Improved results in proteomics by use of local and peptide-class specific false discovery rates

<p>Abstract</p> <p>Background</p> <p>Proteomic protein identification results need to be compared across laboratories and platforms, and thus a reliable method is needed to estimate false discovery rates. The target-decoy strategy is a platform-independent and thus a prime candidate for standardized reporting of data. In its current usage based on global population parameters, the method does not utilize individual peptide scores optimally.</p> <p>Results</p> <p>Here we show that proteomic analyses largely benefit from using separate treatment of peptides matching to proteins alone or in groups based on locally estimated false discovery rates. Our implementation reduces the number of false positives and simultaneously increases the number of proteins identified. Importantly, single peptide identifications achieve defined confidence and the sequence coverage of proteins is optimized. As a result, we improve the number of proteins identified in a human serum analysis by 58% without compromising identification confidence.</p> <p>Conclusion</p> <p>We show that proteins can reliably be identified with a single peptide and the sequence coverage for multi-peptide proteins can be increased when using an improved estimation of false discovery rates.</p

Cross-Sample Validation Provides Enhanced Proteome Coverage in Rat Vocal Fold Mucosa

The vocal fold mucosa is a biomechanically unique tissue comprised of a densely cellular epithelium, superficial to an extracellular matrix (ECM)-rich lamina propria. Such ECM-rich tissues are challenging to analyze using proteomic assays, primarily due to extensive crosslinking and glycosylation of the majority of high Mr ECM proteins. In this study, we implemented an LC-MS/MS-based strategy to characterize the rat vocal fold mucosa proteome. Our sample preparation protocol successfully solubilized both proteins and certain high Mr glycoconjugates and resulted in the identification of hundreds of mucosal proteins. A straightforward approach to the treatment of protein identifications attributed to single peptide hits allowed the retention of potentially important low abundance identifications (validated by a cross-sample match and de novo interpretation of relevant spectra) while still eliminating potentially spurious identifications (global single peptide hits with no cross-sample match). The resulting vocal fold mucosa proteome was characterized by a wide range of cellular and extracellular proteins spanning 12 functional categories

Efeito de substratos no secretoma e subproteoma de células estaminais mesenquimais

Mestrado em Biologia Molecular e CelularMesenchymal stem cells (MSCs) are adult multipotent cells that possess self-renewal capacity, have a high proliferative ability and are able to differentiate into mesodermal cell types. MSCs can be isolated from mesenchymal and extraembryonic tissues such as the umbilical cord matrix. The latter constitutes an attractive and alternative source of MSCs since these cells seem to be more naïve and possess higher proliferation capacity. In vivo, MSCs reside in a specialized microenvironment that is essential for the regulation of signaling, proliferation, migration and differentiation. Through mechanotransduction, mechanical forces of microenvironment are transduced into biochemical responses, which can lead to alterations in phenotype and lineage-specific differentiation, and changes in protein synthesis of MSCs. Based on these observations, this study aimed at exploring the effect of physical and biochemical substrate composition on the secretome and cellular subproteomes of MSCs derived from umbilical cord matrix. The present study revealed the modulation of the secretome and cellular subproteome profiles (soluble and membrane fractions) of MSCs cultured on soft substrates, with several proteins being modulated, namely the up-regulation of antioxidant proteins. Hence, we propose that MSCs cultured on soft substrates may constitute a population of cells with increased antioxidant properties, in principle allowing the cells to cope better with the stressful and hostile environments that they may encounter in vivo in a transplantation context.As células estaminais mesenquimais (MSCs) são células estaminais adultas, multipotentes, capazes de se auto-renovar e diferenciar em diferentes tipos celulares mesodermais. O isolamento destas células pode ser feito a partir tecidos mesenquimais, bem como de extra embrionários, como por exemplo da matriz do cordão umbilical. Este último constitui uma fonte alternativa e atrativa de MSCs uma vez que estas apresentam características mais naïve e uma maior taxa de proliferação. In vivo, as células mesenquimais têm um microambiente especializado que é essencial à regulação da sinalização, proliferação, migração e diferenciação celular. Através da mecanotransdução, as forças mecânicas do ambiente extracelular são traduzidas em respostas bioquímicas que podem conduzir à alteração de fenótipo e diferenciação em diferentes tipos celulares e, além disso, à alteração da síntese de proteínas. A partir destas observações, este trabalho teve como objetivo o estudo do efeito das propriedades do substrato, tais como a rigidez e composição bioquímica, no secretoma e subproteomas de células estaminais mesenquimais isoladas a partir da matriz do cordão umbilical. Este estudo revelou uma modulação do secretoma e subproteomas celulares (frações solúvel e membranar) quando as MSCs foram mantidas em cultura sobre substratos com menor rigidez, demonstrando a modulação de múltiplas proteínas e nomeadamente o aumento dos níveis de proteínas anti-oxidantes. Deste modo, propomos que células cultivadas em substratos com menor rigidez possuam uma maior atividade anti-oxidante, o que irá permitir que estas apresentem uma melhor resposta face a ambientes hostis in vivo num contexto de transplantação

