Dissertations / Theses on the topic 'Comprehensive proteomics'

Reversible protein phosphorylation, the best studied post-translational modification, regulates most cellular processes, including signaling, migration, cell cycle progression, DNA damage repair, stress response and modulaton of the activities of metabolic enzymes. Therefore, it has emerged as a key therapeutic target in diseases in which these processes are deregulated. Unlike kinases, protein phosphatase 1 (PP1) is a promiscuous enzyme that gains its substrate specificity from a large group of “regulatory subunits” with which it associates to form a range of holoenzyme complexes targeted to specific subcellular localizations and substrates. Inhibition of a specific dephosphorylation event therefore relies on targeting the regulatory rather than the catalytic subunit. The present study uses GFP as a molecular reporter to assess the localization of PP1c and identify the underlying binding events that govern it via a combination of fluorescence imaging, cellular fractionation, affinity purification and quantitative mass spectrometry. While there is some overlap in their targeting and intracellular roles, the three PP1 isoforms show distinct localizations based on relative preferences for particular regulatory subunits. In this study we assembled a comprehensive map of isoform- and compartment- specific phosphatase complexes in three different cultured human cell lines, using the data to extrapolate, with confidence, the distribution of each PP1 isoform between a large pool of known/predicted and novel regulatory subunits. Network analysis also highlighted key multiprotein complexes to which PP1 is targeted by these regulatory subunits, and identified a novel regulatory subunit that links phosphatase activity to regulation of protein degradation. Our work confirmed that Mypt1, the regulatory subunit that targets PP1 activity to the myosin light chain, preferentially associates with the beta isoform of PP1c. We further demonstrated that they are in complex in both the cytoplasm and nucleus, and represent ~30% of the total PP1β holoenzyme complexes in both interaphase and mitotic cells. Further investigation of these complexes led to the discovery of Specc1 and Specc1L, which associate with Mypt1/PP1β via direct binding to Mypt1. Specc1/1L are microtubule binding proteins that can also associate with actin filaments, and we demonstrated that they mediate the distribution of Mypt1/PP1β complexes between these two cytoskeletal networks. Given that disruption of this balance has been implicated in disease states including cancer and hypertension, the Specc1/L family represents a novel therapeutic target for the regulation of Mypt1/PP1 activity. With PP1 now emerging as a promising therapeutic target and the first PP1-targeted therapeutic drug, Sephin 1, in clinical trials, a better understanding of PP1’s in vivo distribution between holoenzyme complexes is essential. Our work establishes an initial “snapshot” of this distribution against which changes can be assessed, as we demonstrated here by showing its re-distribution in mitotic cells. Dynamic redistributions during specific cell processes such as differentiation or in response to perturbations or disease states can be assessed in a similar fashion in future, facilitating both identification of the relevant complexes and the design of specific strategies to target them therapeutically.

The plant endoplasmic reticulum is the location of storage oil and membrane lipid assembly, and for fatty acid modifying reactions (desaturation, elongation, hydroxylation). It therefore represents a source of enzymes involved in these processes. Many of these defy traditional purification strategies. In this study, ER membranes have been isolated biochemically pure and in milligram quanties from the endosperm of developing and germinating castor bean. One-dimensional SDS- PAGE, used to routinely assess sample integrity, showed resolution limitations. Two-dimensional gel electrophoresis was optimized regarding sample preparation and solubilization, and reproducible profiles confirmed its suitability as a sound basis for analysis of stage-specific ER components. In large format 2-D experiments, preparative loadings were reproducibly resolved. MALDI TOP mass spectrometry was evaluated for high throughput peptide signature generation with individual ER components. Resolution problems were again highlighted with 1-D separations, although some functional assignments were made. Subsequently analysis of selected spots from a preparative 2-D gel of germinating ER was used to establish the limitations of the procedure. Database matching of a single component at very low levels of mass error tolerance also demonstrated the power and accuracy of the technology. Membranes were subfractionated to simplify protein patterns. It is proposed that an organellar approach, including subfractionation, provides enrichment of specific subsets of cellular components. A putative plant phosphatidic acid phosphatase gene has been investigated following identification from the EST database. The aim of this research is the identification of proteins involved in storage lipid synthesis in castor bean in reactions specific to the endoplasmic reticulum.

