Tissue-based absolute quantification using large-scale TMT and LFQ experiments

Relative and absolute intensity-based protein quantification across cell lines, tissue atlases and tumour datasets is increasingly available in public datasets. These atlases enable researchers to explore fundamental biological questions, such as protein existence, expression location, quantity and correlation with RNA expression. Most studies provide MS1 feature-based label-free quantitative (LFQ) datasets; however, growing numbers of isobaric tandem mass tags (TMT) datasets remain unexplored. Here, we compare traditional intensity-based absolute quantification (iBAQ) proteome abundance ranking to an analogous method using reporter ion proteome abundance ranking with data from an experiment where LFQ and TMT were measured on the same samples. This new TMT method substitutes reporter ion intensities for MS1 feature intensities in the iBAQ framework. Additionally, we compared LFQ-iBAQ values to TMT-iBAQ values from two independent large-scale tissue atlas datasets (one LFQ and one TMT) using robust bottom-up proteomic identification, normalisation and quantitation workflows

From tsunami risk assessment to disaster risk reduction the case of Oman

Oman is located in an area of high seismicity, facing the Makran Subduction Zone, which is the major source of earthquakes in the eastern border of the Arabian plate. These earthquakes, as evidenced by several past events, may trigger a tsunami event. The aim of this work is to minimize the consequences that tsunami events may cause in coastal communities by integrating tsunami risk assessment and risk reduction measures as part of the risk-management preparedness strategy. An integrated risk assessment approach and the analysis of site-specific conditions permitted to propose target-oriented risk reduction measures. The process included a participatory approach, involving a panel of local stakeholders and international experts. One of the main concerns of this work was to obtain a useful outcome for the actual improvement of tsunami risk management in Oman. This goal was achieved through the development of comprehensive and functional management tools such as the Tsunami Hazard, Vulnerability and Risk Atlas and the Risk Reduction Measures Handbook, which will help to design and plan a roadmap towards risk reduction. The integrated tsunami risk assessment performed showed that the northern area of Oman would be the most affected, considering both the hazard and vulnerability components. This area also concentrates nearly 50% of the hot spots identified throughout the country, 70% of them are located in areas with a very high risk class, in which risk reduction measures were selected and prioritized.The authors thank the Ministry of Transport and Communications of the Government of the Sultanate of Oman (MOTC), the Public Authority for Civil Aviation (PACA) and the Directorate General of Meteorology (DGMET), for supporting and funding the project “Assessment of Coastal Hazards, Vulnerability and Risk for the Coast of Oman” during the period 2014–2016. We also thank and appreciate the collaboration of the International Oceanographic Commission of the United Nations Educational, Scientific and Cultural Organization personnel (IOC-UNESCO)

Evaluación del riesgo por tsunami en zonas costeras y estratégicas de adaptación y mitigación

Los eventos de tsunami suponen una amenaza natural de baja frecuencia pero con un gran poder destructivo, que ha causado la pérdida de miles de vidas humanas y ha provocado cuantiosos daños en infraestructuras y comunidades costeras en todo el mundo. En el período comprendido entre el año 2004 y 2013, se estima la pérdida de cerca de 240.000 vidas y de más de 250.000 millones de dólares americanos (GAR-UNISDR, 2015). Los avances en el conocimiento sobre los mecanismos de generación y propagación proporcionan una mejora en el pronóstico de los impactos que pueden causar los tsunamis sobre las comunidades afectadas. La evaluación del riesgo permite identificar estrategias de gestión y medidas de reducción de riesgos apropiadas y específicas para cada lugar, integrando de esta manera la reducción del riesgo de desastres en las políticas, la planificación y la programación en todos los niveles, incluyendo la prevención, mitigación, preparación y reducción de la vulnerabilidad, como se subraya en el Marco de Acción de Hyogo (UNISDR, 2005). El objetivo de este trabajo es la evaluación del riesgo por tsunami y la dentificación, recomendación y priorización de medidas de reducción de riesgo específicas. Al mismo tiempo, los resultados de esta evaluación se han integrado en un sistema de alerta temprana multi-riesgo. El trabajo se ha desarrollado en la costa de Omán, localizada frente a una zona de subducción de alta sismicidad. La metodología desarrollada se centra principalmente en los impactos sobre las personas, integrando el análisis de la peligrosidad, exposición y vulnerabilidad para determinar el riesgo ante tsunami. Para realizar dichos análisis se consideran dos dimensiones: humana e infraestructuras; y dos resoluciones espaciales: escala nacional, incluyendo toda la zona costera del país y escala local, comprendiendo nueve ciudades costeras y una distancia mínima de 20 kilómetros a cada lado de la ciudad. Este trabajo ha permitido vincular los resultados de la evaluación del riesgo por tsunami con una gestión del riesgo, a diferentes niveles administrativos y a diferentes escalas, enfocada a minimizar los posibles impactos derivados de un tsunami potencial

