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The OpenProt proteogenomic resource (https://www.openprot.org/) provides users with a complete and freely accessible set of non-canonical or alternative open reading frames (AltORFs) within the transcriptome of various species, as well as functional annotations of the corresponding protein sequences not found in standard databases. Enhancements in this update are largely the result of user feedback and include the prediction of structure, subcellular localization, and intrinsic disorder, using cutting-edge algorithms based on machine learning techniques. The mass spectrometry pipeline now integrates a machine learning-based peptide rescoring method to improve peptide identification. We continue to help users explore this cryptic proteome by providing OpenCustomDB, a tool that enables users to build their own customized protein databases, and OpenVar, a genomic annotator including genetic variants within AltORFs and protein sequences. A new interface improves the visualization of all functional annotations, including a spectral viewer and the prediction of multicoding genes. All data on OpenProt are freely available and downloadable. Overall, OpenProt continues to establish itself as an important resource for the exploration and study of new proteins.
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Bases de Datos de Proteínas , Péptidos , Proteómica , Secuencia de Aminoácidos , Genómica , Internet , Péptidos/genética , Proteoma/genética , Proteómica/métodos , HumanosRESUMEN
Conventionally, eukaryotic mRNAs were thought to be monocistronic, leading to the translation of a single protein. However, large-scale proteomics has led to the identification of proteins translated from alternative open reading frames (AltORFs) in mRNAs. AltORFs are found in addition to predicted reference ORFs and noncoding RNA. Alternative proteins are not represented in the conventional protein databases, and this 'Ghost proteome' was not considered until recently. Some of these proteins are functional, and there is growing evidence that they are involved in central functions in physiological and physiopathological contexts. Here, we review how this Ghost proteome fills the gap in our understanding of signaling pathways, establishes new markers of pathologies, and highlights therapeutic targets.
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Biosíntesis de Proteínas , Proteoma , Bases de Datos de Proteínas , Sistemas de Lectura Abierta , Proteoma/genética , ProteómicaRESUMEN
Artificial intelligence (AI) platforms have emerged as pivotal tools in genetics and molecular medicine, as in many other fields. The growth in patient data, identification of new diseases and phenotypes, discovery of new intracellular pathways, availability of greater sets of omics data, and the need to continuously analyse them have led to the development of new AI platforms. AI continues to weave its way into the fabric of genetics with the potential to unlock new discoveries and enhance patient care. This technology is setting the stage for breakthroughs across various domains, including dysmorphology, rare hereditary diseases, cancers, clinical microbiomics, the investigation of zoonotic diseases, omics studies in all medical disciplines. AI's role in facilitating a deeper understanding of these areas heralds a new era of personalised medicine, where treatments and diagnoses are tailored to the individual's molecular features, offering a more precise approach to combating genetic or acquired disorders. The significance of these AI platforms is growing as they assist healthcare professionals in the diagnostic and treatment processes, marking a pivotal shift towards more informed, efficient, and effective medical practice. In this review, we will explore the range of AI tools available and show how they have become vital in various sectors of genomic research supporting clinical decisions.
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Inteligencia Artificial , Medicina Molecular , Humanos , Medicina Molecular/métodos , Genética Médica/tendencias , Genética Médica/métodos , Medicina de Precisión/métodos , Genómica/métodosRESUMEN
Traumatic brain injury (TBI) represents one of the major public health concerns worldwide due to the increase in TBI incidence as a result of injuries from daily life accidents such as sports and motor vehicle transportation as well as military-related practices. This type of central nervous system trauma is known to predispose patients to several neurological disorders such as Parkinson's disease, Alzheimer's disease, chronic trauamatic encephalopathy, and age-related Dementia. Recently, several proteomic and lipidomic platforms have been applied on different TBI studies to investigate TBI-related mechanisms that have broadened our understanding of its distinct neuropathological complications. In this study, we provide an updated comprehensive overview of the current knowledge and novel perspectives of the spatially resolved microproteomics and microlipidomics approaches guided by mass spectrometry imaging used in TBI studies and its applications in the neurotrauma field. In this regard, we will discuss the use of the spatially resolved microproteomics and assess the different microproteomic sampling methods such as laser capture microdissection, parafilm assisted microdissection, and liquid microjunction extraction as accurate and precise techniques in the field of neuroproteomics. Additionally, we will highlight lipid profiling applications and their prospective potentials in characterizing molecular processes involved in the field of TBI. Specifically, we will discuss the phospholipid metabolism acting as a precursor for proinflammatory molecules such as eicosanoids. Finally, we will survey the current state of spatial neuroproteomics and microproteomics applications and present the various studies highlighting their findings in these fields.
