Fallopian tube lesions as potential precursors of early ovarian cancer: a comprehensive proteomic analysis.

Ovarian cancer is the leading cause of death from gynecologic cancer worldwide. High-grade serous carcinoma (HGSC) is the most common and deadliest subtype of ovarian cancer. While the origin of ovarian tumors is still debated, it has been suggested that HGSC originates from cells in the fallopian tube epithelium (FTE), specifically the epithelial cells in the region of the tubal-peritoneal junction. Three main lesions, p53 signatures, STILs, and STICs, have been defined based on the immunohistochemistry (IHC) pattern of p53 and Ki67 markers and the architectural alterations of the cells, using the Sectioning and Extensively Examining the Fimbriated End Protocol. In this study, we performed an in-depth proteomic analysis of these pre-neoplastic epithelial lesions guided by mass spectrometry imaging and IHC. We evaluated specific markers related to each preneoplastic lesion. The study identified specific lesion markers, such as CAVIN1, Emilin2, and FBLN5. We also used SpiderMass technology to perform a lipidomic analysis and identified the specific presence of specific lipids signature including dietary Fatty acids precursors in lesions. Our study provides new insights into the molecular mechanisms underlying the progression of ovarian cancer and confirms the fimbria origin of HGSC.

Heimdall, an alternative protein issued from a ncRNA related to kappa light chain variable region of immunoglobulins from astrocytes: a new player in neural proteome.

The dogma "One gene, one protein" is clearly obsolete since cells use alternative splicing and generate multiple transcripts which are translated into protein isoforms, but also use alternative translation initiation sites (TISs) and termination sites on a given transcript. Alternative open reading frames for individual transcripts give proteins originate from the 5'- and 3'-UTR mRNA regions, frameshifts of mRNA ORFs or from non-coding RNAs. Longtime considered as non-coding, recent in-silico translation prediction methods enriched the protein databases allowing the identification of new target structures that have not been identified previously. To gain insight into the role of these newly identified alternative proteins in the regulation of cellular functions, it is crucial to assess their dynamic modulation within a framework of altered physiological modifications such as experimental spinal cord injury (SCI). Here, we carried out a longitudinal proteomic study on rat SCI from 12 h to 10 days. Based on the alternative protein predictions, it was possible to identify a plethora of newly predicted protein hits. Among these proteins, some presented a special interest due to high homology with variable chain regions of immunoglobulins. We focus our interest on the one related to Kappa variable light chains which is similarly highly produced by B cells in the Bence jones disease, but here expressed in astrocytes. This protein, name Heimdall is an Intrinsically disordered protein which is secreted under inflammatory conditions. Immunoprecipitation experiments showed that the Heimdall interactome contained proteins related to astrocyte fate keepers such as "NOTCH1, EPHA3, IPO13" as well as membrane receptor protein including "CHRNA9; TGFBR, EPHB6, and TRAM". However, when Heimdall protein was neutralized utilizing a specific antibody or its gene knocked out by CRISPR-Cas9, sprouting elongations were observed in the corresponding astrocytes. Interestingly, depolarization assays and intracellular calcium measurements in Heimdall KO, established a depolarization effect on astrocyte membranes KO cells were more likely that the one found in neuroprogenitors. Proteomic analyses performed under injury conditions or under lipopolysaccharides (LPS) stimulation, revealed the expression of neuronal factors, stem cell proteins, proliferation, and neurogenesis of astrocyte convertor factors such as EPHA4, NOTCH2, SLIT3, SEMA3F, suggesting a role of Heimdall could regulate astrocytic fate. Taken together, Heimdall could be a novel member of the gatekeeping astrocyte-to-neuroprogenitor conversion factors.

Protocol to identify human subcellular alternative protein interactions using cross-linking mass spectrometry.

Since the start of mass-spectrometry-based proteomics, proteins from non-referenced open reading frames or alternative proteins (AltProts) have been overlooked. Here, we present a protocol to identify human subcellular AltProt and decipher some interactions using cross-linking mass spectrometry. We describe steps for cell culture, in cellulo cross-link, subcellular extraction, and sequential digestion. We then detail both liquid chromatography-tandem mass spectrometry and cross-link data analyses. The implementation of a single workflow allows the non-targeted identification of signaling pathways involving AltProts. For complete details on the use and execution of this protocol, please refer to Garcia-del Rio et al.1.

Employing non-targeted interactomics approach and subcellular fractionation to increase our understanding of the ghost proteome.