The Hidden Story of Heterogeneous B-raf V600E Mutation Quantitative Protein Expression in Metastatic Melanoma-Association with Clinical Outcome and Tumor Phenotypes

In comparison to other human cancer types, malignant melanoma exhibits the greatest amount of heterogeneity. After DNA-based detection of the BRAF V600E mutation in melanoma patients, targeted inhibitor treatment is the current recommendation. This approach, however, does not take the abundance of the therapeutic target, i.e., the B-raf V600E protein, into consideration. As shown by immunohistochemistry, the protein expression profiles of metastatic melanomas clearly reveal the existence of inter-and intra-tumor variability. Nevertheless, the technique is only semi-quantitative. To quantitate the mutant protein there is a fundamental need for more precise techniques that are aimed at defining the currently non-existent link between the levels of the target protein and subsequent drug efficacy. Using cutting-edge mass spectrometry combined with DNA and mRNA sequencing, the mutated B-raf protein within metastatic tumors was quantitated for the first time. B-raf V600E protein analysis revealed a subjacent layer of heterogeneity for mutation-positive metastatic melanomas. These were characterized into two distinct groups with different tumor morphologies, protein profiles and patient clinical outcomes. This study provides evidence that a higher level of expression in the mutated protein is associated with a more aggressive tumor progression. Our study design, comprised of surgical isolation of tumors, histopathological characterization, tissue biobanking, and protein analysis, may enable the eventual delineation of patient responders/non-responders and subsequent therapy for malignant melanoma

CIGB-300, a synthetic peptide-based drug that targets the CK2 phosphoaceptor domain. Translational and clinical research

CK2 represents an oncology target scientifically validated. However, clinical research with inhibitors of the CK2-mediated phosphorylation event is still insufficient to recognize it as a clinically validated target. CIGB-300, an investigational peptide-based drug that targets the phosphoaceptor site, binds to a CK2 substrate array in vitro but mainly to B23/nucleophosmin in vivo. The CIGB-300 proapoptotic effect is preceded by its nucleolar localization, inhibition of the CK2-mediated phosphorylation on B23/nucleophosmin and nucleolar disassembly. Importantly, CIGB-300 shifted a protein array linked to apoptosis, ribosome biogenesis, cell proliferation, glycolisis, and cell motility in proteomic studies which helped to understand its mechanism of action. In the clinical ground, CIGB-300 has proved to be safe and well tolerated in a First-in-Human trial in women with cervical malignancies who also experienced signs of clinical benefit. In a second Phase 1 clinical trial in women with cervical cancer stage IB2/II, the MTD and DLT have been also identified in the clinical setting. Interestingly, in cervical tumors the B23/nucleophosmin protein levels were significantly reduced after CIGB-300 treatment at the nucleus compartment. In addition, expanded use of CIGB-300 in case studies has evidenced antitumor activity when administered as compassional option. Collectively, our data outline important clues on translational and clinical research from this novel peptide-based drug reinforcing its perspectives to treat cancer and paving the way to validate CK2 as a promising target in oncology.Fil: Perea, Silvio E.. Center for Genetic Engineering and Biotechnology; CubaFil: Baladron, Idania. Center for Genetic Engineering and Biotechnology; CubaFil: Garcia, Yanelda. Center for Genetic Engineering and Biotechnology; CubaFil: Perera, Yasser. Center for Genetic Engineering and Biotechnology; CubaFil: Lopez, Adlin. Center for Genetic Engineering and Biotechnology; CubaFil: Soriano, Jorge L.. Center for Genetic Engineering and Biotechnology; Cuba. General Hospital ‘‘Hermanos Ameijeiras’; CubaFil: Batista, Noyde. Center for Genetic Engineering and Biotechnology; Cuba. General Hospital ‘‘Hermanos Ameijeiras’; CubaFil: Palau, Aley. Center for Genetic Engineering and Biotechnology; Cuba. General Hospital ‘‘Hermanos Ameijeiras’; CubaFil: Hernández, Ignacio. Center for Genetic Engineering and Biotechnology; CubaFil: Farina, Hernán Gabriel. Universidad Nacional de Quilmes; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Garcia, Idrian. Center for Genetic Engineering and Biotechnology; CubaFil: Gonzalez, Lidia. Center for Genetic Engineering and Biotechnology; CubaFil: Gil, Jeovanis. Center for Genetic Engineering and Biotechnology; CubaFil: Rodriguez, Arielis. Center for Genetic Engineering and Biotechnology; CubaFil: Solares, Margarita. Center for Genetic Engineering and Biotechnology; CubaFil: Santana, Agueda. Center for Genetic Engineering and Biotechnology; CubaFil: Cruz, Marisol. Center for Genetic Engineering and Biotechnology; CubaFil: Lopez, Matilde. Center for Genetic Engineering and Biotechnology; CubaFil: Valenzuela, Carmen. Center for Genetic Engineering and Biotechnology; CubaFil: Reyes, Osvaldo. Center for Genetic Engineering and Biotechnology; CubaFil: López Saura, Pedro A.. Center for Genetic Engineering and Biotechnology; CubaFil: González, Carlos A.. Center for Genetic Engineering and Biotechnology; CubaFil: Diaz, Alina. Center for Genetic Engineering and Biotechnology; CubaFil: Castellanos, Lila. Center for Genetic Engineering and Biotechnology; CubaFil: Sanchez, Aniel. Center for Genetic Engineering and Biotechnology; CubaFil: Betancourt, Lazaro. Center for Genetic Engineering and Biotechnology; CubaFil: Besada, Vladimir. Center for Genetic Engineering and Biotechnology; CubaFil: González, Luis J.. Center for Genetic Engineering and Biotechnology; CubaFil: Garay, Hilda. Center for Genetic Engineering and Biotechnology; CubaFil: Gómez, Roberto. Center for Genetic Engineering and Biotechnology; CubaFil: Gomez, Daniel Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes; ArgentinaFil: Alonso, Daniel Fernando. Universidad Nacional de Quilmes; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Perrin, Phillipe. No especifíca;Fil: Renualt, Jean Yves. No especifíca;Fil: Sigman, Hugo. No especifíca;Fil: Herrera, Luis. Center for Genetic Engineering and Biotechnology; CubaFil: Acevedo, Boris. Center for Genetic Engineering and Biotechnology; Cub