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Enfermedad de Alzheimer , Lesiones Traumáticas del Encéfalo , Humanos , Espectrometría de Masas , Proteómica/métodos , Lesiones Traumáticas del Encéfalo/complicaciones , Lesiones Traumáticas del Encéfalo/metabolismoRESUMEN
BACKGROUND: Cancer heterogeneity is a main obstacle for the development of effective therapies, as its replication in in vitro preclinical models is challenging. Around 96% of developed drugs are estimated to fail from discovery to the clinical trial phase probably because of the unsuitability and unreliability of current preclinical models (Front Pharmacol 9:6, 2018; Nat Rev Cancer 8: 147-56, 2008) in replicating the overall biology of tumors, for instance the tumor microenvironment. Breast cancer is the most frequent cancer among women causing the greatest number of cancer-related deaths. Breast cancer can typically be modeled in vitro through the use of tumoroids; however, current approaches using mouse tumoroids fail to reproduce crucial aspect of human breast cancer, while access to human cells is limited and the focus of ethical concerns. New models of breast cancer, such as companion dogs, have emerged given the resemblance of developed spontaneous mammary tumors to human breast cancer in many clinical and molecular aspects; however, they have so far failed to replicate the tumor microenvironment. The present work aimed at developing a robust canine mammary tumor model in the form of tumoroids which recapitulate the tumor diversity and heterogeneity. RESULTS: We conducted a complete characterization of canine mammary tumoroids through histologic, molecular, and proteomic analysis, demonstrating their strong similarity to the primary tumor. We demonstrated that these tumoroids can be used as a drug screening model. In fact, we showed that paclitaxel, a human chemotherapeutic, could kill canine tumoroids with the same efficacy as human tumoroids with 0.1 to 1 µM of drug needed to kill 50% of the cells. Due to easy tissue availability, canine tumoroids can be produced at larger scale and cryopreserved to constitute a biobank. We have demonstrated that cryopreserved tumoroids keep the same histologic and molecular features (ER, PR, and HER2 expression) as fresh tumoroids. Furthermore, two cryopreservation techniques were compared from a proteomic point of view which showed that tumoroids made from frozen material allowed to maintain the same molecular diversity as from freshly dissociated tumor. CONCLUSIONS: These findings revealed that canine mammary tumoroids can be easily generated and may provide an adequate and more reliable preclinical model to investigate tumorigenesis mechanisms and develop new treatments for both veterinary and human medicine.