Bacillus amyloliquefaciens FZB42 ist ein frei lebendes Bakterium, das Pflanzenwurzeln besiedelt und das Pflanzenwachstum durch viele verschiedene Wirkmechanismen anregt. In dieser Arbeit wurden die molekularen Grundlagen dieser positiven Wirkungen, die dieses „Pflanzenwachstum fördernde Rhizobakterium“ (PGPR) auf seine Wirte ausübt, untersucht. Um den gegenseitigen Austausch von B. amyloliquefaciens und seinen Wirtspflanzen zu entschlüsseln, wurden umfangreiche Proteomstudien durchgeführt. Es wurden Referenzkarten der extrazellulären und zytosolischen Proteinfraktionen erstellt. Die größte Anzahl an ausgeschiedenen Proteinen konnte während der stationären Phase beobachtet werden. Die identifizierten extrazellulären Proteine gehören verschiedenen Funktionsklassen an, wobei die prominentesten Klassen am Kohlenhydrat-Abbau und den Transport von Molekülen durch die Zellwand beteiligt sind. Die zytosolischen Extrakte von Kulturen, die in 1C-Medium bzw. Mineralmedium angezogen wurden, und in der zweidimensionalen Gelelektrophorese (2 DE) aufgetrennt wurden, ergaben 461 und 245 verschiedene Protein-Einträge. Die erstellten Referenz-Karten wurden anschließend verwendet, um Proteine und Prozesse, in an der Interaktion mit Pflanzen beteiligt sind, zu identifizieren. Dafür wurden die Bakterien Wurzelexudaten von Mais (Zea mays L.) ausgesetzt. Die Proteine aus zwei Stämmen, denen die globalen Transkriptionsregulatoren (Degu, AbrB) und vier Sigma-Faktoren (SigB, SigM, SigV, und SigX) fehlen, wurden ebenfalls untersucht, um ihre Beteiligung an den bakteriellen Reaktionen auf die Wurzelausscheidungen zu analysieren. Zusammenfassend ist dies die erste Studie, die umfangreiche Proteomdaten von Gram-positiven PGPR präsentiert, wobei gleichzeitig die Veränderung der Expression von extrazellulären und zytoplasmatischen Proteinen, nach Zugabe von Wurzelexudaten, ausgewertet wurde.
Bacillus amyloliquefaciens strain FZB42 is a free-living bacterium that competitively colonizes plant roots and stimulates plant growth by many different modes of action. The molecular basis of singular beneficial effects that this Plant Growth-Promoting Rhizobacteria (PGPR) exert on their hosts have been studied. To decipher the molecular cross-talk of B. amyloliquefaciens and its’ host plants as a whole system, an extensive proteomic approach was performed. Reference maps of the extracellular and cytosolic protein fractions were established. The highest number of secreted proteins was observed during stationary growth phase. Identified extracellular proteins belong to different functional classes, with the most prominent classes involved in carbohydrate degradation and transportation of molecules across the cell wall. Cytosolic extracts obtained from cultures grown in 1C and minimal media subjected to the 2 Dimensional Electrophoresis (2 DE), revealed 461 and 245 different protein entries, respectively. Created reference maps were subsequently used to identify proteins and processes involved in the interaction with plants, prior to exposure of bacteria to maize (Zea mays L.) root exudates. The proteomics of two strains lacking expression of genes coding for global transcriptional regulators (degU, abrB) and four sigma factors (sigB, sigM, sigV, and sigX) were also inves-tigated, in order to analyse their involvement in bacterial responses to root exudates. In summary, this is the first study presenting comprehensive proteomics of Gram-positive PGPR, evaluating at the same time changes in protein expression caused by addition of root exudates at the extracellular and cytosolic level.

