RESUMEN
Malaria-causing parasites of the Plasmodium genus undergo multiple developmental phases in the human and the mosquito hosts, regulated by various post-translational modifications. While ubiquitination by multi-component E3 ligases is key to regulate a wide range of cellular processes in eukaryotes, little is known about its role in Plasmodium. Here we show that Plasmodium berghei expresses a conserved SKP1/Cullin1/FBXO1 (SCFFBXO1) complex showing tightly regulated expression and localisation across multiple developmental stages. It is key to cell division for nuclear segregation during schizogony and centrosome partitioning during microgametogenesis. It is additionally required for parasite-specific processes including gamete egress from the host erythrocyte, as well as integrity of the apical and the inner membrane complexes (IMC) in merozoite and ookinete, two structures essential for the dissemination of these motile stages. Ubiquitinomic surveys reveal a large set of proteins ubiquitinated in a FBXO1-dependent manner including proteins important for egress and IMC organisation. We additionally demonstrate an interplay between FBXO1-dependent ubiquitination and phosphorylation via calcium-dependent protein kinase 1. Altogether we show that Plasmodium SCFFBXO1 plays conserved roles in cell division and is also important for parasite-specific processes in the mammalian and mosquito hosts.
Asunto(s)
Plasmodium berghei , Humanos , Eritrocitos/metabolismo , Plasmodium berghei/genética , Plasmodium berghei/metabolismo , Unión Proteica , Proteínas Quinasas Asociadas a Fase-S/metabolismo , UbiquitinaciónRESUMEN
Global analysis of protein phosphorylation by mass spectrometry proteomic techniques has emerged in the last decades as a powerful tool in biological and biomedical research. However, there are several factors that make the global study of the phosphoproteome more challenging than measuring non-modified proteins. The low stoichiometry of the phosphorylated species and the need to retrieve residue specific information require particular attention on sample preparation, data acquisition and processing to ensure reproducibility, qualitative and quantitative robustness and ample phosphoproteome coverage in phosphoproteomic workflows. Aiming to investigate the effect of different variables in the performance of proteome wide phosphoprotein analysis protocols, ProteoRed-ISCIII and EuPA launched the Proteomics Multicentric Experiment 11 (PME11). A reference sample consisting of a yeast protein extract spiked in with different amounts of a phosphomix standard (Sigma/Merck) was distributed to 31 laboratories around the globe. Thirty-six datasets from 23 laboratories were analyzed. Our results indicate the suitability of the PME11 reference sample to benchmark and optimize phosphoproteomics strategies, weighing the influence of different factors, as well as to rank intra and inter laboratory performance.
Asunto(s)
Proteoma , Proteómica , Laboratorios , Fosfoproteínas/análisis , Fosforilación , Proteoma/análisis , Proteómica/métodos , Estándares de Referencia , Reproducibilidad de los ResultadosRESUMEN
Calcium signaling regulated by the cGMP-dependent protein kinase (PKG) controls key life cycle transitions in the malaria parasite. However, how calcium is mobilized from intracellular stores in the absence of canonical calcium channels in Plasmodium is unknown. Here, we identify a multipass membrane protein, ICM1, with homology to transporters and calcium channels that is tightly associated with PKG in both asexual blood stages and transmission stages. Phosphoproteomic analyses reveal multiple ICM1 phosphorylation events dependent on PKG activity. Stage-specific depletion of Plasmodium berghei ICM1 prevents gametogenesis due to a block in intracellular calcium mobilization, while conditional loss of Plasmodium falciparum ICM1 is detrimental for the parasite resulting in severely reduced calcium mobilization, defective egress, and lack of invasion. Our findings suggest that ICM1 is a key missing link in transducing PKG-dependent signals and provide previously unknown insights into atypical calcium homeostasis in malaria parasites essential for pathology and disease transmission.
Asunto(s)
Malaria , Parásitos , Animales , Calcio/metabolismo , Canales de Calcio , Gametogénesis , Malaria/parasitología , Proteínas de la Membrana/metabolismo , Plasmodium berghei/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismoRESUMEN
Cell cycle transitions are generally triggered by variation in the activity of cyclin-dependent kinases (CDKs) bound to cyclins. Malaria-causing parasites have a life cycle with unique cell-division cycles, and a repertoire of divergent CDKs and cyclins of poorly understood function and interdependency. We show that Plasmodium berghei CDK-related kinase 5 (CRK5), is a critical regulator of atypical mitosis in the gametogony and is required for mosquito transmission. It phosphorylates canonical CDK motifs of components in the pre-replicative complex and is essential for DNA replication. During a replicative cycle, CRK5 stably interacts with a single Plasmodium-specific cyclin (SOC2), although we obtained no evidence of SOC2 cycling by transcription, translation or degradation. Our results provide evidence that during Plasmodium male gametogony, this divergent cyclin/CDK pair fills the functional space of other eukaryotic cell-cycle kinases controlling DNA replication.
