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During megakaryopoiesis, megakaryocytes (MK) undergo cellular morphological changes with strong modification of membrane composition and lipid signaling. Here we adopt a lipid-centric multiomics approach to create a quantitative map of the MK lipidome during maturation and proplatelet formation. Data reveal that MK differentiation is driven by an increased fatty acyl import and de novo lipid synthesis, resulting in an anionic membrane phenotype. Pharmacological perturbation of fatty acid import and phospholipid synthesis blocked membrane remodeling and directly reduced MK polyploidization and proplatelet formation resulting in thrombocytopenia. The anionic lipid shift during megakaryopoiesis was paralleled by lipid-dependent relocalization of the scaffold protein CKIP-1 and recruitment of the kinase CK2α to the plasma membrane, which seems to be essential for sufficient platelet biogenesis. Overall, this study provides a framework to understand how the MK lipidome is altered during maturation and the impact of MK membrane lipid remodeling on MK kinase signaling involved in thrombopoiesis.
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Pancreatic cancer has the worst prognosis among all cancers. Cancer screening of body fluids may improve the survival time prognosis of patients, who are often diagnosed too late at an incurable stage. Several studies report the dysregulation of lipid metabolism in tumor cells, suggesting that changes in the blood lipidome may accompany tumor growth. Here we show that the comprehensive mass spectrometric determination of a wide range of serum lipids reveals statistically significant differences between pancreatic cancer patients and healthy controls, as visualized by multivariate data analysis. Three phases of biomarker discovery research (discovery, qualification, and verification) are applied for 830 samples in total, which shows the dysregulation of some very long chain sphingomyelins, ceramides, and (lyso)phosphatidylcholines. The sensitivity and specificity to diagnose pancreatic cancer are over 90%, which outperforms CA 19-9, especially at an early stage, and is comparable to established diagnostic imaging methods. Furthermore, selected lipid species indicate a potential as prognostic biomarkers.
Assuntos
Biomarcadores Tumorais/sangue , Ceramidas/sangue , Metabolismo dos Lipídeos/genética , Lisofosfatidilcolinas/sangue , Neoplasias Pancreáticas/diagnóstico , Esfingomielinas/sangue , Biomarcadores Tumorais/genética , Antígeno CA-19-9/sangue , Estudos de Casos e Controles , Feminino , Humanos , Lipidômica/métodos , Masculino , Análise Multivariada , Neoplasias Pancreáticas/sangue , Neoplasias Pancreáticas/mortalidade , Neoplasias Pancreáticas/patologia , Modelos de Riscos Proporcionais , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Neoplasias PancreáticasRESUMO
AIMS: Cardiac immune-related adverse events (irAEs) from immune checkpoint inhibition (ICI) targeting programmed death 1 (PD1) are of growing concern. Once cardiac irAEs become clinically manifest, fatality rates are high. Cardio-oncology aims to prevent detrimental effects before manifestation of severe complications by targeting early pathological changes. We therefore aimed to investigate early consequences of PD1 inhibition for cardiac integrity to prevent the development of overt cardiac disease. METHODS AND RESULTS: We investigated cardiac-specific consequences from anti-PD1 therapy in a combined biochemical and in vivo phenotyping approach. Mouse hearts showed broad expression of the ligand PDL1 on cardiac endothelial cells as a main mediator of immune-crosstalk. Using a novel melanoma mouse model, we assessed that anti-PD1 therapy promoted myocardial infiltration with CD4+ and CD8+ T cells, the latter being markedly activated. Left ventricular (LV) function was impaired during pharmacological stress, as shown by pressure-volume catheterization. This was associated with a dysregulated myocardial metabolism, including the proteome and the lipidome. Analogous to the experimental approach, in patients with metastatic melanoma (n = 7) receiving anti-PD1 therapy, LV function in response to stress was impaired under therapy. Finally, we identified that blockade of tumour necrosis factor alpha (TNFα) preserved LV function without attenuating the anti-cancer efficacy of anti-PD1 therapy. CONCLUSIONS: Anti-PD1 therapy induces a disruption of cardiac immune homeostasis leading to early impairment of myocardial functional integrity, with potential prognostic effects on the growing number of treated patients. Blockade of TNFα may serve as an approach to prevent the manifestation of ICI-related cardiotoxicity.