Tato práce je zaměřena jak na cílený tak na přehledný přístup ve studiu proteomiky. Cílená proteomika přináší informace o přítomnosti proteinu a jeho lokalizaci v buňce či tkáni pomocí luminiscenčních značek na bázi kvantových teček, zatímco přehledná proteomika se zabývá identifikací změn v proteomu dvou nebo více jedinců stejného druhu vystavených různým podmínkám. Protože proteomika vyžaduje vysoce citlivé separační a identifikační techniky, byly v této práci ověřeny různé metody zlepšení citlivosti kapilární elektroforézy s hmotnostní detekcí. Použití rozhraní s kapalinovým spojem pro spojení těchto dvou technik, které zajišťuje vyšší citlivost analýz, bylo také ověřeno analýzou metabolitů etanolu a kokainu v lidské moči. Zavedené techniky instrumentace jsou využitelné při posouzení vlivu významných faktorů životního prostředí na živé systémy jak na buněčné tak na molekulární úrovni.

Malignant tumors in children and adolescents are one of the leading causes of death from disease in this population despite being rare events. The main liver tumor in children is Hepatoblastoma (HB) representing two thirds of the total while pediatric Hepatocellular Carcinoma (pHCC) is rarer than HB and is usually diagnosed in older patients. Patient survival at 5 years is higher than 75% for HB and less than 30% for pHCC. At molecular level, 2 different subclasses of HBs have been described based on a 16-gene signature, C1 and C2, being the latest more aggressive. Nowadays patient stratification is based only in clinical and pathological parameters. For this reason, the identification of prognostic markers easy to apply at the clinical practice is mandatory in order to better stratify patients and diminish the side effects of chemotherapy treatment, moving towards a more personalized medicine. The proteomic profile of 16HBs and 8 paired normal liver (NL) tissue was obtained by 2 different techniques, two-dimensional gel electrophoresis and label-free LC-MS. The differential expressed proteins were validated by western blot (WB) in the same patients and the final protein signature was validated by immunohistochemistry in additional 144 patients. Furthermore, RNA sequencing and copy number variation analysis were performed in 31 HB samples, 11 patient-derived xenografts (PDXs) and 5 pHCC. Results were validated by Sanger sequencing, droplet digital PCR or real time PCR and correlated with clinical features. Two hundred and thirty proteins were identified as deregulated in aggressive C2 tumors and 8 of them were selected and validated by WB considering also their expression in NL. After WB, 2 proteins were found significantly upregulated in C2 tumors while 1 was downregulated. A score was calculated for each protein and biomarkers 1 and 2 were considered altered when their staining was at least 2-fold higher than the NL while the biomarker 3 was considered as altered when no staining was observed. The 3-protein signature was defined by the number of altered biomarkers in each tumor and was highly correlated with patient survival and complementary to the current clinical stratification. RNA-sequencing data revealed that fusion proteins are rare events in HB as they were found in only 2% of patients. Mutational analysis allowed us to identify mutations in CTNNB1 (30%), NFE2L2 (7%) and EPHB4 (7%). Interestingly we identified a downregulation of the RNA editing which is correlated with patient outcome. The copy number variation analysis showed that gains are more frequent than losses in HB tumors and that PDXs maintain 78% of the aberrations, being a good model for the study of HB. In contrast, pHCC are characterized by more aberrations than HB and mainly losses. Thus, we stablished a molecular classification that includes 3 HB types: stable (no big CNVs), gains-enriched and losses-enriched classes. This classification is correlated with event-free survival, CTNNB1 mutations and the expression of stem cell markers. As a result of this thesis, we stablished a 3-protein signature that could be easily applied at the clinical practice and increased the molecular knowledge of childhood liver tumors.