A Pathogen and a Non-pathogen Spotted Fever Group Rickettsia Trigger Differential Proteome Signatures in Macrophages

We have previously reported that Rickettsia conorii and Rickettsia montanensis have distinct intracellular fates within THP-1 macrophages, suggesting that the ability to proliferate within macrophages may be a distinguishable factor between pathogenic and non-pathogenic Spotted fever group (SFG) members. To start unraveling the molecular mechanisms underlying the capacity (or not) of SFG Rickettsia to establish their replicative niche in macrophages, we have herein used quantitative proteomics by SWATH-MS to profile the alterations resulted by the challenge of THP-1 macrophages with R. conorii and R. montanensis. We show that the pathogenic, R. conorii, and the non-pathogenic, R. montanensis, member of SFG Rickettsia trigger differential proteomic signatures in macrophage-like cells upon infection. R. conorii specifically induced the accumulation of several enzymes of the tricarboxylic acid cycle, oxidative phosphorylation, fatty acid β-oxidation, and glutaminolysis, as well as of several inner and outer membrane mitochondrial transporters. These results suggest a profound metabolic rewriting of macrophages by R. conorii toward a metabolic signature of an M2-like, anti-inflammatory activation program. Moreover, several subunits forming the proteasome and immunoproteasome are found in lower abundance upon infection with both rickettsial species, which may help bacteria to escape immune surveillance. R. conorii-infection specifically induced the accumulation of several host proteins implicated in protein processing and quality control in ER, suggesting that this pathogenic Rickettsia may be able to increase the ER protein folding capacity. This work reveals novel aspects of macrophage-Rickettsia interactions, expanding our knowledge of how pathogenic rickettsiae explore host cells to their advantage