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Neoplasias de la Mama , Neoplasias Mamarias Animales , Animales , Perros , Femenino , Humanos , Neoplasias de la Mama/patología , Neoplasias Mamarias Animales/diagnóstico , Neoplasias Mamarias Animales/metabolismo , Neoplasias Mamarias Animales/patología , Proteómica , Investigación Biomédica Traslacional , Microambiente TumoralRESUMEN
Liquid chromatography-mass spectrometry (LC-MS) is a powerful method for cell profiling. The use of LC-MS technology is a tool of choice for cancer research since it provides molecular fingerprints of analyzed tissues. However, the ubiquitous presence of noise, the peaks shift between acquisitions, and the huge amount of information owing to the high dimensionality of the data make rapid and accurate cancer diagnosis a challenging task. Deep learning (DL) models are not only effective classifiers but are also well suited to jointly learn feature representation and classification tasks. This is particularly relevant when applied to raw LC-MS data and hence avoid the need for costly preprocessing and complicated feature selection. In this study, we propose a new end-to-end DL methodology that addresses all of the above challenges at once, while preserving the high potential of LC-MS data. Our DL model is designed to early discriminate between tumoral and normal tissues. It is a combination of a convolutional neural network (CNN) and a long short-term memory (LSTM) Network. The CNN network allows for significantly reducing the high dimensionality of the data while learning spatially relevant features. The LSTM network enables our model to capture temporal patterns. We show that our model outperforms not only benchmark models but also state-of-the-art models developed on the same data. Our framework is a promising strategy for improving early cancer detection during a diagnostic process.
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Benchmarking , Detección Precoz del Cáncer , Cromatografía Liquida , Espectrometría de Masas , Redes Neurales de la ComputaciónRESUMEN
In this study, we conducted a direct comparison of water-assisted laser desorption ionization (WALDI) and matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging, with MALDI serving as the benchmark for label-free molecular tissue analysis in biomedical research. Specifically, we investigated the lipidomic profiles of several biological samples and calculated the similarity of detected peaks and Pearson's correlation of spectral profile intensities between the two techniques. We show that, overall, MALDI MS and WALDI MS present very close lipidomic analyses and that the highest similarity is obtained for the norharmane MALDI matrix. Indeed, for norharmane in negative ion mode, the lipidomic spectra revealed 100% similarity of detected peaks and over 0.90 intensity correlation between both technologies for five samples. The MALDI-MSI positive ion lipid spectra displayed more than 83% similarity of detected peaks compared to those of WALDI-MSI. However, we observed a lower percentage (77%) of detected peaks when comparing WALDI-MSI with MALDI-MSI due to the rich WALDI-MSI lipid spectra. Despite this difference, the global lipidomic spectra showed high consistency between the two technologies, indicating that they are governed by similar processes. Thanks to this similarity, we can increase datasets by including data from both modalities to either co-train classification models or obtain cross-interrogation.
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OpenProt (www.openprot.org) is the first proteogenomic resource supporting a polycistronic annotation model for eukaryotic genomes. It provides a deeper annotation of open reading frames (ORFs) while mining experimental data for supporting evidence using cutting-edge algorithms. This update presents the major improvements since the initial release of OpenProt. All species support recent NCBI RefSeq and Ensembl annotations, with changes in annotations being reported in OpenProt. Using the 131 ribosome profiling datasets re-analysed by OpenProt to date, non-AUG initiation starts are reported alongside a confidence score of the initiating codon. From the 177 mass spectrometry datasets re-analysed by OpenProt to date, the unicity of the detected peptides is controlled at each implementation. Furthermore, to guide the users, detectability statistics and protein relationships (isoforms) are now reported for each protein. Finally, to foster access to deeper ORF annotation independently of one's bioinformatics skills or computational resources, OpenProt now offers a data analysis platform. Users can submit their dataset for analysis and receive the results from the analysis by OpenProt. All data on OpenProt are freely available and downloadable for each species, the release-based format ensuring a continuous access to the data. Thus, OpenProt enables a more comprehensive annotation of eukaryotic genomes and fosters functional proteomic discoveries.