Els tumors malignes en nens i adolescents són una de les principals causes de mort per malaltia en aquesta població malgrat ser poc freqüents. El principal tumor de fetge en la infància és l’Hepatoblastoma (HB) mentre que el Carcinoma Hepatocel·lular pediàtric (pHCC) és menys freqüent i normalment es diagnostica en pacients més grans. La supervivència als 5 anys és superior al 75% per l’HB i menor del 30% pel pHCC. A nivell molecular, s’han descrit 2 subclasses d’HB en base a una signatura de 16 gens, C1 i C2, essent la segona més agressiva. Actualment l’estratificació dels pacients es basa només en paràmetres clínics i patològics. Per aquesta raó, la identificació de marcadors pronòstic que siguin fàcilment aplicables a la pràctica clínica és imprescindible per a una millor estratificació els pacients per tal de disminuir els efectes secundaris de la quimioteràpia, avançant cap a una medicina personalitzada. Es va estudiar el perfil proteòmic de 16 HBs i 8 teixits no tumorals (NL) mitjançant 2 tècniques, l’electroforesi bidimensional amb fluorescència i una tècnica sense marcatge LC-MS. Les proteïnes amb expressió diferencial es van validar per western blot (WB) en els mateixos pacients i la signatura final es va validar per immunohistoquímica en 144 pacients. A més, es va realitzar seqüenciació de RNA i un array genòmic en 31HBs, 11 xenògrafts derivats de pacients (PDXs) i 5 pHCC. Els resultats es van validar per seqüenciació Sanger, droplet digital PCR o PCR a temps real i correlacionar amb característiques clíniques. Es van identificar 230 proteïnes desregulades en els tumors agressius C2 i 8 d’aquestes es van seleccionar per ser validades per WB considerant també la seva expressió en NL. Els resultats del WB van confirmar que 2 de les proteïnes estaven sobreexpressades en els tumors C2 mentre que una d’elles estava infraexpressada. Es va calcular una puntuació per cada proteïna, de manera que els biomarcadors 1 i 2 es consideren desregulats si la seva expressió és superior a 2 vegades l’expressió del NL mentre que BM3 es considera alterat si no es detecta expressió. La firma de 3 proteïnes, que es va definir com el número de biomarcadors alterats, estava fortament correlacionada amb la supervivència i era complementària a l’actual estratificació clínica. Les dades de seqüenciació de RNA van revelar que les proteïnes de fusió són poc freqüents en HB i l’anàlisi de mutacions ens va permetre identificar mutacions en CTNNB1 (30%), NFE2L2 (7%) i EPHB4 (7%). A més, vam identificar una infra-regulació del mecanisme d’edició del RNA correlacionat amb el pronòstic. L’anàlisi genòmic va mostrar que els guanys cromosòmics són més freqüents que les pèrdues en l’HB i que els PDXs mantenen un 78% de les alteracions, representant un bon model per a l’estudi del HB. Per contra, el pHCC presenta més alteracions que l’HB i majoritàriament pèrdues. Finalment, vam establir una classificació molecular que inclou 3 classes d’HB: estable, enriquida en guanys i enriquida en pèrdues. Aquesta classificació està correlacionada amb la supervivència, mutacions de CTNNB1 i expressió de marcadors de cèl·lules progenitores. Amb aquesta tesi, hem establert una signatura de 3 proteïnes fàcilment aplicable a la pràctica clínica i augmentat el coneixement molecular dels tumors hepàtics pediàtrics.

Presented in this dissertation are proteomic analysis studies focused on identifying proteins to be used as vaccine candidates against Coccidioidomycosis, a potentially fatal human pulmonary disease caused by inhalation of a spore from the soil-dwelling pathogenic fungi Coccidioides posadasii and C. immitis. A method of tandem mass spectrometry data analysis using dual protein sequence search algorithms for increasing the total protein identifications from an analysis is described. This method was utilized in a comprehensive proteomic analysis of cell walls isolated from the dimorphic fungal pathogen C. posadasii. A strategy of tandem mass spectrometry-based protein identification coupled with bioinformatic sequence analysis was used to produce a list of protein vaccine candidates for further testing. A differential proteome analysis using stable isotope protein labeling was undertaken to identify vaccine candidate proteins that are more highly expressed in the spherule, or pathogenic phase, of C. posadasii. The results of these analyses are 9 previously undescribed protein vaccine candidates isolated from spherule cell walls that have sequence indications of extracellular association such as GPI anchors and N-terminal signal sequences and antigen potential based on homology to known antigenic or secreted proteins. An additional 14 proteins identified from spherule cell walls are potential vaccine candidates based on extracellular sequence predictions without any indications of antigenic potential. The stable isotope labeling study has identified 3 more proteins that are preferentially expressed in spherules and exhibit antigenic potential based on extracellular localization or homology to known antigenic proteins. Additionally, there were 5 unknown function proteins identified by stable isotope labeling that are more highly expressed in spherules that may be good vaccine candidates but cannot be identified or localized by sequence analysis.The dual algorithm protein identification method presented here is a new technique to address some common shortcomings associated with a proteomic analysis. The comprehensive proteomic analyses of Coccidioides posadasii presented here have provided new targets for Coccidioidomycosis vaccine development as well as insights into the proteome of this pathogen, such as the sequence comparison of C.posadasii proteins to human proteins, as well as a comprehensive analysis of predicted protein function in the Coccidioides proteome.