Asunto(s)
Quinasa 5 Dependiente de la Ciclina/genética , Plasmodium berghei/genética , Proteínas Protozoarias/genética , Transducción de Señal , Quinasa 5 Dependiente de la Ciclina/metabolismo , Malaria/transmisión , Plasmodium berghei/crecimiento & desarrollo , Proteínas Protozoarias/metabolismoRESUMEN
Despite the maturity reached by targeted proteomic strategies, reliable and standardized protocols are urgently needed to enhance reproducibility among different laboratories and analytical platforms, facilitating a more widespread use in biomedical research. To achieve this goal, the use of dimensionless relative retention times (iRT), defined on the basis of peptide standard retention times (RT), has lately emerged as a powerful tool. The robustness, reproducibility and utility of this strategy were examined for the first time in a multicentric setting, involving 28 laboratories that included 24 of the Spanish network of proteomics laboratories (ProteoRed-ISCIII). According to the results obtained in this study, dimensionless retention time values (iRTs) demonstrated to be a useful tool for transferring and sharing peptide retention times across different chromatographic set-ups both intra- and inter-laboratories. iRT values also showed very low variability over long time periods. Furthermore, parallel quantitative analyses showed a high reproducibility despite the variety of experimental strategies used, either MRM (multiple reaction monitoring) or pseudoMRM, and the diversity of analytical platforms employed. BIOLOGICAL SIGNIFICANCE: From the very beginning of proteomics as an analytical science there has been a growing interest in developing standardized methods and experimental procedures in order to ensure the highest quality and reproducibility of the results. In this regard, the recent (2012) introduction of the dimensionless retention time concept has been a significant advance. In our multicentric (28 laboratories) study we explore the usefulness of this concept in the context of a targeted proteomics experiment, demonstrating that dimensionless retention time values is a useful tool for transferring and sharing peptide retention times across different chromatographic set-ups.
Asunto(s)
Investigación Biomédica/métodos , Cromatografía Liquida/métodos , Proteómica/métodos , Investigación Biomédica/normas , Cromatografía Liquida/normas , Variaciones Dependientes del Observador , Proteómica/organización & administración , Proteómica/normas , Estándares de Referencia , Reproducibilidad de los Resultados , Investigación/normasRESUMEN
Approaches aimed at deciphering the proteome have illustrated the need for relatively complex and highly sensitive methodologies. The major elements of proteome analysis, such as powerful protein separation and enzymatic processing, mass spectrometry and dedicated bioinformatics have been assembled in the development of the molecular scanner. This highly flexible and data-rich approach has combined the power of electrophoretic protein separation, the simultaneous digestion and transfer of proteins through an enzymatic membrane, the immediate use of the MALDI mass spectrometer to scan a collecting membrane, and the development of dedicated bioinformatics tools to perform protein identification and molecular imaging of the proteome. Clinical applications of the molecular scanner have also started to be developed for disease diagnosis in biological material.
Asunto(s)
Electroforesis en Gel Bidimensional/métodos , Perfilación de la Expresión Génica/métodos , Proteínas/análisis , Proteínas/química , Proteómica/métodos , Análisis de Secuencia de Proteína/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Animales , Biotecnología/instrumentación , Biotecnología/métodos , Biotecnología/tendencias , Electroforesis en Gel Bidimensional/instrumentación , Diseño de Equipo , Perfilación de la Expresión Génica/instrumentación , Perfilación de la Expresión Génica/tendencias , Humanos , Proteómica/instrumentación , Proteómica/tendencias , Análisis de Secuencia de Proteína/instrumentación , Análisis de Secuencia de Proteína/tendencias , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/instrumentación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/tendencias , Integración de Sistemas , Interfaz Usuario-ComputadorRESUMEN
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was evaluated for both qualitative and quantitative analysis of insulin entrapped within poly(D,L-lactic-co-glycolic acid) nanoparticles. Quantitation was performed by adding an internal standard (arg-insulin) to defined and unknown sample solutions, in order to reduce point-to-point and sample-to-sample variability. The ratio of the peak height of insulin to the peak height of arg-insulin was plotted against the insulin concentration. In this way, an excellent linear relationship was found (R2 > 0.99). This method of quantitation was compared with classical UV spectroscopy and reverse-phase high-performance liquid chromatography measurements. All methods provided close final drug loading values for the insulin-loaded nanoparticle batches tested. Additionally, with respect to molecular stability, covalent insulin dimers were found only at trace levels in those nanoparticles. Compared with other methods, MALDI-TOF MS is a valuable tool for the characterization of proteins from nanoparticles, because no extensive extraction and complex sampling procedures are required.