Assuntos
Inibidores de Checkpoint Imunológico , Melanoma , Animais , Cardiotoxicidade/etiologia , Células Endoteliais , Humanos , Inibidores de Checkpoint Imunológico/efeitos adversos , Melanoma/tratamento farmacológico , Camundongos , Receptor de Morte Celular Programada 1/uso terapêuticoRESUMO
We introduce STAMPS, a pathway-centric web service for the development of targeted proteomics assays. STAMPS guides the user by providing several intuitive interfaces for a rapid and simplified method design. Applying our curated framework to signaling and metabolic pathways, we reduced the average assay development time by a factor of â¼150 and revealed that the insulin signaling is actively controlled by protein abundance changes in insulin-sensitive and -resistance states. Although at the current state STAMPS primarily contains mouse data, it was designed for easy extension with additional organisms.
Assuntos
Redes e Vias Metabólicas , Proteômica/métodos , Transdução de Sinais , Animais , Cromatografia Líquida de Alta Pressão , Bases de Dados de Proteínas , Insulina/metabolismo , Redes e Vias Metabólicas/genética , Camundongos , Peptídeos/análise , Transdução de Sinais/genética , Espectrometria de Massas em TandemRESUMO
Stress response pathways are critical for cellular homeostasis, promoting survival through adaptive changes in gene expression and metabolism. They play key roles in numerous diseases and are implicated in cancer progression and chemoresistance. However, the underlying mechanisms are only poorly understood. We have employed a multi-omics approach to monitor changes to gene expression after induction of a stress response pathway, the unfolded protein response (UPR), probing in parallel the transcriptome, the proteome, and changes to translation. Stringent filtering reveals the induction of 267 genes, many of which have not previously been implicated in stress response pathways. We experimentally demonstrate that UPR-mediated translational control induces the expression of enzymes involved in a pathway that diverts intermediate metabolites from glycolysis to fuel mitochondrial one-carbon metabolism. Concomitantly, the cells become resistant to the folate-based antimetabolites Methotrexate and Pemetrexed, establishing a direct link between UPR-driven changes to gene expression and resistance to pharmacological treatment.
Assuntos
Antimetabólitos/farmacologia , Ácido Fólico/farmacologia , Regulon/genética , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Resposta a Proteínas não Dobradas/genética , Animais , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Humanos , Metotrexato/farmacologia , Pemetrexede/farmacologia , Proteoma/efeitos dos fármacos , Proteoma/genética , Regulon/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Transcriptoma/genéticaRESUMO
PKR-like kinase (PERK) plays a significant role in inducing angiogenesis in various cancer types including glioblastoma. By proteomics analysis of the conditioned medium from a glioblastoma cell line treated with a PERK inhibitor, we showed that peptidylglycine α-amidating monooxygenase (PAM) expression is regulated by PERK under hypoxic conditions. Moreover, PERK activation via CCT020312 (a PERK selective activator) increased the cleavage and thus the generation of PAM cleaved cytosolic domain (PAM sfCD) that acts as a signaling molecule from the cytoplasm to the nuclei. PERK was also found to interact with PAM, suggesting a possible involvement in the generation of PAM sfCD. Knockdown of PERK or PAM reduced the formation of tubes by HUVECs in vitro. Furthermore, in vivo data highlighted the importance of PAM in the growth of glioblastoma with reduction of PAM expression in engrafted tumor significantly increasing the survival in mice. In summary, our data revealed PAM as a potential target for antiangiogenic therapy in glioblastoma.
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Many cellular events are driven by changes in protein expression, measurable by mass spectrometry or antibody-based assays. However, using conventional technology, the analysis of transcription factor or membrane receptor expression is often limited by an insufficient sensitivity and specificity. To overcome this limitation, we have developed a high-resolution targeted proteomics strategy, which allows quantification down to the lower attomol range in a straightforward way without any prior enrichment or fractionation approaches. The method applies isotope-labeled peptide standards for quantification of the protein of interest. As proof of principle, we applied the improved workflow to proteins of the unfolded protein response (UPR), a signaling pathway of great clinical importance, and could for the first time detect and quantify all major UPR receptors, transducers and effectors that are not readily detectable via antibody-based-, SRM- or conventional PRM assays. As transcription and translation is central to the regulation of UPR, quantification and determination of protein copy numbers in the cell is important for our understanding of the signaling process as well as how pharmacologic modulation of these pathways impacts on the signaling. These questions can be answered using our newly established workflow as exemplified in an experiment using UPR perturbation in a glioblastoma cell lines.