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Bases de Datos de Proteínas , Eucariontes/genética , Genoma , Anotación de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Espectrometría de Masas , Isoformas de Proteínas/genética , Proteogenómica , Ribosomas/metabolismo , Interfaz Usuario-ComputadorRESUMEN
It has been recently shown that many proteins are lacking from reference databases used in mass spectrometry analysis, due to their translation templated on alternative open reading frames. This questions our current understanding of gene annotation and drastically expands the theoretical proteome complexity. The functions of these alternative proteins (AltProts) still remain largely unknown. We have developed a large-scale and unsupervised approach based on cross-linking mass spectrometry (XL-MS) followed by shotgun proteomics to gather information on the functional role of AltProts by mapping them back into known signalling pathways through the identification of their reference protein (RefProt) interactors. We have identified and profiled AltProts in a cancer cell reprogramming system: NCH82 human glioma cells after 0, 16, 24 and 48 h Forskolin stimulation. Forskolin is a protein kinase A activator inducing cell differentiation and epithelial-mesenchymal transition. Our data show that AltMAP2, AltTRNAU1AP and AltEPHA5 interactions with tropomyosin 4 are downregulated under Forskolin treatment. In a wider perspective, Gene Ontology and pathway enrichment analysis (STRING) revealed that RefProts associated with AltProts are enriched in cellular mobility and transfer RNA regulation. This study strongly suggests novel roles of AltProts in multiple essential cellular functions and supports the importance of considering them in future biological studies.
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Reprogramación Celular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Mapeo de Interacción de Proteínas , Línea Celular Tumoral , Reprogramación Celular/efectos de los fármacos , Colforsina/farmacología , Activación Enzimática , Humanos , Espectrometría de Masas , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Proteómica , Proteínas de Unión al ARN/metabolismo , Receptor EphA5/metabolismo , Transducción de Señal , Tropomiosina/metabolismoRESUMEN
Expansion microscopy (EM) is an emerging approach for morphological examination of biological specimens at nanoscale resolution using conventional optical microscopy. To achieve physical separation of cell structures, tissues are embedded in a swellable polymer and expanded several fold in an isotropic manner. This work shows the development and optimization of physical tissue expansion as a new method for spatially resolved large-scale proteomics. Herein we established a novel method to enlarge the tissue section to be compatible with manual microdissection on regions of interest and MS-based proteomic analysis. A major issue in expansion microscopy is the loss of protein information during the mechanical homogenization phase due to the use of proteinase K. For isotropic expansion, different homogenization agents were investigated, both to maximize protein identification and to minimize protein diffusion. Best results were obtained with SDS for homogenization. Using our modified protocol, we were able to enlarge a tissue section more than 3-fold and identified up to 655 proteins from 1 mm in size after expansion, equivalent to 330 µm in their real size corresponding thus to an average of 260 cells. This approach can be performed easily without any expensive sampling instrument. We demonstrated the compatibility of sample preparation for expansion microscopy and proteomic study in a spatial context.
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Microscopía , Proteómica , Polímeros , Proteínas , Manejo de EspecímenesRESUMEN
Mass spectrometry imaging (MSI) has shown to bring invaluable information for biological and clinical applications. However, conventional MSI is generally performed ex vivo from tissue sections. Here, we developed a novel MS-based method for in vivo mass spectrometry imaging. By coupling the SpiderMass technology, that provides in vivo minimally invasive analysis-to a robotic arm of high accuracy, we demonstrate that images can be acquired from any surface by moving the laser probe above the surface. By equipping the robotic arm with a sensor, we are also able to both get the topography image of the sample surface and the molecular distribution, and then and plot back the molecular data, directly to the 3D topographical image without the need for image fusion. This is shown for the first time with the 3D topographic MS-based whole-body imaging of a mouse. Enabling fast in vivo MSI bridged to topography paves the way for surgical applications to excision margins.