Mesenchymal stem cells secretome in Parkinson’s disease regenerative medicine

Dissertação de mestrado em Ciências da SaúdeParkinson’s disease (PD) represents the second most common neurodegenerative brain disorder, which is clinically characterized by the progressive degeneration of dopaminergic neurons (DAergic neurons), mainly in the nigrostriatal pathway, leading to the appearance of characteristic motor and non-motor symptoms. Currently, pharmacological and surgical treatments are the most common approaches for the treatment of PD. However, so far, all of these treatments are focused on reducing the symptoms. In fact, they do not slow down or reverse the degenerative process, imposing the need for innovative therapeutical approaches. The use of adult stem cells cell-based strategy has emerged as a potential alternative therapy for PD, in which, among a number of promising stem cell sources, human mesenchymal stem cells (hMSCs) and neural progenitors cells (hNPCs) have stand out as a valid therapeutic option. Indeed, over the last years, a substantial effort has been performed in order to address the impact of hMSCs and hNPCs in central nervous system repair. Recently, and from an application point of view, several studies have claimed that the therapeutical effects of stem cells is mainly mediated by their trophic action namely, through their capacity of secreting a wide panel of neuroregulatory molecules (e.g. neurotrophic factors, cytokines, vesicles), which is defined as secretome. Thus, based in all these concepts, in this thesis we aimed to: 1) Characterize the secretome of hMSCs and hNPCs through proteomic-based approaches; 2) Determine the role of hMSCs and hNPCs secretome as a modulator of neuronal differentiation and 3) Investigate the effects of the hMSCs and hNPCs secretome in a rat model of PD, in comparison with cell transplantation. In vitro, experiments revealed that the secretome of hMSCs induced a more robust neuronal differentiation when compared to the one obtained from hNPCs. Additionally, it was also possible to observe that the injection of the secretome of both hMSCs and hNPCs in a 6-hydroxydopamine (6-OHDA)-rat model of PD potentiated the recovery of DAergic neurons (estimated by neuronal densities in substantia nigra and striatum) when compared to the untreated group 6-OHDA, and those transplanted with cells (hMSCs and hNPCs). Similar outcomes were observed in the motor performance of these animals as assessed by the rotarod and staircase tests. Finally, proteomic characterization of hMSCs and hNPCs secretome revealed that these cells were able to secrete important molecules with neuroregulatory actions such as, Galectin-1, 14-3-3 proteins, PEDF, DJ-1, whereby may support the effects observed both in vitro and in vivo. Overall, we concluded that the use of secretome per se was able to partially revert the motor phenotype and the neuronal structure of PD animals, indicating that the secretome of stem cells could represent a novel therapeutic tool for the treatment of PD.A doença de Parkinson (DP) é clinicamente caracterizada pela degeneração progressiva dos neurónios dopaminérgicos (ND), principalmente na via nigroestriatal, levando ao aparecimento dos sintomas motores e não motores da doença. Atualmente, os tratamentos farmacológicos e cirúrgicos representam a abordagem mais comum no tratamento da DP. Contudo, estes estão apenas focados na redução sintomática da doença, não retardando ou revertendo o processo degenerativo, sendo assim necessária a criação de abordagens terapêuticas inovadoras. O uso de células estaminais adultas tem emergido como uma potencial terapia alternativa para a DP. Dentro destas, as células humanas estaminais mesenquimatosas (hMSCs) e as células progenitoras neurais (hNPCs) têm emergido como uma válida opção terapêutica. Do ponto de vista de aplicação destas duas populações de células estaminais na DP, diversos estudos demonstraram que o seu efeito terapêutico é essencialmente mediado pela sua ação trófica, isto é, através da sua capacidade de segregar um vasto painel de moléculas neuroreguladoras (p.ex. fatores neurotróficos, citoquinas e vesículas), definido como secretoma. Assim, a presente tese teve como principais objetivos: 1) Caraterizar o secretoma de hMSCs e hNPCs através de análises de proteómica; 2) Determinar o efeito do secretoma de hMSCs e hNPCs como um modulador da diferenciação neuronal e 3) Investigar os efeitos do secretoma de hMSCs e hNPCs num modelo de rato da DP (6-OHDA), em comparação com a transplantação de células. In vitro, verificou–se uma maior diferenciação neuronal promovida pelo secretoma de hMSCs quando comparado com o das hNPCs. In vivo, observou-se que a injeção do secretoma quer de hMSCs quer de hNPCs num modelo de DP em ratos (6-OHDA) potenciou a recuperação dos ND (avaliado por densidades neuronais na substância negra e estriado) quando comparado com o grupo não tratado, e com os grupos transplantados com células (hMSCs e hNPCs). Resultados semelhantes foram observados no desempenho motor destes animais, avaliado pelos testes rotarod e staircase. Por último, a caracterização proteómica do secretoma de hMSCs e hNPCs revelou que estas células são capazes de segregar moléculas com importantes ações neuroreguladoras tais como, Galactina-1, proteínas 14-3-3, PEDF, DJ-1, suportando desta forma os efeitos observados tanto in vitro como in vivo. Em suma, podemos concluir que a utilização de secretoma por si só foi capaz de reverter parcialmente o fenótipo motor e a estrutura neuronal de animais parkinsonianos, indicando que o secretoma das células estaminais pode representar uma nova abordagem terapêutica para o tratamento da DP