Assuntos
Glioblastoma/metabolismo , Proteínas de Membrana/metabolismo , Proteômica/métodos , Fatores de Transcrição/metabolismo , Resposta a Proteínas não Dobradas , Linhagem Celular Tumoral , Dosagem de Genes , Glioblastoma/química , Glioblastoma/patologia , Humanos , Marcação por Isótopo , Proteínas de Membrana/análise , Proteínas de Membrana/normas , Peptídeos/normas , Proteômica/normas , Fatores de Transcrição/análise , Fatores de Transcrição/normasRESUMO
Glioblastomas strongly invade the brain by infiltrating into the white matter along myelinated nerve fiber tracts even though the myelin protein Nogo-A prevents cell migration by activating inhibitory RhoA signaling. The mechanisms behind this long-known phenomenon remained elusive so far, precluding a targeted therapeutic intervention. This study demonstrates that the prevalent activation of AKT in gliomas increases the ER protein-folding capacity and enables tumor cells to utilize a side effect of RhoA activation: the perturbation of the IRE1α-mediated decay of SPARC mRNA. Once translation is initiated, glioblastoma cells rapidly secrete SPARC to block Nogo-A from inhibiting migration via RhoA. By advanced ultramicroscopy for studying single-cell invasion in whole, undissected mouse brains, we show that gliomas require SPARC for invading into white matter structures. SPARC depletion reduces tumor dissemination that significantly prolongs survival and improves response to cytostatic therapy. Our finding of a novel RhoA-IRE1 axis provides a druggable target for interfering with SPARC production and underscores its therapeutic value.
Assuntos
Neoplasias Encefálicas/genética , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Proteínas de Neoplasias/fisiologia , Proteínas Nogo/biossíntese , Osteonectina/biossíntese , Biossíntese de Proteínas , Substância Branca/patologia , Proteína rhoA de Ligação ao GTP/fisiologia , Animais , Ligação Competitiva , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Invasividade Neoplásica , Proteínas Nogo/genética , Osteonectina/genética , Domínios Proteicos , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Receptores de Esfingosina-1-Fosfato/fisiologia , Células Tumorais Cultivadas , Substância Branca/metabolismoRESUMO
Serum concentrations of lathosterol, the plant sterols campesterol and sitosterol and the cholesterol metabolite 5α-cholestanol are widely used as surrogate markers of cholesterol synthesis and absorption, respectively. Increasing numbers of laboratories utilize a broad spectrum of well-established and recently developed methods for the determination of cholesterol and non-cholesterol sterols (NCS). In order to evaluate the quality of these measurements and to identify possible sources of analytical errors our group initiated the first international survey for cholesterol and NCS. The cholesterol and NCS survey was structured as a two-part survey which took place in the years 2013 and 2014. The first survey part was designed as descriptive, providing information about the variation of reported results from different laboratories. A set of two lyophilized pooled sera (A and B) was sent to twenty laboratories specialized in chromatographic lipid analysis. The different sterols were quantified either by gas chromatography-flame ionization detection, gas chromatography- or liquid chromatography-mass selective detection. The participants were requested to determine cholesterol and NCS concentrations in the provided samples as part of their normal laboratory routine. The second part was designed as interventional survey. Twenty-two laboratories agreed to participate and received again two different lyophilized pooled sera (C and D). In contrast to the first international survey, each participant received standard stock solutions with defined concentrations of cholesterol and NCS. The participants were requested to use diluted calibration solutions from the provided standard stock solutions for quantification of cholesterol and NCS. In both surveys, each laboratory used its own internal standard (5α-cholestane, epicoprostanol or deuterium labelled sterols). Main outcome of the survey was, that unacceptably high interlaboratory variations for cholesterol and NCS concentrations are reported, even when the individual laboratories used the same calibration material. We discuss different sources of errors and recommend all laboratories analysing cholesterol and NCS to participate in regular quality control programs.