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Robótica , Animales , Imagenología Tridimensional , Ratones , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
BACKGROUND: Formalin-fixed paraffin-embedded (FFPE) tissue has been the gold standard for routine pathology for general and cancer postoperative diagnostics. Despite robust histopathology, immunohistochemistry, and molecular methods, accurate diagnosis remains difficult for certain cases. Overall, the entire process can be time consuming, labor intensive, and does not reach over 90% diagnostic sensitivity and specificity. There is a growing need in onco-pathology for adjunct novel rapid, accurate, reliable, diagnostically sensitive, and specific methods for high-throughput biomolecular identification. Lipids have long been considered only as building blocks of cell membranes or signaling molecules, but have recently been introduced as central players in cancer. Due to sample processing, which limits their detection, lipid analysis directly from unprocessed FFPE tissues has never been reported. METHODS: We present a proof-of-concept with direct analysis of tissue-lipidomic signatures from FFPE tissues without dewaxing and minimal sample preparation using water-assisted laser desorption ionization mass spectrometry and deep-learning. RESULTS: On a cohort of difficult canine and human sarcoma cases, classification for canine sarcoma subtyping was possible with 99.1% accuracy using "5-fold" and 98.5% using "leave-one-patient out," and 91.2% accuracy for human sarcoma using 5-fold and 73.8% using leave-one-patient out. The developed classification model enabled stratification of blind samples in <5 min and showed >95% probability for discriminating 2 human sarcoma blind samples. CONCLUSION: It is possible to create a rapid diagnostic platform to screen clinical FFPE tissues with minimal sample preparation for molecular pathology.
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Lipidómica , Sarcoma , Animales , Perros , Formaldehído/química , Humanos , Rayos Láser , Adhesión en Parafina , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Fijación del Tejido/métodos , AguaRESUMEN
Traumatic brain injury (TBI) represents a major health concerns with no clinically-approved FDA drug available for therapeutic intervention. Several genomics and neuroproteomics studies have been employed to decipher the underlying pathological mechanisms involved that can serve as potential neurotherapeutic targets and unveil a possible underlying relation of TBI to other secondary neurological disorders. In this work, we present a novel high throughput systems biology approach using a spatially resolved microproteomics platform conducted on different brain regions in an experimental rat model of moderate of controlled cortical injury (CCI) at a temporal pattern postinjury (1 day, 3 days, 7 days, and 10 days). Mapping the spatiotemporal landscape of signature markers in TBI revealed an overexpression of major protein families known to be implicated in Parkinson's disease (PD) such as GPR158, HGMB1, synaptotagmin and glutamate decarboxylase in the ipsilateral substantia nigra. In silico bioinformatics docking experiments indicated the potential correlation between TBI and PD through alpha-synuclein. In an in vitro model, stimulation with palmitoylcarnitine triggered an inflammatory response in macrophages and a regeneration processes in astrocytes which also further confirmed the in vivo TBI proteomics data. Taken together, this is the first study to assess the microproteomics landscape in TBI, mainly in the substantia nigra, thus revealing a potential predisposition for PD or Parkinsonism post-TBI.
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Lesiones Traumáticas del Encéfalo/metabolismo , Enfermedad de Parkinson/metabolismo , Animales , Encéfalo/metabolismo , Línea Celular , Modelos Animales de Enfermedad , Masculino , Proteómica , Ratas Sprague-DawleyRESUMEN
Adult stem cells have become prominent candidates for treating various diseases in veterinary practice. The main goal of our study was therefore to provide a comprehensive study of canine bone marrow-derived mesenchymal stem cells (BMMSC) and conditioned media, isolated from healthy adult dogs of different breeds. Under well-defined standardized isolation protocols, the multipotent differentiation and specific surface markers of BMMSC were supplemented with their gene expression, proteomic profile, and their biological function. The presented data confirm that canine BMMSC express important genes for differentiation toward osteo-, chondro-, and tendo-genic directions, but also genes associated with angiogenic, neurotrophic, and immunomodulatory properties. Furthermore, using proteome profiling, we identify for the first time the dynamic release of various bioactive molecules, such as transcription and translation factors and osteogenic, growth, angiogenic, and neurotrophic factors from canine BMMSC conditioned medium. Importantly, the relevant genes were linked to their proteins as detected in the conditioned medium and further associated with angiogenic activity in chorioallantoic membrane (CAM) assay. In this way, we show that the canine BMMSC release a variety of bioactive molecules, revealing a strong paracrine component that may possess therapeutic potential in various pathologies. However, extensive experimental or preclinical trials testing canine sources need to be performed in order to better understand their paracrine action, which may lead to novel therapeutic strategies in veterinary medicine.