Includes bibliographical references.
Tuberculosis is an airborne infectious disease caused by the bacillus known as Mycobacterium tuberculosis. Despite limited genetic variability, Mycobacterium tuberculosis strains exhibit vast discrepancies in phenotypic presentation in terms of virulence, elicited immune response and transmissibility. This study aims to use Mass Spectrometry (MS) tools to quantitatively and qualitatively investigate the total proteome expressed by various epidemiologically significant strains within the Mycobacterium tuberculosis complex (MTBC) as well as a clinically relevant non-tuberculous Mycobacteria (NTM) strain when cultured in vitro. We aim to use the experimental data obtained using discovery mass spectrometry to identify candidate proteins to use in the design of multiple reaction monitoring (MRM) MS experiments for targeted biomarker validation in patient derived biological samples such as sputum. Liquid chromatography mass spectrometry (LC MS/MS) and data capture were carried out using the LTQ Orbitrap Velos. 1D LC was carried out on gel fractionated samples to increase proteome coverage. This allowed a significant increase in the number of protein identifications of up to 80% proteome coverage per strain. Comparative analysis of the datasets was carried out to identify and define the core-proteome expressed across all strains as well as to identify differentially expressed proteins amongst the strains.

The outstanding material properties of spider dragline silk fibers have been attributed to two spidroins, MaSp1 and MaSp2. Although dragline silk fibers have been treated with different chemical solvents to elucidate the relationship between protein structure and fiber mechanics, there has not been a comprehensive proteomic analysis of the major ampullate (MA) gland, its spinning dope, and dragline silk using a wide range of chaotropic agents, inorganic salts, and fluorinated alcohols to elucidate their complete molecular constituents. In these studies, we perform in-solution tryptic digestions of solubilized MA glands, spinning dope and dragline silk fibers using 5 different solvents, followed by nanoLC-MS/MS analysis with an Orbitrap Fusion™ Tribrid™ mass spectrometer. To improve protein identification, we employed three different tryptic peptide fragmentation modes, which included CID, HCD, and ETD to discover proteins involved in the silk assembly pathway and silk fiber. In addition to MaSp1 and MaSp2, we confirmed the presence of a third spidroin AcSp1 in dragline silk, a spidroin widely recognized as the major constituent of wrapping silk. Our findings also reveal that MA glands, spinning dope, and dragline silk contain at least 7 common proteins: major ampullate spidroin 1 and 2 (MaSp1 and MaSp2), 3 members of the Cysteine-Rich Protein Family (CRP1, CRP2 and CRP4), and two uncharacterized proteins, CRISP3 and fasciclin. In summary, this study provides a proteomic blueprint to construct synthetic silk fibers that most closely mimic natural fibers

Dissertação de Mestrado Integrado em Engenharia Biomédica apresentada à Faculdade de Ciências e Tecnologia
Parkinson's disease (PD) is a common neurodegenerative disorder associated with motor and cognitive impairments. It is characterized by dopaminergic neurodegeneration in the nigrostriatal budle, which has repercussions to other brain regions producing the motor (parkinsonism) and non-motor symptomatology characteristic of PD. Research efforts have been directed toward understanding the etiology and pathogenesis of PD in the hope of developing a more effective therapy that will slow or halt the natural progression of PD. In this sense, study of structural, as well as, functional modifications in the different brain areas is fundamental. A similar dopaminergic neuronal loss in the substantia nigra (SN) and motor symptoms can be achieved on intracerebral administration of the neurotoxin 6-hydroxydopamine (6-OHDA). In order to gain a better understanding of the molecular changes relevant to PD, it was performed a comparatively analysis of the proteome changes in four brain regions (cortex, cerebellum, hippocampus and striatum) using a 6-OHDA-induced PD rat model with the objective of: i) identifying region specific protein abundance changes; ii) identifying which major pathways are altered by those proteins and at the same time are characteristic of the pathology; and iii) evaluating the viability of 6-OHDA model to “mimic” PD characteristics and identification of new potential mechanisms