Eukaryotic mRNA has long been considered monocistronic, but nowadays, alternative proteins (AltProts) challenge this tenet. The alternative or ghost proteome has largely been neglected and the involvement of AltProts in biological processes. Here, we used subcellular fractionation to increase the information about AltProts and facilitate the detection of protein-protein interactions by the identification of crosslinked peptides. In total, 112 unique AltProts were identified, and we were able to identify 220 crosslinks without peptide enrichment. Among these, 16 crosslinks between AltProts and Referenced Proteins (RefProts) were identified. We further focused on specific examples such as the interaction between IP_2292176 (AltFAM227B) and HLA-B, in which this protein could be a potential new immunopeptide, and the interactions between HIST1H4F and several AltProts which can play a role in mRNA transcription. Thanks to the study of the interactome and the localization of AltProts, we can reveal more of the importance of the ghost proteome.

Neurotrauma investigation through spatial omics guided by mass spectrometry imaging: Target identification and clinical applications.

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.

Spatial analysis of the glioblastoma proteome reveals specific molecular signatures and markers of survival.

Molecular heterogeneity is a key feature of glioblastoma that impedes patient stratification and leads to large discrepancies in mean patient survival. Here, we analyze a cohort of 96 glioblastoma patients with survival ranging from a few months to over 4 years. 46 tumors are analyzed by mass spectrometry-based spatially-resolved proteomics guided by mass spectrometry imaging. Integration of protein expression and clinical information highlights three molecular groups associated with immune, neurogenesis, and tumorigenesis signatures with high intra-tumoral heterogeneity. Furthermore, a set of proteins originating from reference and alternative ORFs is found to be statistically significant based on patient survival times. Among these proteins, a 5-protein signature is associated with survival. The expression of these 5 proteins is validated by immunofluorescence on an additional cohort of 50 patients. Overall, our work characterizes distinct molecular regions within glioblastoma tissues based on protein expression, which may help guide glioblastoma prognosis and improve current glioblastoma classification.

Surfaceome Proteomic of Glioblastoma Revealed Potential Targets for Immunotherapy.

Glioblastoma (GBM) is the most common and devastating malignant brain tumor in adults. The mortality rate is very high despite different treatments. New therapeutic targets are therefore highly needed. Cell-surface proteins represent attractive targets due to their accessibility, their involvement in essential signaling pathways, and their dysregulated expression in cancer. Moreover, they are potential targets for CAR-based immunotherapy or mRNA vaccine strategies. In this context, we investigated the GBM-associated surfaceome by comparing it to astrocytes cell line surfaceome to identify new specific targets for GBM. For this purpose, biotinylation of cell surface proteins has been carried out in GBM and astrocytes cell lines. Biotinylated proteins were purified on streptavidin beads and analyzed by shotgun proteomics. Cell surface proteins were identified with Cell Surface Proteins Atlas (CSPA) and Gene Ontology enrichment. Among all the surface proteins identified in the different cell lines we have confirmed the expression of 66 of these in patient's glioblastoma using spatial proteomic guided by MALDI-mass spectrometry. Moreover, 87 surface proteins overexpressed or exclusive in GBM cell lines have been identified. Among these, we found 11 specific potential targets for GBM including 5 mutated proteins such as RELL1, CYBA, EGFR, and MHC I proteins. Matching with drugs and clinical trials databases revealed that 7 proteins were druggable and under evaluation, 3 proteins have no known drug interaction yet and none of them are the mutated form of the identified proteins. Taken together, we discovered potential targets for immune therapy strategies in GBM.

In-depth proteomics analysis of sentinel lymph nodes from individuals with endometrial cancer.

Endometrial cancer (EC) is one of the most common gynecological cancers worldwide. Sentinel lymph node (SLN) status could be a major prognostic factor in evaluation of EC, but several prospective studies need to be performed. Here we report an in-depth proteomics analysis showing significant variations in the SLN protein landscape in EC. We show that SLNs are correlated to each tumor grade, which strengthens evidence of SLN involvement in EC. A few proteins are overexpressed specifically at each EC tumor grade and in the corresponding SLN. These proteins, which are significantly variable in both locations, should be considered potential markers of overall survival. Five major proteins for EC and SLN (PRSS3, PTX3, ASS1, ALDH2, and ANXA1) were identified in large-scale proteomics and validated by immunohistochemistry. This study improves stratification and diagnosis of individuals with EC as a result of proteomics profiling of SLNs.

Protein Kinase C Activation Drives a Differentiation Program in an Oligodendroglial Precursor Model through the Modulation of Specific Biological Networks.

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.

Path to Clonal Theranostics in Luminal Breast Cancers.