Assuntos
Colesterol/sangue , Fitosteróis/sangue , Colestanol/sangue , Colesterol/análogos & derivados , Cromatografia Gasosa/métodos , Cromatografia Líquida/métodos , Humanos , Sitosteroides/sangue , Inquéritos e QuestionáriosRESUMO
The UDP-glucose ceramide glucosyltransferase (UGCG) is a key enzyme in the synthesis of glycosylated sphingolipids, since this enzyme generates the precursor for all complex glycosphingolipids (GSL), the GlcCer. The UGCG has been associated with several cancer-related processes such as maintaining cancer stem cell properties or multidrug resistance induction. The precise mechanisms underlying these processes are unknown. Here, we investigated the molecular mechanisms occurring after UGCG overexpression in breast cancer cells. We observed alterations of several cellular properties such as morphological changes, which enhanced proliferation and doxorubicin resistance in UGCG overexpressing MCF-7 cells. These cellular effects seem to be mediated by an altered composition of glycosphingolipid-enriched microdomains (GEMs), especially an accumulation of globotriaosylceramide (Gb3) and glucosylceramide (GlcCer), which leads to an activation of Akt and ERK1/2. The induction of the Akt and ERK1/2 signaling pathway results in an increased gene expression of multidrug resistance protein 1 (MDR1) and anti-apoptotic genes and a decrease of pro-apoptotic gene expression. Inhibition of the protein kinase C (PKC) and phosphoinositide 3 kinase (PI3K) reduced MDR1 gene expression. This study discloses how changes in UGCG expression impact several cellular signaling pathways in breast cancer cells resulting in enhanced proliferation and multidrug resistance.
Assuntos
Proliferação de Células , Resistencia a Medicamentos Antineoplásicos , Glucosiltransferases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Apoptose/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Colesterol/análise , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Glucosiltransferases/genética , Humanos , Células MCF-7 , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Transdução de Sinais/genética , Esfingolipídeos/análise , Esfingolipídeos/metabolismo , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/metabolismo , Esfingosina N-Aciltransferase/genética , Esfingosina N-Aciltransferase/metabolismoRESUMO
Computational proteomics is a constantly growing field to support end users with powerful and reliable tools for performing several computational steps within an analytics workflow for proteomics experiments. Typically, after capturing with a mass spectrometer, the proteins have to be identified and quantified. After certain follow-up analyses, an optional targeted approach is suitable for validating the results. The de.NBI (German network for bioinformatics infrastructure) service center in Dortmund provides several software applications and platforms as services to meet these demands. In this work, we present our tools and services, which is the combination of SearchGUI and PeptideShaker. SearchGUI is a managing tool for several search engines to find peptide spectra matches for one or more complex MS2 measurements. PeptideShaker combines all matches and creates a consensus list of identified proteins providing statistical confidence measures. In a next step, we are planning to release a web service for protein identification containing both tools. This system will be designed for high scalability and distributed computing using solutions like the Docker container system among others. As an additional service, we offer a web service oriented database providing all necessary high-quality and high-resolution data for starting targeted proteomics analyses. The user can easily select proteins of interest, review the according spectra and download both protein sequences and spectral library. All systems are designed to be intuitively and user-friendly operable.
Assuntos
Proteômica , Controle de Qualidade , Software , Bases de Dados de Proteínas , Peptídeos/análise , Peptídeos/química , Proteínas/análise , Proteínas/química , Proteômica/métodos , Proteômica/normas , Reprodutibilidade dos TestesRESUMO
In response to the urgent need for analysis software that is capable of handling data from targeted high-throughput lipidomics experiments, we here present a systematic workflow for the straightforward method design and analysis of selected reaction monitoring data in lipidomics based on lipid building blocks. Skyline is a powerful software primarily designed for proteomics applications where it is widely used. We adapted this tool to a "Plug and Play" system for lipid research. This extension offers the unique capability to assemble targeted mass spectrometry methods for complex lipids easily by making use of building blocks. With simple yet tailored modifications, targeted methods to analyze main lipid classes such as glycerophospholipids, sphingolipids, glycerolipids, cholesteryl-esters, and cholesterol can be quickly introduced into Skyline for easy application by end users without distinct bioinformatics skills. To illustrate the benefits of our novel strategy, we used Skyline to quantify sphingolipids in mesenchymal stem cells. We demonstrate a simple method building procedure for sphingolipids screening, collision energy optimization, and absolute quantification of sphingolipids. In total, 72 sphingolipids were identified and absolutely quantified at the fatty acid scan species level by utilizing Skyline for data interpretation and visualization.