associated with the disease. To achieve these goals, SWATH-MS analysis was performed for quantitative comparison of the four tissues referred above between control animals (Sham group) and PD animals (6-OHDA group). It was possible to quantify 1998, 2573, 2277, 1790 proteins for cortex, cerebellum, hippocampus and striatum, respectively. From those proteins, a total of 202, 341, 477 and 183 proteins in cortex, cerebellum, hippocampus and striatum, respectively, passed the quality filters and statistical tests (50% alteration). Bioinformatics tools allowed an overall characterization of the identified proteins for pathway enrichment and expression profiles. From the altered pathways observed in clustering analysis, eight main pathways were analysed due its involvement in PD pathologic mechanisms: Parkinson’s disease, oxidative phosphorylation, calcium signalling, synaptic vesicle cycle, dopaminergic synapses, gluthathione metabolism, protein processing in endoplasmatic reticulum and PI3k/Akt signalling. Altogether, these findings highlight several pathways affected in PD that present molecular changes in this model, confirming the validity of this model to study molecular alterations associated with PD. In the future, more studies need to be performed in order to confirm the alteration at the protein expression level identified in this work, and to clarify the potential function or dysfunction induced by the alterations observed in this screening.
A doença de Parkinson (PD) é uma desordem neurodegenerativa comum associada a impedimentos cognitivos e motores. É caracterizada pela neurodegeneração dopaminérgica da via nigrostriatal, tendo repercussões em outras zonas do cérebro, produzindo assim a sintomatologia motora (parkinsonismo) e não motora, que caracterizam a PD. A sua investigação tem feito esforços para tentar entender a etiologia e patogénese da PD na esperança de desenvolver uma terapia mais eficiente que possa abrandar ou parar a progressão natural da PD. Neste sentido, o estudo das mudanças estruturais, assim como funcionais nas diferentes áreas do cérebro é fundamental. Através da administração intracerebral da neurotoxina 6-hidroxidopamina (6-OHDA) é possível atingir uma perda dopaminérgica neuronal na substantia nigra (SN) e sintomas motores similares. Para atingir um melhor entendimento das mudanças moleculares relevantes à PD, foi executada uma análise comparativa das alterações no proteoma em quatro zonas do cérebro (córtex, cerebelo, hipocampo e estriado) utilizando um modelo de Parkinson induzido pelo 6-OHDA em ratos com o objectivo de: i) identificar mudanças de abundancia de proteínas em regiões específicas; ii) identificar que vias principais são alteradas por essas proteínas e ao mesmo tempo são características da patologia; e iii) avaliar a capacidade do modelo 6-OHDA de simular a PD, para poder entender a sua viabilidade de ser utilizado como um modelo PD e para identificação de novos potenciais mecanismos associados com a doença. Para atingir estes objetivos, foi utilizada uma análise SWATH-MS para comparação quantitativa dos quatro tecidos referidos acima entre animais controlo (grupo SHAM) e animais PD (grupo 6-OHDA). Foi possível quantificar 1998, 2573, 2277 e 1790 proteínas para o córtex, cerebelo, hipocampo e estriado, respetivamente. Dessas proteínas, um total de 202, 341, 477 e 183 no córtex, cerebelo, hipocampo e estriado respetivamente, passaram os filtros de qualidade e testes estatísticos (alteração de 50%) e foram consideradas como sendo alteradas entre os dois grupos. As ferramentas bioinformáticas utilizadas permitiram-nos uma caracterização geral das proteínas identificadas para análise das vias e perfis de expressão. Das vias alteradas observadas na análise cluster, foram analisadas oito vias principais devido ao seu envolvimento com os mecanismos patológicos da PD: doença de Parkinson, fosforilação oxidativa, sinalização de cálcio, ciclo das vesiculas sinápticas, sinapses dopaminérgicas, metabolismo glutatione, processamento de proteínas no reticulo endoplasmático e sinalização Pl3k/Akt. Em conjunto, estas descobertas evidenciam várias vias afectadas na PD que estão presentes neste modelo, confirmando a validade deste modelo para estudar alterações moleculares associadas com a PD. No futuro, terão de ser executados mais estudos para que se possa confirmar a alteração a nível de proteínas identificada neste trabalho, assim com para clarificar a potencial função ou disfunção induzida pelas alterações observadas neste estudo.