Proteomic approach to the caracterization of unknown origin male infertility

Infertility affects around 50 million couples worldwide, where male infertility contributes for half of all cases. When no clear causesfor infertilitycanbe found, it is designated of unknown origin(UOMI), including idiopathic(ID)and unexplained male infertility (UMI). Diagnosis of male infertility isfirstlybased on routine semen analysis, an evaluationthat has been shown to havepoor prognostic value. This is especially evident in men with UMI that, despite presenting a normal seminal analysis, arestill infertile,stressing the need to deepen the analysis of sperm functionality in these patients to clarify what might be behind their infertility state.In this project,wefocusedon a detailed characterization of sperm function,that goes far beyondthe conventional analysis, to clarifythis issue. Besidesanalysing functional and bioenergetic parameters such as spermcapacitation, acrosome reaction, chromatin status and mitochondrial functionality, a sperm proteomic analysis was alsoperformedusing sequential window acquisition of all theoretical mass spectra (SWATH-MS). In addition, lifestyle and medical history of patients were assessed by survey. Symptomsof anxiety and depression were also evaluated through proper surveys.In this study, ID patients besides having significantly decreased sperm concentration, motility and morphology, also presented significantly decreased sperm viability, chromatin integrity and percentage of capacitated cells,comparing to healthy men, with the proteomic results further supporting the differences among these groups.Furthermore, ID patients had significantly increased incidence of urogenital infections and varicocele. Regarding UMI patients, we observedthat their sperm functionality is very similar to that of the healthy individuals, but significantly different from ID patients, whichwas also mirroredat the proteomic level.UMI patients were also observed to have increased incidence of diagnosed depression, when comparing to healthy individuals and ID patients. Finally, the proteins annexin A5, α-crystallin B chain, apolipoprotein H, destrin, NADH-cytochrome b5 reductase 3, platelet-activating factor acetylhydrolase IB subunit α1and transthyretinwere found, for the first time, to significantly differentiate the 3patient groups,hence being good candidates for further studies on UOMI.Overall, this study entailinga unique complete and integrated analysis of 3 groups of individuals’ sperm functionality and proteome, accuratelycategorized, providednew insights and add knowledge on these patients’ infertility unknown aetiology, opening road to future studies in the field