Assuntos
Metabolismo dos Lipídeos , Células-Tronco Mesenquimais/metabolismo , Metabolômica/métodos , Software , Animais , Linhagem Celular , Cromatografia de Fase Reversa , Ácidos Graxos/química , Ácidos Graxos/isolamento & purificação , Ácidos Graxos/metabolismo , Limite de Detecção , Camundongos , Esfingolipídeos/química , Esfingolipídeos/isolamento & purificação , Esfingolipídeos/metabolismo , Espectrometria de Massas em TandemRESUMO
As cells develop and differentiate, they change in function and morphology, which often precede earlier changes in signaling and metabolic control. Here we present a selected reaction monitoring (SRM) approach which allows for the parallel quantification of metabolic regulators and their downstream targets.In particular we explain and describe how to monitor abundance changes of glycolytic enzymes upon PPARγ activation by using a label-free or a stable isotope-labeled standard peptide (SIS peptides) approach applying triple-quadrupole mass spectrometry. We further outline how to fractionate the cell lysate into cytosolic and nuclear fractions to enhance the sensitivity of the measurements and to investigate the dynamic concentration changes in those compartments.
Assuntos
Glicólise , PPAR gama/metabolismo , Proteoma , Proteômica/métodos , Animais , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Humanos , Espectrometria de Massas , PeptídeosRESUMO
Gli proteins are transcriptional effectors of the Hedgehog (Hh) pathway in both normal development and cancer. We describe a program of multisite phosphorylation that regulates the conversion of Gli proteins into transcriptional activators. In the absence of Hh ligands, Gli activity is restrained by the direct phosphorylation of six conserved serine residues by protein kinase A (PKA), a master negative regulator of the Hh pathway. Activation of signaling leads to a global remodeling of the Gli phosphorylation landscape: the PKA target sites become dephosphorylated, while a second cluster of sites undergoes phosphorylation. The pattern of Gli phosphorylation can regulate Gli transcriptional activity in a graded fashion, suggesting a phosphorylation-based mechanism for how a gradient of Hh signaling in a morphogenetic field can be converted into a gradient of transcriptional activity.
Assuntos
Proteínas Hedgehog/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais , Animais , Embrião de Galinha , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células HEK293 , Humanos , Fatores de Transcrição Kruppel-Like/química , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Células NIH 3T3 , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , Serina/metabolismo , Proteína Gli2 com Dedos de Zinco , Proteína Gli3 com Dedos de ZincoRESUMO
Adipogenesis, or the conversion of proliferating preadipocytes into nondividing adipocytes, is an important part of the vertebrate weight-maintenance program. It is not yet understood how and when an irreversible transition occurs into a distinct state capable of accumulating lipid. Here, we use single-cell fluorescence imaging to show that an all-or-none switch is induced before lipid accumulation occurs. Conversion begins by glucocorticoid and cAMP signals raising C/EBPß levels above a critical threshold, triggering three consecutive positive feedback loops: from PPARγ to C/EBPα, then to C/EBPß, and last to the insulin receptor. Experiments and modeling show that these feedbacks create a robust, irreversible transition to a terminally differentiated state by rejecting short- and low-amplitude stimuli. After the differentiation switch is triggered, insulin controls fat accumulation in a graded fashion. Altogether, our study introduces a regulatory motif that locks cells in a differentiated state by engaging a sequence of positive feedback loops.