Outro - This work was financed by the European Regional Development Fund (ERDF) through the COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation and Portuguese national funds via FCT – Fundação para a Ciência e a Tecnologia, I.P., OE FCT/MCTES (PIDDAC) under projects: POCI-01-0145-FEDER-029311, POCI-01-0145-FEDER-007440 (strategic project UID/NEU/04539/2019), POCI-01-0145-FEDER-016428 (ref.: SAICTPAC/0010/2015), and POCI-01-0145-FEDER-016795 (ref.: PTDC/NEU-SCC/7051/2014); POCI-01-0145-FEDER-029311 (ref.: PTDC/BTM-TEC/29311/2017); POCI-01-0145-FEDER-30943 (ref.: PTDC/MEC-PSQ/30943/2017); PTDC/MED-NEU/27946/2017; and by The National Mass Spectrometry Network (RNEM) under the contract POCI-01-0145-FEDER-402-022125 (ref.: ROTEIRO/0028/2013)

Le long bio-polymère d'ADN est condensé à l’intérieur du noyau des cellules eukaryotes à l'aide de petites protéines appelées histones. En plus de leurs fonctions condensatrices,ces histones sont également la cible de nombreuses modifications post-traductionnelles(MPT), particulièrement au niveau de leur section N-terminale. Ces modifications réversibles font partie d’un code d’histones épi-génétique transmissible qui orchestre et module dynamiquement certains événements impliquant la chromatine, tels l’activation et la désactivation de gènes ainsi que la duplication et la réparation d’ADN. Ces modifications sont impliquées subséquemment dans la signalisation et la progression de cancers, tels que la leucémie. En conséquence, l'élucidation des modifications d’histones est importante pour comprendre leurs fonctions biologiques. Une méthodologie analytique a été mise au point en laboratoire pour isoler, détecter, et quantifier les MPT d’histones en utilisant une approche rapide à deux volets à l’aide d’outils bioinformatiques spécialisés. La méthodologie développée en laboratoire a été validée en utilisant des histones de souche sauvage ainsi que deux types d’histones mutants déficients en enzymes acétyltransferase. Des trois sources d’histones utilisées, la seule MPT qui a démontré un changement significatif est l’acétylation de l’histone H3 à lysine 56 (H3K56ac). L’expression et la stoechiométrie de cette MPT, issue de cellules de souche sauvage et de cellules mutantes, ont été déterminées avec précision et comparées. Les fonctions de balayage polyvalentes d'un instrument à trappe ionique quadrupôle linéaire hybride ont été utilisées pour améliorer la détection de protéines intactes. Le mode de balayage « enhanced multiply charged » (EMC) a été modifié pour contenir et détecter les ions de protéines intactes situées dans la trappe ionique linéaire. Ce mode de balayage nommé « targeted EMC » (tEMC) a permis de quadrupler le niveau de sensibilité (signal/interférence), et quintupler la résolution du mode de balayage conventionnel. De plus, la capacité de séparation des charges du tEMC a réduit de façon significative les effets de « space charge » dans la trappe ionique linéaire. La résolution supérieure du mode tEMC a permis de différencier plusieurs isoformes modifiées, particulièrement pour l’histone H3. L’analyse des peptides d’histones trypsiques à l’aide du mode de balayage « MRM » a permis le séquençage et la quantification de MPT avec un haut degré de précision. La seule MPT qui était sous-exprimée entre l’histone de souche sauvage et le mutant DOT1L fut la méthylation de l’histone H3 lysine 79(H3K79me1). Les effets de deux inhibiteurs d’enzymes HDAC (HDACi) sur l’expression de MPT d’histone ont été évalués en utilisant la méthodologie analytique mentionnée. Les histones extraites de cellules normales et cancéreuses ont été exposées à du Vorinostat(SAHA) ou du Entinostat (MS-275) pour une période de 24 à 72 heures. Deux histones furent principalement affectées, soit H3 et H4. Étonnamment, les mêmes effets n'ont pas été détectés lorsque les cellules normales ont été traitées avec le HDACi pour une période de 48 à 72 heures. Une méthode absolue de quantification avec une courbe d’étalonnage a été développée pour le peptide H3K56ac. Contrairement à certaines publications, nos résultats démontrent que cette MPT est présente dans les cellules mammifères avec une stoechiométrie très basse (< 0,1%) et n'est pas surexprimée de façon significative après le traitement au HDACi.
In eukaryotic cells, the lengthy DNA biopolymer is condensed into the cell nucleus with the aid of small packaging proteins called histones. In addition to their packing functions,histones are also targets of numerous post translational modifications (PTMs), especially on their N-terminus. These reversible modifications are believed to be constituents of a heritable epigenetic “histone code” that dynamically orchestrate and modulate chromatin based events such as gene activation and silencing, DNA replication and repair, and are also involved in the downstream signaling and progression of cancers, such as leukemia. Thus, the elucidation of histone PTMs is important in understanding their biological function. An analytical workflow was designed and set-up in the laboratory to isolate, detect, and quantitate histone PTM, using a two-pronged, unbiased, and rapid approach with specialized bioinformatic tools. The workflow was validated using histones from wildtype, and 2 mutants deficient in acetyltransferase activity. Between the three histone sources, the only PTM that demonstrated any change was acetylation at histone H3 lysine 56 (H3K56ac). The down-regulation and stoichiometry of this PTM was accurately assessed between wild-type and mutant cells. The versatile scan functions of a hybrid quadrupole-linear ion trap instrument were exploited to enhance the detection of intact histone proteins. The enhanced multiply charged (EMC) scan was modified in order to contain and detect intact protein ions within the linear ion trap. This targeted EMC (or tEMC) resulted in not only a 4-fold increase in signal-to-noise, but also a 5-fold increase in resolution. Furthermore, the charge separation capability of the tEMC dramatically reduced space charge effects within the linear ion trap. The superior resolution of the tEMC mode allowed for the discimination of many modified histone isoforms, especially for histone H3. Using the bottom-up strategy with multiple reaction monitoring (MRM), histone peptides were quantified and sequenced with a high degree of precision. The only PTM that was down-regulated between wild-type and DOT1L mutant histones was methylation at histone H3 lysine 79 (H3K79me1). The effects of two clinically relevant small molecule HDAC inhibitors (HDACi) on histone PTMs patterns were assessed using the analytical workflow developed. Histones derived from both normal and cancer cells were exposed to either Vorinostat (SAHA) or Entinostat (MS-275) over a 24- to 72 hour period. The two core histones primarily affected were H3 and H4. Surprisingly, the same effects were not observed when normal cells were treated with three doses of SAHA at 24-hour intervals over a 72-hour period. An absolute quantitation method using a calibration curve was developed for H3K56ac. In opposition to other published literature, our findings demonstrate that this PTM is present in very low stoichiometry (< 0.1%) in mammalian cells, and exhibits no significant up-regulation in different cell lines treated with several types of HDACi.