The immunome and embryo quality in sea bream and sea bass

Gilthead sea bream (Sparus aurata) and European sea bass (Dicentrarchus labrax) are teleosts belonging to Eupercaria and are the most important aquaculture fish species in the Mediterranean region. These two species are ranked second after the Atlantic salmon (Salmo salar) in production volume and value in the European Union (EU) aquaculture sector. Unpredictable fertilized egg/embryo quality and performance remain a bottleneck that threatens sustainability of sea bream and sea bass aquaculture, impeding the increased productivity of aquaculture that entirely depends on hatchery production. To address this issue, criteria and molecular markers linked to embryo quality that could be used to monitor and manage hatchery production were procured. Comparative molecular approaches using molecular biology, proteomics and transcriptomics were performed to analyze embryo performance and immunity in samples from several European commercial hatcheries. The core achievements were the: a) identification and characterization of lysozyme and complement 5 (C5) gene families and embryo and larval gene expression and enzyme activity from a diversity of hatcheries, b) characterization of the embryo proteome from three Mediterranean fish species [white sea bream (Diplodus sargus), meagre (Argyrosomus regius) and sea bream] 24h before hatch and at hatch and identification of common and species specific molecular patterns linked to biological function and putative quality-related proteins, c) comparative transcriptomics of good and poor quality sea bream embryos from several Mediterranean hatcheries. Qualitylinked transcripts and some elements of the regulatory epitranscriptome (non-coding RNA) were identified as well as the contribution of maternal proteins to embryos. Taken together, the results provide a comprehensive description of the molecular basis of sea bream and sea bass embryo development and reveal that immune-related molecules in fertilized eggs are low abundance. The development (quality)-related candidate markers identified will be of value for management of fish embryos in aquaculture hatcheries.O mar Mediterrâneo cobre apenas 0,7% da área oceânica mundial, mas é um dos principais reservatórios de biodiversidade marinha e costeira com cerca de 28% de espécies endémicas. A dourada (Sparus aurata) e o robalo (Dicentrarchus labrax), são duas espécies de peixe que pertencem à série Eupercaria e das mais comercializadas pela indústria de aquicultura nesta região. No sector da aquicultura da União Europeia (EU), estas duas espécies ocupam o segundo lugar no “ranking” da cadeia de valor, depois do salmão do Atlântico. Contudo, as suas características morfológicas e de crescimento são os parâmetros de qualidade relevantes considerados pela indústria. Esta abordagem deve-se à sua comercialização em formato de peixe inteiro, o que reduz o valor de mercado na cadeia de valor e também à ausência de critérios de qualidade para seleção de ovos e embriões, bem como, de marcadores moleculares de qualidade com maior grau de sensibilidade. Todos estes obstáculos, limitam substancialmente o desenvolvimento das indústrias associadas á comercialização destas duas espécies de peixe, impedindo a expansão da aquicultura e das “hatcheries” (maternidades incubadoras de ovos/embriões controlados artificialmente para fins comerciais). Para mitigar este problema, utilizou-se técnicas de biologia molecular e tecnologias ómicas e estabeleceu-se uma abordagem comparativa direcionada á descoberta de moléculas e vias metabólicas funcionais de importância crítica para o sistema imune dos peixes associada á “performance” de desenvolvimento de ovos e embriões. Esta tese está organizada em seis capítulos. Inicia-se com uma visão geral dos critérios morfológicos, físico-químicos e moleculares existentes para avaliar a qualidade de ovos e embriões para melhorar a gestão da aquicultura de peixes (Capítulo 1). Subsequentemente, caracterizou-se a família de genes do sistema imune: a) a das lisozimas em peixes teleósteos com enfase na sua caracterização molecular e funcional em dourada (Capítulo 2) e análises moleculares estruturais e evolutivas e b) a do complemento C5 (C5) em peixes especialmente em espécies da faamilia Cyprinidae (Capítulo 3). Os capítulos 4 e 5, integram abordagens de proteómica e transcriptómica em espécies de peixes mediterrânicos [pargo (Diplodus sargus), corvina (Argyrosomus regius) e dourada], focando os processos de desenvolvimento e de eclosão e na função da enzima “hatching enzyme” em dourada e robalo. Foi feita uma associação entre os capítulos e a análise integrada dos dados do transcritoma do embrião (Capítulo 5) revelou um padrão de expressão significativamente diferente (p-valor < 0,05) para o C5 (Capítulo 3) em diferentes lotes de embriões de dourada nas comparações entre graus de qualidade (Boa vrs Má) e entre estágios de desenvolvimento (Pré- eclosão vrs Eclosão). A variação do C5 em relação ao lote de embriões não foi afetada pela origem da “hatchery”, indicando que as prática de manejo ou os próprios reprodutores não influenciam a sua expressão. Os resultados sugerem que este gene e o seu produto proteico, são provavelmente importantes na proteção imunológica precoce e também em outras funções ainda não descritas na dourada ou em outras espécies de peixes. Também a integração dos resultados do proteoma (Capítulo 4) e do transcritoma (Capítulo 5) do embrião de dourada nos mesmos estágios de desenvolvimento, identificou um grupo de proteínas que se especula serem de origem materna. No último capítulo, sumarizou-se os resultados e são apresentadas perspetivas baseadas nos avanços e desafios atuais e propostas para o desenvolvimento de uma ferramenta integrada de monitorização da qualidade dos embriões e uma base biológica do desenvolvimento de ovos e embriões de peixes (Capítulo 6). Neste projeto foram: 1) identificadas duas importantes famílias de genes associadas à imunidade inata em peixes, a das lisozimas e a do C5. Caracterizou-se pela primeira vez a função das lisozimas através da sua expressão e atividade enzimática em embriões e em diferentes estágios larvares de uma diversidade de reprodutores de dourada. Estudou-se a função do C5 em peixes, através da construção de redes génicas, modelação por homologia e “docking” molecular entre o C5 e o seu receptor (C5R/CD88); 2) mapeou-se e caracterizou-se o proteoma do embrião de três espécies de peixes mediterrânicos (sargo, corvina e dourada) em duas fases do seu desenvolvimento (24h antes da eclosão e na eclosão) e identificou-se um grupo de proteínas potencialmente relacionadas com a imunidade e a qualidade dos embriões. Avaliou-se a função do gene para enzima “hatching enzyme”, com base na sua expressão em embriões de dourada e robalo nos estágios acima referidos; 3) mapeou-se e caracterizou-se o transcritoma de embriões de dourada com origem em diferentes “hatcheries” na região mediterrânica através de uma abordagem comparativa entre qualidade e estágios de desenvolvimento (qualidade- Boa vrs Má; estágios- Pré- eclosão vrs Eclosão) e identificou-se uma diversidade de transcritos, vias metabólicas e elementos do epitranscriptoma regulatório do RNA-não codificante. Foram identificados em comum 42 candidatos a marcadores de qualidade e enriquecidas duas vias metabólicas relacionadas com o sistema imunológico e associadas às “hatcheries”: a via de infeção por Salmonella (constituída por 7 genes relacionados com o sistema imune) e a via de sinalização MAPK (mitogen-activated protein kinase). Foram identificadas 543 proteínas que são expressas apenas no proteoma, sugerindo que podem ter origem materna e destas, 7 (diferencialmente expressas) estão potencialmente relacionadas com o sistema imune. Globalmente, os resultados forneceram um grupo de marcadores relacionados à imunidade e ao desenvolvimento (qualidade), com potencial de se traduzirem em critérios de qualidade de ovos e embriões para a indústria da aquicultura. Estes resultados, foram amplamente estudados para descrever a base molecular biológica entre os diferentes estágios de desenvolvimento de ovos e embriões de peixes e também, entre diferentes lotes de embriões de diferente qualidade. As ferramentas biológicas e critérios desenvolvidos neste trabalho, oferecem uma orientação para as “hatcheries” de peixes e a sua aplicação contribuirá para melhorar no futuro o sector da aquicultura.O apoio financeiro ao trabalho relatado na presente tese de doutoramento é reconhecido com gratidão, pois foi crucial para o progresso positivo e sucesso do trabalho científico. Uma diversidade de fontes de financiamento apoiou o trabalho desenvolvido nesta tese, quer diretamente através da compra de consumíveis/materiais (PerformFISH a European Union’s Horizon 2020 research and innovation grant, agreement Nº 727610), quer indiretamente através do financiamento do CCMAR de apoio a serviços e equipamentos utilizados durante a execução do trabalho (projetos da Fundação para a Ciência e Tecnologia (FCT) - UIDB/04326/2020, UIDP/04326/2020 e dos programas operacionais CRESC Algarve 2020 e COMPETE 2020 através do projeto EMBRC.PT ALG-01-0145-FEDER-022121)

