo , Perfilação da Expressão Gênica , Redes e Vias Metabólicas/fisiologia , Infecções por Mycobacterium não Tuberculosas/metabolismo , Mycobacterium smegmatis/metabolismo , Proteoma/análise , Proteômica
11.
Nat Commun ; 4: 2848, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24270157

RESUMO

Telomere composition changes during tumourigenesis, aging and in telomere syndromes in a poorly defined manner. Here we develop a quantitative telomeric chromatin isolation protocol (QTIP) for human cells, in which chromatin is cross-linked, immunopurified and analysed by mass spectrometry. QTIP involves stable isotope labelling by amino acids in cell culture (SILAC) to compare and identify quantitative differences in telomere protein composition of cells from various states. With QTIP, we specifically enrich telomeric DNA and all shelterin components. We validate the method characterizing changes at dysfunctional telomeres, and identify and validate known, as well as novel telomere-associated polypeptides including all THO subunits, SMCHD1 and LRIF1. We apply QTIP to long and short telomeres and detect increased density of SMCHD1 and LRIF1 and increased association of the shelterins TRF1, TIN2, TPP1 and POT1 with long telomeres. Our results validate QTIP to study telomeric states during normal development and in disease.


Assuntos
Bioquímica/métodos , Heterocromatina/isolamento & purificação , Proteínas/isolamento & purificação , Telômero/química , DNA/genética , DNA/metabolismo , Células HeLa , Heterocromatina/química , Heterocromatina/metabolismo , Humanos , Espectrometria de Massas , Ligação Proteica , Proteínas/genética , Proteínas/metabolismo , Complexo Shelterina , Telômero/metabolismo , Encurtamento do Telômero , Proteínas de Ligação a Telômeros
12.
J Biol Chem ; 286(14): 12172-88, 2011 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-21300794

RESUMO

Tissue transglutaminase (TGase) has been implicated in a number of cellular processes and disease states, where the enzymatic actions of TGase may serve in both, cell survival and apoptosis. To date, the precise functional properties of TGase in cell survival or cell death mechanisms still remain elusive. TGase-mediated cross-linking has been reported to account for the formation of insoluble lesions in conformational diseases. We report here that TGase induces intramolecular cross-linking of ß-amyloid peptide (Aß), resulting in structural changes of monomeric Aß. Using high resolution mass spectrometry (MS) of cross-linked Aß peptides, we observed a shift in mass, which is, presumably associated with the loss of NH3 due to enzymatic transamidation activity and hence intramolecular peptide cross-linking. We have observed that a large population of Aß monomers contained an 0.984 Da increase in mass at a glutamine residue, indicating that glutamine 15 serves as an indispensable substrate in TGase-mediated deamidation to glutamate 15. We provide strong analytical evidence on TGase-mediated Aß peptide dimerization, through covalent intermolecular cross-linking and hence the formation of Aß1-40 dimers. Our in depth analyses indicate that TGase-induced post-translational modifications of Aß peptide may serve as an important seed for aggregation.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Transglutaminases/metabolismo , Peptídeos beta-Amiloides/química , Animais , Células CHO , Cromatografia Líquida , Dicroísmo Circular , Cricetinae , Cricetulus , Eletroforese em Gel de Poliacrilamida , Focalização Isoelétrica , Microscopia Eletrônica de Transmissão , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem
13.
J Neurosci ; 30(9): 3184-98, 2010 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-20203178

RESUMO

Increasing evidence suggests that phosphorylation may play an important role in the oligomerization, fibrillogenesis, Lewy body (LB) formation, and neurotoxicity of alpha-synuclein (alpha-syn) in Parkinson disease. Herein we demonstrate that alpha-syn is phosphorylated at S87 in vivo and within LBs. The levels of S87-P are increased in brains of transgenic (TG) models of synucleinopathies and human brains from Alzheimer disease (AD), LB disease (LBD), and multiple system atrophy (MSA) patients. Using antibodies against phosphorylated alpha-syn (S129-P and S87-P), a significant amount of immunoreactivity was detected in the membrane in the LBD, MSA, and AD cases but not in normal controls. In brain homogenates from diseased human brains and TG animals, the majority of S87-P alpha-syn was detected in the membrane fractions. A battery of biophysical methods were used to dissect the effect of S87 phosphorylation on the structure, aggregation, and membrane-binding properties of monomeric alpha-syn. These studies demonstrated that phosphorylation at S87 expands the structure of alpha-syn, increases its conformational flexibility, and blocks its fibrillization in vitro. Furthermore, phosphorylation at S87, but not S129, results in significant reduction of alpha-syn binding to membranes. Together, our findings provide novel mechanistic insight into the role of phosphorylation at S87 and S129 in the pathogenesis of synucleinopathies and potential roles of phosphorylation in alpha-syn normal biology.