The Human Melanoma Proteome Atlas—Complementing the melanoma transcriptome

The MM500 meta‐study aims to establish a knowledge basis of the tumor proteome to serve as a complement to genome and transcriptome studies. Somatic mutations and their effect on the transcriptome have been extensively characterized in melanoma. However, the effects of these genetic changes on the proteomic landscape and the impact on cellular processes in melanoma remain poorly understood. In this study, the quantitative mass‐spectrometry‐based proteomic analysis is interfaced with pathological tumor characterization, and associated with clinical data. The melanoma proteome landscape, obtained by the analysis of 505 well‐annotated melanoma tumor samples, is defined based on almost 16 000 proteins, including mutated proteoforms of driver genes. More than 50 million MS/MS spectra were analyzed, resulting in approximately 13,6 million peptide spectrum matches (PSMs). Altogether 13 176 protein‐coding genes, represented by 366 172 peptides, in addition to 52 000 phosphorylation sites, and 4 400 acetylation sites were successfully annotated. This data covers 65% and 74% of the predicted and identified human proteome, respectively. A high degree of correlation (Pearson, up to 0.54) with the melanoma transcriptome of the TCGA repository, with an overlap of 12 751 gene products, was found. Mapping of the expressed proteins with quantitation, spatiotemporal localization, mutations, splice isoforms, and PTM variants was proven not to be predicted by genome sequencing alone. The melanoma tumor molecular map was complemented by analysis of blood protein expression, including data on proteins regulated after immunotherapy. By adding these key proteomic pillars, the MM500 study expands the knowledge on melanoma disease

Metodologías para la identificación y la cuantificación relativa de proteínas por espectrometría de masas mediante la modificación química de grupos amino primarios.

<p></p><p>Se describen tres metodologías para la caracterización e identificación de proteínas presentes en mezclas complejas. La combinación de métodos cromatográficos y reacciones químicas simples permite por primera vez reconocer al péptido <i>N-</i>terminal en un espectro de masas en presencia de otros péptidos proteolíticos mediante la observación de su distribución isotópica. Las otras dos metodologías permiten el aislamiento selectivo de un tipo determinado de péptidos denominados <b>RRnK</b> y <b>RH. </b> El aislamiento selectivo de estos péptidos permite simplificar la mezcla compleja sin renunciar a la cobertura teórica de más del 80% de las proteínas presentes en la base de datos. </p><br><p></p

Tissue-based absolute quantification using large-scale TMT and LFQ experiments

Relative and absolute intensity-based protein quantification across cell lines, tissue atlases and tumour datasets is increasingly available in public datasets. These atlases enable researchers to explore fundamental biological questions, such as protein existence, expression location, quantity and correlation with RNA expression. Most studies provide MS1 feature-based label-free quantitative (LFQ) datasets; however, growing numbers of isobaric tandem mass tags (TMT) datasets remain unexplored. Here, we compare traditional intensity-based absolute quantification (iBAQ) proteome abundance ranking to an analogous method using reporter ion proteome abundance ranking with data from an experiment where LFQ and TMT were measured on the same samples. This new TMT method substitutes reporter ion intensities for MS1 feature intensities in the iBAQ framework. Additionally, we compared LFQ-iBAQ values to TMT-iBAQ values from two independent large-scale tissue atlas datasets (one LFQ and one TMT) using robust bottom-up proteomic identification, normalisation and quantitation workflows