The role and regulation of nuclear myosins in DNA damage

Unconventional myosins have often been characterised within the cytoplasm of the cell, however little work has been done to characterise their role within the nucleus. The first nuclear myosin was identified within the 1990s and since then eight types of myosin have been found within the nucleus. The first to be found and the one myosin that has been heavily characterised is nuclear myosin 1. This myosin has a role in transcription, as well as chromatin movement after DNA damage. Myosin VI another myosin recently found in the nucleus has also been attributed to stable transcription, and, like nuclear myosin 1 can also bind to the RNA polymerase II complex. So far, a full length structure of nuclear myosin I is yet to be defined, here in this thesis, using typical biochemical techniques, an interaction between the N-terminus of the protein and the nuclear localisation sequence has been identified, along with disassociation constants, that finally show a direct interaction between DNA and the myosin. As well as this, MVI has had another role in the nucleus, which is observed after the induction of double strand breaks within DNA. This thesis discusses why MVI is vital for double strand break signalling, as cellular biology techniques have shown the lack of γH2AX signalling the motor domain of MVI is inhibited. Not only is a working MVI necessary for when signalling DNA damage, this thesis has also found direct interactions with histones and their modifiers. As both nuclear myosins are involved in transcription, this thesis has looked at how these two myosins interact, and looks further into their nuclear import, roles in the DNA damage response, and how they are regulated within these roles

Expanding the immune self : impact of non-canonical translation on the repertoire of MHC I-associated peptides