Assuntos
Encéfalo/metabolismo , Membrana Celular/metabolismo , Corpos de Lewy/metabolismo , Doenças Neurodegenerativas/metabolismo , Neurônios/metabolismo , alfa-Sinucleína/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/fisiopatologia , Sequência de Aminoácidos/fisiologia , Animais , Encéfalo/patologia , Creatina Quinase/genética , Creatina Quinase/metabolismo , Modelos Animais de Doenças , Humanos , Corpos de Lewy/genética , Corpos de Lewy/patologia , Doença por Corpos de Lewy/genética , Doença por Corpos de Lewy/metabolismo , Doença por Corpos de Lewy/fisiopatologia , Masculino , Camundongos , Camundongos Transgênicos , Atrofia de Múltiplos Sistemas/genética , Atrofia de Múltiplos Sistemas/metabolismo , Atrofia de Múltiplos Sistemas/fisiopatologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/fisiopatologia , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/fisiopatologia , Fosforilação , Polímeros/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratos , Ratos Wistar , Serina/metabolismo , alfa-Sinucleína/química
14.
J Biol Chem ; 284(19): 13128-42, 2009 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-19164286

RESUMO

Tissue transglutaminase (tTG) has been implicated in the pathogenesis of Parkinson disease (PD). However, exactly how tTG modulates the structural and functional properties of alpha-synuclein (alpha-syn) and contributes to the pathogenesis of PD remains unknown. Using site-directed mutagenesis combined with detailed biophysical and mass spectrometry analyses, we sought to identify the exact residues involved in tTG-catalyzed cross-linking of wild-type alpha-syn and alpha-syn mutants associated with PD. To better understand the structural consequences of each cross-linking reaction, we determined the effect of tTG-catalyzed cross-linking on the oligomerization, fibrillization, and membrane binding of alpha-syn in vitro. Our findings show that tTG-catalyzed cross-linking of monomeric alpha-syn involves multiple cross-links (specifically 2-3). We subjected tTG-catalyzed cross-linked monomeric alpha-syn composed of either wild-type or Gln --> Asn mutants to sequential proteolysis by multiple enzymes and peptide mapping by mass spectrometry. Using this approach, we identified the glutamine and lysine residues involved in tTG-catalyzed intramolecular cross-linking of alpha-syn. These studies demonstrate for the first time that Gln(79) and Gln(109) serve as the primary tTG reactive sites. Mutating both residues to asparagine abolishes tTG-catalyzed cross-linking of alpha-syn and tTG-induced inhibition of alpha-syn fibrillization in vitro. To further elucidate the sequence and structural basis underlying these effects, we identified the lysine residues that form isopeptide bonds with Gln(79) and Gln(109). This study provides mechanistic insight into the sequence and structural basis of the inhibitory effects of tTG on alpha-syn fibrillogenesis in vivo, and it sheds light on the potential role of tTG cross-linking on modulating the physiological and pathogenic properties of alpha-syn.


Assuntos
Reagentes de Ligações Cruzadas/farmacologia , Doença de Parkinson/etiologia , Transglutaminases/metabolismo , alfa-Sinucleína/metabolismo , Benzotiazóis , Dicroísmo Circular , Dimerização , Glutamina/metabolismo , Humanos , Immunoblotting , Mutagênese Sítio-Dirigida , Mutação , Conformação Proteica , Espectrometria de Massas por Ionização por Electrospray , Tiazóis/metabolismo
15.
J Am Soc Mass Spectrom ; 20(4): 567-75, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19112028