Assuntos
Adipócitos/citologia , Células 3T3 , Adipócitos/metabolismo , Adipogenia/efeitos dos fármacos , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/antagonistas & inibidores , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/antagonistas & inibidores , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Linhagem Celular , AMP Cíclico/farmacologia , Glucocorticoides/farmacologia , Insulina/farmacologia , Lipídeos/biossíntese , Camundongos , PPAR gama/antagonistas & inibidores , PPAR gama/genética , PPAR gama/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptor de Insulina/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Enzymes secreted by Legionella pneumophila, such as phospholipases A (PLAs) and glycerophospholipid:cholesterol acyltransferases (GCATs), may target host cell lipids and therefore contribute to the establishment of Legionnaires disease. L. pneumophila possesses three proteins, PlaA, PlaC, and PlaD, belonging to the GDSL family of lipases/acyltransferases. We have shown previously that PlaC is the major GCAT secreted by L. pneumophila and that the zinc metalloproteinase ProA is essential for GCAT activity. Here we characterized the mode of PlaC GCAT activation and determined that ProA directly processes PlaC. We further found that not only cholesterol but also ergosterol present in protozoa was palmitoylated by PlaC. Such ester formations were not induced by either PlaA or PlaD. PlaD was shown here to possess lysophospholipase A activity, and interestingly, all three GDSL enzymes transferred short chain fatty acids to sterols. The three single putative catalytic amino acids (Ser-37, Asp-398, and His-401) proved essential for all PlaC-associated PLA, lysophospholipase A, and GCAT activities. A further four cysteine residues are important for the PLA/GCAT activities as well as their oxidized state, and we therefore conclude that PlaC likely forms at least one disulfide loop. Analysis of cleavage site and loop deletion mutants suggested that for GCAT activation deletion of several amino acids within the loop is necessary rather than cleavage at a single site. Our data therefore suggest a novel enzyme inhibition/activation mechanism where a disulfide loop inhibits PlaC GCAT activity until the protein is exported to the external space where it is ProA-activated.
Assuntos
Aciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Legionella pneumophila/enzimologia , Metaloproteases/metabolismo , Aciltransferases/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sítios de Ligação/genética , Western Blotting , Domínio Catalítico/genética , Colesterol/metabolismo , Cisteína/genética , Cisteína/metabolismo , Ativação Enzimática , Técnicas de Inativação de Genes , Legionella pneumophila/genética , Legionella pneumophila/metabolismo , Metaloproteases/genética , Dados de Sequência Molecular , Mutação , Ácido Palmítico/metabolismo , Propionatos/metabolismo , Homologia de Sequência de Aminoácidos , Esteróis/metabolismo , Especificidade por Substrato , Zinco/metabolismoRESUMO
Quantitative proteomics has become an important method in modern life sciences. Besides protein identification, the aspect of quantification is of rapidly increasing relevance. MeCAT (metal-coded affinity tagging) is able to provide a tool that enables relative as well as absolute quantification. For structural elucidation, knowledge on the fragmentation behavior of MeCAT-modified peptides is highly beneficial. Therefore, the fragmentation behavior of MeCAT-labeled peptides under collision induced dissociation (CID), electron capture dissociation (ECD) and infrared multiphoton dissociation (IRMPD) conditions was studied. Application of CID and ECD allowed a straight-forward sequence elucidation of MeCAT-labeled peptides. During IRMPD all MeCAT-labeled peptides form characteristic fragments resulting from the fragmentational cleavage of the tagging group, thus, providing a screening method for the identification of labeled compounds. Furthermore, occurring side reactions during the labeling process were investigated. By-products were structurally characterized and reaction conditions were optimized in order to prevent the formation of these.