Asunto(s)
Insulina/análisis , Ácido Láctico/análisis , Nanoestructuras/análisis , Ácido Poliglicólico/análisis , Polímeros/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Animales , Portadores de Fármacos/análisis , Estabilidad de Medicamentos , Estudios de Evaluación como Asunto , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , PorcinosRESUMEN
Proteomic technologies based on mass spectrometry (MS) have greatly evolved in the past years, and nowadays it is possible to routinely identify thousands of peptides from complex biological samples in a single LC-MS/MS experiment. Despite the advancements in proteomic technologies, the scientific community still faces important challenges in terms of depth and reproducibility of proteomics analyses. Here, we present a multicenter study designed to evaluate long-term performance of LC-MS/MS platforms within the Spanish Proteomics Facilities Network (ProteoRed-ISCIII). The study was performed under well-established standard operating procedures, and demonstrated that it is possible to attain qualitative and quantitative reproducibility over time. Our study highlights the importance of deploying quality assessment metrics routinely in individual laboratories and in multi-laboratory studies. The mass spectrometry data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD000205.This article is part of a Special Issue entitled: HUPO 2014.
Asunto(s)
Espectrometría de Masas/métodos , Espectrometría de Masas/normas , Péptidos/análisis , Proteínas de Saccharomyces cerevisiae/análisis , Saccharomyces cerevisiae/química , Cromatografía Liquida/métodos , Cromatografía Liquida/normas , Control de CalidadRESUMEN
High throughput protein identification and quantification analysis based on mass spectrometry are fundamental steps in most proteomics projects. Here, we present EasyProt (available at http://easyprot.unige.ch), a new platform for mass spectrometry data processing, protein identification, quantification and unexpected post-translational modification characterization. EasyProt provides a fully integrated graphical experience to perform a large part of the proteomic data analysis workflow. Our goal was to develop a software platform that would fulfill the needs of scientists in the field, while emphasizing ease-of-use for non-bioinformatician users. Protein identification is based on OLAV scoring schemes and protein quantification is implemented for both, isobaric labeling and label-free methods. Additional features are available, such as peak list processing, isotopic correction, spectra filtering, charge-state deconvolution and spectra merging. To illustrate the EasyProt platform, we present two identification and quantification workflows based on isobaric tagging and label-free methods.
Asunto(s)
Proteómica/métodos , Análisis de Secuencia de Proteína/métodos , Programas Informáticos , Espectrometría de Masas/métodos , Procesamiento Proteico-Postraduccional , Proteínas/análisisRESUMEN
Isobaric tagging, via TMT or iTRAQ, is widely used in quantitative proteomics. To date, tandem mass spectrometric analysis of isobarically-labeled peptides with hybrid ion trap-orbitrap (LTQ-OT) instruments has been mainly carried out with higher-energy C-trap dissociation (HCD) or pulsed q dissociation (PQD). HCD provides good fragmentation of the reporter-ions, but peptide sequence-ion recovery is generally poor compared to collision-induced dissociation (CID). Herein, we describe an approach where CID and HCD spectra are combined. The approach ensures efficiently both identification and relative quantification of proteins. Tandem mass tags (TMTs) were used to label digests of human plasma and LC-MS/MS was performed with an LTQ-OT instrument. Different HCD collision energies were tested. The benefits to use CID and HCD with respect to HCD alone were demonstrated in terms of number of identifications, subsequent number of quantifiable proteins, and quantification accuracy. A program was developed to merge the peptide sequence-ion m/z range from CID spectra and the reporter-ion m/z range from HCD spectra, and alternatively to separate both spectral data into different files. As parallel CID in the LTQ almost doesn't affect the analysis duty cycle, the procedure should become a standard for quantitative analyses of proteins with isobaric tagging using LTQ-OT instruments.