RESUMO

We decoupled electron-transfer dissociation (ETD) and collision-induced dissociation of charge-reduced species (CRCID) events to probe the lifetimes of intermediate radical species in ETD-based ion trap tandem mass spectrometry of peptides. Short-lived intermediates formed upon electron transfer require less energy for product ion formation and appear in regular ETD mass spectra, whereas long-lived intermediates require additional vibrational energy and yield product ions as a function of CRCID amplitude. The observed dependencies complement the results obtained by double-resonance electron-capture dissociation (ECD) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and ECD in a cryogenic ICR trap. Compared with ECD FT-ICR MS, ion trap MS offers lower precursor ion internal energy conditions, leading to more abundant charge-reduced radical intermediates and larger variation of product ion abundance as a function of vibrational post-activation amplitude. In many cases decoupled CRCID after ETD exhibits abundant radical c-type and even-electron z-type ions, in striking contrast to predominantly even-electron c-type and radical z-type ions in ECD FT-ICR MS and especially activated ion-ECD, thus providing a new insight into the fundamentals of ECD/ETD.


Assuntos
Elétrons , Peptídeos/química , Espectrometria de Massas em Tandem , Análise de Fourier
16.
Anal Chem ; 77(2): 596-606, 2005 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-15649059

RESUMO

Relative quantitation is key to enable differential proteomics and hence answer biological questions by comparing samples. Classical approaches involve stable isotope labeling with/without spiked standards. Although stable isotopes may lead to precise results, their application is not straightforward. In Proteomics, 2004, 4, 2333-2351, we proposed an approach where we summed peptide identification scores to derive a semiquantitative abundance indicator. In this study, we combine such an indicator with a statistical test to detect differentially expressed proteins. We demonstrate the effectiveness of this method by using mixtures of purified proteins and human plasma spiked with proteins at low-nanomolar concentrations. The impact of the number of repeated experiments is discussed, and we show that the statistical test we use performs well with two to three repetitions, whereas a classical t-test would require at least four repetitions to achieve the same performance. Typically, 2.5-5-fold changes are detected with 90-95% confidence in human plasma. The method is finally characterized by deriving estimates of its false positive and negative rates. This new characterization is valid for a wider class of methods such as spectrum sampling (Liu, H.; Sadygov, R. G.; Yates, J. R. III. Anal. Chem. 2004, 76, 4193-4201).


Assuntos
Modelos Estatísticos , Peptídeos/sangue , Plasma/química , Proteômica/métodos , Animais , Bases de Dados como Assunto , Humanos , Leptina/sangue , Mioglobina/análise , Reprodutibilidade dos Testes
17.
Proteomics ; 4(8): 2333-51, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15274127

RESUMO

We present an integrated proteomics platform designed for performing differential analyses. Since reproducible results are essential for comparative studies, we explain how we improved reproducibility at every step of our laboratory processes, e.g. by taking advantage of the powerful laboratory information management system we developed. The differential capacity of our platform is validated by detecting known markers in a real sample and by a spiking experiment. We introduce an innovative two-dimensional (2-D) plot for displaying identification results combined with chromatographic data. This 2-D plot is very convenient for detecting differential proteins. We also adapt standard multivariate statistical techniques to show that peptide identification scores can be used for reliable and sensitive differential studies. The interest of the protein separation approach we generally apply is justified by numerous statistics, complemented by a comparison with a simple shotgun analysis performed on a small volume sample. By introducing an automatic integration step after mass spectrometry data identification, we are able to search numerous databases systematically, including the human genome and expressed sequence tags. Finally, we explain how rigorous data processing can be combined with the work of human experts to set high quality standards, and hence obtain reliable (false positive < 0.35%) and nonredundant protein identifications.


Assuntos
Líquidos Corporais/química , Perfilação da Expressão Gênica , Gestão da Informação/métodos , Proteínas/análise , Proteínas/química , Proteômica/métodos , Cromatografia/instrumentação , Cromatografia/métodos , Biologia Computacional , Bases de Dados Factuais , Humanos , Gestão da Informação/instrumentação , Espectrometria de Massas/instrumentação , Espectrometria de Massas/métodos , Peptídeos/análise , Proteínas/genética , Proteínas/metabolismo , Reprodutibilidade dos Testes , Interface Usuário-Computador
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