Assuntos
Metais/química , Peptídeos/química , Coloração e Rotulagem , Cromatografia Líquida de Alta Pressão , Fragmentos de Peptídeos/química , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Quantitative peptide and protein analysis is one of the most promising fields in modern life science. Besides stable isotope coded labeling, metal chelate complexes are an alternative tool for quantification. The development of metal-coded affinity tags (MeCAT) was aimed to provide a robust tool for the quantification of peptides and proteins by utilizing lanthanide-harboring metal tags. It was shown that MeCAT is suited for relative quantification of proteins via standard mass spectrometric methods. The approach of tagging biomolecules with MeCAT offers the unique advantage of absolute quantification via inductively coupled plasma mass spectrometry (ICPMS), a well-established technique for assessing concentrations down to low attomole ranges. This work investigates the compatibility of MeCAT labeling to analysis workflows such as nano liquid chromatography/electrospray ionization tandem mass spectrometry (nano-LC/ESI-MS(n)). Focus was given toward the separation behavior of labeled peptides and the dynamic range of detection and peptide charge distribution. Furthermore, the stability of MeCAT under harsh analytical conditions was investigated. With the application of the MeCAT technique to a standard analysis scheme in proteomics, such as the investigation of changes in an Escherichia coli proteome, we successfully addressed the suitability to utilize MeCAT on biological samples. Furthermore, we demonstrated that MeCAT complexes are stable under a variety of conditions and that by applying LC/ESI-MS it is possible to cover a dynamic range of 2 orders of magnitude down to the low femtomole range with an average standard deviation below 15%. Therefore, this technique is suitable to common proteomic workflows and enables relative as well as absolute differential peptide quantification.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Complexos de Coordenação/química , Elementos da Série dos Lantanídeos/química , Peptídeos/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Sequência de Aminoácidos , Animais , Escherichia coli/metabolismo , Marcação por Isótopo , Proteoma/análise , Soroalbumina Bovina/química , Soroalbumina Bovina/metabolismo , Tripsina/metabolismoRESUMO
The quantitative analysis of protein mixtures is pivotal for the understanding of variations in the proteome of living systems. Therefore, approaches have been recently devised that generally allow the relative quantitative analysis of peptides and proteins. Here we present proof of concept of the new metal-coded affinity tag (MeCAT) technique, which allowed the quantitative determination of peptides and proteins. A macrocyclic metal chelate complex (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)) loaded with different lanthanides (metal(III) ions) was the essential part of the tag. The combination of DOTA with an affinity anchor for purification and a reactive group for reaction with amino acids constituted a reagent that allowed quantification of peptides and proteins in an absolute fashion. For the quantitative determination, the tagged peptides and proteins were analyzed using flow injection inductively coupled plasma MS, a technique that allowed detection of metals with high precision and low detection limits. The metal chelate complexes were attached to the cysteine residues, and the course of the labeling reaction was followed using SDS-PAGE and MALDI-TOF MS, ESI MS, and inductively coupled plasma MS. To limit the width in isotopic signal spread and to increase the sensitivity for ESI analysis, we used the monoisotopic lanthanide macrocycle complexes. Peptides tagged with the reagent loaded with different metals coelute in liquid chromatography. In first applications with proteins, the calculated detection limit for bovine serum albumin for example was 110 amol, and we have used MeCAT to analyze proteins of the Sus scrofa eye lens as a model system. These data showed that MeCAT allowed quantification not only of peptides but also of proteins in an absolute fashion at low concentrations and in complex mixtures.
Assuntos
Compostos Heterocíclicos com 1 Anel/química , Elementos da Série dos Lantanídeos/química , Peptídeos/análise , Proteínas/análise , Proteômica/métodos , Sequência de Aminoácidos , Animais , Bovinos , Quelantes/química , Cristalinas/análise , Eletroforese em Gel de Poliacrilamida , Cristalino/química , Dados de Sequência Molecular , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Sus scrofaRESUMO
To investigate protein-protein interaction sites in the DNA mismatch repair system we developed a crosslinking/mass spectrometry technique employing a commercially available trifunctional crosslinker with a thiol-specific methanethiosulfonate group, a photoactivatable benzophenone moiety and a biotin affinity tag. The XACM approach combines photocrosslinking (X), in-solution digestion of the crosslinked mixtures, affinity purification via the biotin handle (A), chemical coding of the crosslinked products (C) followed by MALDI-TOF mass spectrometry (M). We illustrate the feasibility of the method using a single-cysteine variant of the homodimeric DNA mismatch repair protein MutL. Moreover, we successfully applied this method to identify the photocrosslink formed between the single-cysteine MutH variant A223C, labeled with the trifunctional crosslinker in the C-terminal helix and its activator protein MutL. The identified crosslinked MutL-peptide maps to a conserved surface patch of the MutL C-terminal dimerization domain. These observations are substantiated by additional mutational and chemical crosslinking studies. Our results shed light on the potential structures of the MutL holoenzyme and the MutH-MutL-DNA complex.