Asunto(s)
Péptidos/análisis , Coloración y Etiquetado/métodos , Espectrometría de Masas en Tándem/métodos , Proteínas Sanguíneas/análisis , Proteínas Sanguíneas/química , Cromatografía Liquida , Humanos , Marcaje Isotópico , Péptidos/química , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Espectrometría de Masa por Ionización de Electrospray , TiazolesRESUMEN
We generated a murine monoclonal antibody (B28p) detecting an antigenic determinant shared by the immunoglobulin superfamily receptor translocation-associated 1 (IRTA1) receptor (the immunogen used to raise B28p) and an unrelated 28-kDa protein that was subsequently subjected to extensive characterization. The expression of the 28-kDa protein in normal lymphohematopoietic tissues was restricted to B cells and plasma cells and clearly differed from that expected for IRTA1 (selectively expressed by mucosa-associated lymphoid tissue [MALT] marginal zone B cells). Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE)/mass-spectrometry analysis identified the 28-kDa protein as human tumor protein D52 (TPD52), whose expression had been previously described only in normal and neoplastic epithelia. Specific B28p reactivity with TPD52 was confirmed by immunostaining/immunoblotting of TPD52-transfected cells. TPD52 expression pattern in normal and neoplastic B cells was unique. In fact, unlike other B-cell molecules (paired box 5 [PAX5], CD19, CD79a, CD20, CD22), which are down-regulated during differentiation from B cells to plasma cells, TPD52 expression reached its maximum levels at the plasma cell stage. In the Thiel myeloma cell line, TPD52 bound to annexin VI in a Ca(2+)-dependent manner, suggesting that these molecules may act in concert to regulate secretory processes in plasma cells, similarly to what was observed in pancreatic acinar cells. Finally, the anti-TPD52 monoclonal antibody served as a valuable tool for the diagnosis of B-cell malignancies.
Asunto(s)
Anexina A6/metabolismo , Calcio/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Células Plasmáticas/fisiología , Animales , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Linfocitos B/fisiología , Línea Celular Tumoral , Electroforesis en Gel Bidimensional , Epítopos de Linfocito B/inmunología , Regulación Leucémica de la Expresión Génica/inmunología , Leucemia de Células B/diagnóstico , Leucemia de Células B/fisiopatología , Espectrometría de Masas , Ratones , Ratones Endogámicos BALB C , Peso Molecular , Proteínas de Neoplasias/química , Proteínas de Neoplasias/inmunología , Receptores de Superficie Celular/inmunología , Receptores FcRESUMEN
The synthesis and application of two new alkylating reagents, N-tert-butyl-2-iodoacetamide (N-t-butyliodoacetamide) and 2-iodo-N-phenylacetamide (iodoacetanilide), are described. N-t-Butyliodoacetamide and iodoacetanilide were synthesised to purity in their d(0)-light and in their respective d(9)- and d(5)-heavy forms. The newly synthesised reagents are covalently bound to peptides containing cysteines via an alkylation reaction. The mass differences of 5 and 9 Da avoid possible problems of overlapping isotope distribution. For each alkylated cysteine a peptide mass increases, respectively, by a multiple of 113 and 133 Da for the d(0)-light form of N-t-butyliodoacetamide and iodoacetanilide. These reagents can therefore replace common alkylating reagents in existing proteomics-based applications. Alkylated peptides increase in mass in the same mass range as amino acids and remain suitable for tandem mass spectrometry (MS/MS) data acquisition and analysis. The compounds are simple to use and derivatisation is based on widely applied alkylating procedures. Preliminary results show that these reagents can be applied for both protein quantitation and identification by peptide mass finger printing and/or MS/MS techniques. Using these chemicals and the suggested workflow enables the quantitative analysis of the whole protein sample and realises access to peptides that may contain potential post-translational modifications. Other approaches that incorporate a matrix-assisted laser desorption/ionisation (MALDI) interface prior to MS can take advantage of these chemicals, such as the molecular scanner.
Asunto(s)
Acetanilidas/química , Cisteína/química , Yodoacetamida/química , Mapeo Peptídico/métodos , Proteínas/análisis , Proteínas/química , Proteómica/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Alquilantes/química , Alquilación , Indicadores y Reactivos/química , Lactoperoxidasa/análisis , Lactoperoxidasa/química , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Although peptide mass fingerprinting is currently the method of choice to identify proteins, the number of proteins available in databases is increasing constantly, and hence, the advantage of having sequence data on a selected peptide, in order to increase the effectiveness of database searching, is more crucial. Until recently, the ability to identify proteins based on the peptide sequence was essentially limited to the use of electrospray ionization tandem mass spectrometry (MS) methods. The recent development of new instruments with matrix-assisted laser desorption/ionization (MALDI) sources and true tandem mass spectrometry (MS/MS) capabilities creates the capacity to obtain high quality tandem mass spectra of peptides. In this work, using the new high resolution tandem time of flight MALDI-(TOF/TOF) mass spectrometer from Applied Biosystems, examples of successful identification and characterization of bovine heart proteins (SWISS-PROT entries: P02192, Q9XSC6, P13620) separated by two-dimensional electrophoresis and blotted onto polyvinylidene difluoride membrane are described. Tryptic protein digests were analyzed by MALDI-TOF to identify peptide masses afterward used for MS/MS. Subsequent high energy MALDI-TOF/TOF collision-induced dissociation spectra were recorded on selected ions. All data, both MS and MS/MS, were recorded on the same instrument. Tandem mass spectra were submitted to database searching using MS-Tag or were manually de novo sequenced. An interesting modification of a tryptophan residue, a "double oxidation", came to light during these analyses.