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Células Madre Mesenquimatosas/fisiología , Comunicación Paracrina , Proteínas/metabolismo , Adipogénesis/fisiología , Animales , Biomarcadores/metabolismo , Células de la Médula Ósea/citología , Diferenciación Celular/genética , Linaje de la Célula/fisiología , Embrión de Pollo , Membrana Corioalantoides/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Perros , Regulación de la Expresión Génica , Masculino , Células Madre Mesenquimatosas/metabolismo , Neovascularización Fisiológica/genética , Osteogénesis/fisiología , Proteómica/métodosRESUMEN
Protein kinase C (PKC) activation induces cellular reprogramming and differentiation in various cell models. Although many effectors of PKC physiological actions have been elucidated, the molecular mechanisms regulating oligodendrocyte differentiation after PKC activation are still unclear. Here, we applied a liquid chromatography-mass spectrometry (LC-MS/MS) approach to provide a comprehensive analysis of the proteome expression changes in the MO3.13 oligodendroglial cell line after PKC activation. Our findings suggest that multiple networks that communicate and coordinate with each other may finally determine the fate of MO3.13 cells, thus identifying a modular and functional biological structure. In this work, we provide a detailed description of these networks and their participating components and interactions. Such assembly allows perturbing each module, thus describing its physiological significance in the differentiation program. We applied this approach by targeting the Rho-associated protein kinase (ROCK) in PKC-activated cells. Overall, our findings provide a resource for elucidating the PKC-mediated network modules that contribute to a more robust knowledge of the molecular dynamics leading to this cell fate transition.
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Diferenciación Celular/fisiología , Oligodendroglía/citología , Oligodendroglía/metabolismo , Proteína Quinasa C/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Humanos , Espectrometría de Masas/métodos , Oligodendroglía/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Quinasas Asociadas a rho/metabolismoRESUMEN
The epithelial mesenchymal transition (EMT) program is defined as a cellular transition from an epithelial to a mesenchymal state. This process occurs to provide the cell with new phenotypic assets and new skills to perform complex processes. EMT is regulated at multilayer levels, including transcriptional control of gene expression, regulation of RNA splicing, and translational/post-translational control. Although transcriptional regulation by EMT-inducing transcription factors (EMT-TFs), including Zeb, Snail and Slug members, is generally considered the master step in this process, emerging data indicate that all these regulatory networks may have a role in the control of EMT. There is a sort of parallelism between the biological and still unrevealed EMT complexity and the cosmological hypothesis that sustains the universe may exist as a multiverse. The presence of different EMT transition states together with the occurrence of multiple layers of regulation support the idea that EMT is just one on many out there. Is the activation of a single layer of regulation sufficient to initiate the whole EMT program? Can we postulate the activation of different EMT "dimensions"? If we think about these layers as multiple separate "universes", various scenarios can be revealed.
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Células Epiteliales/patología , Transición Epitelial-Mesenquimal/genética , Animales , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Factores de Transcripción/genética , Transcripción Genética/genéticaRESUMEN
Large scale proteomic strategies rely on database interrogation. Thus, only referenced proteins can be identified. Recently, Alternative Proteins (AltProts) translated from nonannotated Alternative Open reading frame (AltORFs) were discovered using customized databases. Because of their small size which confers them peptide-like physicochemical properties, they are more difficult to detect using standard proteomics strategies. In this study, we tested different preparation workflows for improving the identification of AltProts in NCH82 human glioma cell line. The highest number of identified AltProts was achieved with RIPA buffer or boiling water extraction followed by acetic acid precipitation.