Integrating tumor heterogeneity in the drug discovery process is a key challenge to tackle breast cancer resistance. Identifying protein targets for functionally distinct tumor clones is particularly important to tailor therapy to the heterogeneous tumor subpopulations and achieve clonal theranostics. For this purpose, we performed an unsupervised, label-free, spatially resolved shotgun proteomics guided by MALDI mass spectrometry imaging (MSI) on 124 selected tumor clonal areas from early luminal breast cancers, tumor stroma, and breast cancer metastases. 2868 proteins were identified. The main protein classes found in the clonal proteome dataset were enzymes, cytoskeletal proteins, membrane-traffic, translational or scaffold proteins, or transporters. As a comparison, gene-specific transcriptional regulators, chromatin related proteins or transmembrane signal receptor were more abundant in the TCGA dataset. Moreover, 26 mutated proteins have been identified. Similarly, expanding the search to alternative proteins databases retrieved 126 alternative proteins in the clonal proteome dataset. Most of these alternative proteins were coded mainly from non-coding RNA. To fully understand the molecular information brought by our approach and its relevance to drug target discovery, the clonal proteomic dataset was further compared to the TCGA breast cancer database and two transcriptomic panels, BC360 (nanoString®) and CDx (Foundation One®). We retrieved 139 pathways in the clonal proteome dataset. Only 55% of these pathways were also present in the TCGA dataset, 68% in BC360 and 50% in CDx. Seven of these pathways have been suggested as candidate for drug targeting, 22 have been associated with breast cancer in experimental or clinical reports, the remaining 19 pathways have been understudied in breast cancer. Among the anticancer drugs, 35 drugs matched uniquely with the clonal proteome dataset, with only 7 of them already approved in breast cancer. The number of target and drug interactions with non-anticancer drugs (such as agents targeting the cardiovascular system, metabolism, the musculoskeletal or the nervous systems) was higher in the clonal proteome dataset (540 interactions) compared to TCGA (83 interactions), BC360 (419 interactions), or CDx (172 interactions). Many of the protein targets identified and drugs screened were clinically relevant to breast cancer and are in clinical trials. Thus, we described the non-redundant knowledge brought by this clone-tailored approach compared to TCGA or transcriptomic panels, the targetable proteins identified in the clonal proteome dataset, and the potential of this approach for drug discovery and repurposing through drug interactions with antineoplastic agents and non-anticancer drugs.

Unveiling a Ghost Proteome in the Glioblastoma Non-Coding RNAs.

Glioblastoma is the most common brain cancer in adults. Nevertheless, the median survival time is 15 months, if treated with at least a near total resection and followed by radiotherapy in association with temozolomide. In glioblastoma (GBM), variations of non-coding ribonucleic acid (ncRNA) expression have been demonstrated in tumor processes, especially in the regulation of major signaling pathways. Moreover, many ncRNAs present in their sequences an Open Reading Frame (ORF) allowing their translations into proteins, so-called alternative proteins (AltProt) and constituting the "ghost proteome." This neglected world in GBM has been shown to be implicated in protein-protein interaction (PPI) with reference proteins (RefProt) reflecting involvement in signaling pathways linked to cellular mobility and transfer RNA regulation. More recently, clinical studies have revealed that AltProt is also involved in the patient's survival and bad prognosis. We thus propose to review the ncRNAs involved in GBM and highlight their function in the disease.

Shedding Light on the Ghost Proteome.

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.

SARS-Cov-2 Interactome with Human Ghost Proteome: A Neglected World Encompassing a Wealth of Biological Data.

Conventionally, eukaryotic mRNAs were thought to be monocistronic, leading to the translation of a single protein. However, large-scale proteomics have led to a massive identification of proteins translated from mRNAs of alternative ORF (AltORFs), in addition to the predicted proteins issued from the reference ORF or from ncRNAs. These alternative proteins (AltProts) 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 context. Based on our experience with AltProts, we were interested in finding out their interaction with the viral protein coming from the SARS-CoV-2 virus, responsible for the 2020 COVID-19 outbreak. Thus, we have scrutinized the recently published data by Krogan and coworkers (2020) on the SARS-CoV-2 interactome with host cells by affinity purification in co-immunoprecipitation (co-IP) in the perspective of drug repurposing. The initial work revealed the interaction between 332 human cellular reference proteins (RefProts) with the 27 viral proteins. Re-interrogation of this data using 23 viral targets and including AltProts, followed by enrichment of the interaction networks, leads to identify 218 RefProts (in common to initial study), plus 56 AltProts involved in 93 interactions. This demonstrates the necessity to take into account the ghost proteome for discovering new therapeutic targets, and establish new therapeutic strategies. Missing the ghost proteome in the drug metabolism and pharmacokinetic (DMPK) drug development pipeline will certainly be a major limitation to the establishment of efficient therapies.