Les molécules du complexe majeur d’histocompatibilité de classe I (MHC I) sont des glycoprotéines de surface exprimées par la majorité des cellules nucléés de notre organisme. Ces molécules servent à exposer une vue intégrative de l’état interne de nos cellules (soi immunitaire) via la présentation de courts peptides (MAPs) générés lors de la dégradation des protéines cytosoliques par le protéasome. Le répertoire des MAPs de chaque cellule, véritable carte d’identité peptidique, est constamment passé en revue par nos lymphocytes T CD8+, cellules centrales du système immunitaire, afin de débusquer et d’éliminer toute cellule anormale, e.g., celles présentant des MAPs d’origine virale ou tumorale (TSAs). Au vu du nombre grandissant d’articles démontrant que des ARNs autres que les ARNs messagers peuvent être traduits, nous avons décidé d’évaluer l’impact de ces mécanismes de traduction non-canonique sur le répertoire des MAPs présentés par des cellules B. En développant une approche protéogénomique, i.e., combinant spectrométrie de masse et séquençage d’ARN à haut-débit, nous avons pu démontrer qu’environ 10 % des MAPs présentés par nos cellules B dérivent d’évènements de traduction non-canonique incluant (i) la traduction d’ARNs messagers dans un cadre de lecture alternatif ou (ii) la traduction de régions ou ARNs supposés non-codants. L’analyse subséquente des caractéristiques de ces MAPs dits « cryptiques » suggère que leur biogenèse diffère de celle des MAPs conventionnels, les MAPs cryptiques étant principalement encodés par des ARNs instables produisant de courtes protéines dont la dégradation ne semble pas exiger l’intervention du protéasome. Sachant que la déméthylation globale du génome des cellules cancéreuses permet l’expression d’un plus grand bassin d’ARNs non-codants, nous avons supposé que ces cellules pourraient présenter de nombreux MAPs (et TSAs) cryptiques. En adaptant notre approche protéogénomique, nous avons pu analyser le répertoire des MAPs de cellules cancéreuses, incluant celui de deux lignées tumorales de souris (EL4 et CT26) et sept échantillons primaires humains (quatre leucémies aigues lymphoblastiques B et trois biopsies de cancer du poumon). Cette analyse nous a permis de découvrir qu’environ 90% des TSAs sont des TSAs cryptiques. Ayant observé que la plupart de ces TSAs dérivent de séquences normales dont l’expression est restreinte aux cellules tumorales, comme les retroéléments endogènes, il est plausible que ces TSAs soient partagés par plusieurs patients. Enfin, nos études chez la souris nous ont permis de démontrer qu’au moins deux facteurs influencent positivement le potentiel protectif d’un TSA in vivo : l’expression de cet antigène par les cellules cancéreuses et la fréquence des lymphocytes T capables de le reconnaître. En conclusion, le recours à la protéogénomique pour analyser les MAPs présentés par les cellules normales et cancéreuses nous a permis de démontrer que les MAPs cryptiques contribuent significativement au bassin de peptides constituant le soi immunitaire et qu’ils permettent aux lymphocytes T CD8+ d’effectuer une surveillance immunitaire plus efficace.On their surface, nucleated cells present major histocompatibility complex class I (MHC I) molecules in complex with short peptides, that we will refer to as MHC I-associated peptides (MAPs). These MAPs derive from the degradation of cytosolic proteins by the proteasome and provide an integrative view of the inner state of cells to CD8+ T cells, which can, in turn, eliminate abnormal cells, e.g., those presenting viral MAPs or tumor-specific antigens (TSAs). With the growing body of evidence suggesting that translation does occur outside of protein-coding transcripts, we tried to evaluate the impact of non-canonical translation on the repertoire of MAPs. Combining RNA-sequencing and mass spectrometry to analyze the MAP repertoire of B-lymphoblastoid cell lines, we uncovered that ~ 10 % of the MAP repertoire derives from such non-canonical translation events, including (i) the out-of-frame translation of protein-coding transcripts or (ii) the translation of non-coding regions (UTRs, introns, etc.) or transcripts (antisense, pseudogene, etc.). Interestingly, our data suggest that the biogenesis of cryptic and conventional MAPs differs, as cryptic MAPs derive from unstable transcripts generating short proteins that might be degraded in a proteasome-independent fashion. Because the global DNA hypomethylation observed in cancer cells tend to de-repress non-coding transcripts, we developed another proteogenomic approach to probe the cryptic MAP repertoire of two murine cancer cell lines (EL4 and CT26) and seven humor primary tumor samples (four B-lineage acute lymphoblastic leukemias and three lung tumor biopsies). This second analysis revealed that ~ 90% of TSAs are cryptic TSAs. Interestingly, most of those TSAs derived from cancer-restricted yet non-mutated sequences, such as endogenous retroelements, thereby suggesting that such TSAs could be shared between patients. Lastly, our validation study in mice demonstrated that at least two parameters can influence the in vivo protective effect of TSAs, namely TSA expression in cancer cells and the frequency of TSA-specific T cells. Altogether, our proteogenomic studies on the MAP repertoire of normal and cancer cells demonstrate that cryptic MAPs significantly expand the immune self and, consequently, the scope of CD8+ T cell immunosurveillance