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Proteoma/análisis , Extracción en Fase Sólida/métodos , Flujo de Trabajo , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/química , Biomarcadores de Tumor/aislamiento & purificación , Línea Celular Tumoral , Cromatografía Liquida , Humanos , Peso Molecular , Proteoma/química , Proteoma/aislamiento & purificación , Proteómica/métodos , Reproducibilidad de los Resultados , Espectrometría de Masas en TándemRESUMEN
Spinal cord injury (SCI) often leads to irreversible neuro-degenerative changes with life-long consequences. While there is still no effective therapy available, the results of past research have led to improved quality of life for patients suffering from partial or permanent paralysis. In this review we focus on the need, importance and the scientific value of experimental animal models simulating SCI in humans. Furthermore, we highlight modern imaging tools determining the location and extent of spinal cord damage and their contribution to early diagnosis and selection of appropriate treatment. Finally, we focus on available cellular and acellular therapies and novel combinatory approaches with exosomes and active biomaterials. Here we discuss the efficacy and limitations of adult mesenchymal stem cells which can be derived from bone marrow, adipose tissue or umbilical cord blood and its Wharton's jelly. Special attention is paid to stem cell-derived exosomes and smart biomaterials due to their special properties as a delivery system for proteins, bioactive molecules or even genetic material.
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Modelos Animales de Enfermedad , Neuroimagen/métodos , Traumatismos de la Médula Espinal/diagnóstico por imagen , Traumatismos de la Médula Espinal/terapia , Animales , Humanos , Imagen por Resonancia Magnética/métodos , Procedimientos Neuroquirúrgicos/métodos , Tomografía de Emisión de Positrones/métodos , Traumatismos de la Médula Espinal/patología , Trasplante de Células Madre/métodos , Tomografía Computarizada por Rayos X/métodos , Resultado del TratamientoRESUMEN
Remote Infrared Matrix-Assisted Laser Desorption/Ionization (Remote IR-MALDI) system using tissue endogenous water as matrix was shown to enable in vivo real-time mass spectrometry analysis with minimal invasiveness. Initially the system was used to detect metabolites and lipids. Here, we demonstrate its capability to detect and analyze peptides and proteins. Very interestingly, the corresponding mass spectra show ESI-like charge state distribution, opening many applications for structural elucidation to be performed in real-time by Top-Down strategy. The charge states show no dependence toward laser wavelength or length of the transfer tube. Indeed, remote analysis can be performed 5 m away from the mass spectrometer without modification of spectra. On the contrary, addition of glycerol to water shift the charge state distributions toward even higher charge states. The desorption/ionization process is very soft, allowing to maintain protein conformation as in ESI. Observation of proteins and similar spectral features on tissue, when protein standards are deposited on raw tissue pieces, could potentially open the way to their direct analysis from biological samples. This also brings interesting features that could contribute to the understanding of IR MALDI ionization mechanism.
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Presión Atmosférica , Rayos Infrarrojos , Proteínas/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Animales , Concentración de Iones de Hidrógeno , Modelos Biológicos , Procesamiento de Señales Asistido por Computador , TemperaturaRESUMEN
Proteogenomics and ribosome profiling concurrently show that genes may code for both a large and one or more small proteins translated from annotated coding sequences (CDSs) and unannotated alternative open reading frames (named alternative ORFs or altORFs), respectively, but the stoichiometry between large and small proteins translated from a same gene is unknown. MIEF1, a gene recently identified as a dual-coding gene, harbors a CDS and a newly annotated and actively translated altORF located in the 5'UTR. Here, we use absolute quantification with stable isotope-labeled peptides and parallel reaction monitoring to determine levels of both proteins in two human cells lines and in human colon. We report that the main MIEF1 translational product is not the canonical 463 amino acid MiD51 protein but the small 70 amino acid alternative MiD51 protein (altMiD51). These results demonstrate the inadequacy of the single CDS concept and provide a strong argument for incorporating altORFs and small proteins in functional annotations.