Epigenetic Studies Revealed a Ghost Proteome in PC1/3 KD Macrophages under Antitumoral Resistance Induced by IL-10.

Our previous investigation on macrophages has allowed us to show that the inhibition of the enzyme proprotein convertase (PC1/3) controls the activation of macrophages. We demonstrated that PC1/3 knockdown (KD) in macrophages exhibits an increased secretion of proinflammatory and antitumoral factors. In this biological context, we assessed the presence of histone modifications and the presence and contribution of a "ghost proteome" in these macrophages. We identified a set of alternative proteins (AltProts) that have a key role in the regulation of various signaling pathways. In this study, to further investigate the underlying mechanisms involved in the resistance of PC1/3-KD macrophages to anti-inflammatory stimuli, we have conducted a proteomic system biology study to assess the epigenome variation, focusing on histone modifications. Results from our study have indicated the presence of significant variations in histone modifications along with the identification of 28 AltProts, which can be correlated with antitumoral resistance under IL-10 stimulation. These findings highlight a key role of altered epigenome histone modifications in driving resistance and indicate that like the reference proteins, AltProts can have a major impact in the field of epigenetics and regulation of gene expression, as shown in our results.

Reference and Ghost Proteins Identification in Rat C6 Glioma Extracellular Vesicles.

Extracellular vesicles (EVs) mediate intercellular communication and regulate a broad range of biological processes. Novel therapeutic strategies have emerged based on the use of EVs as biological nanoparticles. To separate isolated EVs from protein aggregates and the external part of EVs membrane proteins, we performed a Trypsin/Lys C digestion treatment of EVs pellets, followed by Amicon filtration. After these steps, all the fractions have been subjected to proteomic analyses. Comparison between 6 h Trypsin/Lys C treatment or non-treated EVs revealed a quantitative variation of the surface proteins. Some surface proteins have been demasked after 6 h enzymatic digestion like CD81, CD82, Ust, Vcan, Lamp 1, Rab43, Annexin A2, Synthenin, and VSP37b. Moreover, six ghost proteins have also been identified and one corresponds to a long noncoding RNA. We thus demonstrate the presence of ghost proteins in EVs produced by glioma cells that can contribute to tumorigenesis.

Alternative proteins are functional regulators in cell reprogramming by PKA activation.

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.

Optimized Sample Preparation Workflow for Improved Identification of Ghost Proteins.

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.

Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging of Lipids in Experimental Model of Traumatic Brain Injury Detecting Acylcarnitines as Injury Related Markers.

Identifying new lipid markers linked to traumatic brain injury (TBI) is of major importance in characterizing their central role in the regeneration process and inflammatory response in such an injury model. In the present study, an advanced lipidomics analysis using high spectral resolution matrix-assisted laser desorption/ionization-mass spectrometry imaging was performed on different brain regions in an experimental rat model of moderate controlled cortical impact (CCI) while considering different time points (1 day, 3 days, 7 days, and 10 days) assessing the acute and subacute phase after injury. Our results revealed a new family of lipids, the acylcarnitines, as TBI-lipid related markers, with maximum expression at 3 days after impact and main colocalization within resident microglia of the brain. Furthermore, our experiments highlighted the upregulation of these acylcarnitine lipids, secreted by microglia, in the ipsilateral substantia nigra, the main region in the brain affected in Parkinson's disease (PD).

Distinct Protein Expression Networks are Activated in Microglia Cells after Stimulation with IFN-γ and IL-4.

Microglia cells are the primary immune population of the central nervous system with a role in the regulation of several physiological and pathological conditions. Upon appropriate stimulation, microglia cells can be polarized in a pro-inflammatory M1-like or anti-inflammatory M2-like status. Biological processes and pathways engaged in microglia polarization are starting to be elucidated. To help clarify this, we used a liquid chromatography-mass spectrometry (LC-MS/MS) label free approach to characterize the proteomic profile of human microglia cell line (CHME-5) stimulated with gamma-interferon (IFN-γ) and interleukin-4 (IL-4) to induce a M1 or M2 phenotype, respectively. Outside the classical M1/M2 polarization markers, the M1 status appears to center around the activation of a classical inflammatory response and through the activation of multiple signaling pathways. M2 polarization resulted in a different pattern of protein modulation related to RNA and cellular metabolic processes. Together, our findings provide information regarding the protein changes specific to M1 and M2 activation states, and potentially link the polarization of microglia cells to the acquisition of a specific proteomic profile.