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[EN] BACKGROUND & AIMS: Cholangiocarcinoma (CCA) comprises a heterogeneous group of malignant tumors associated with dismal prognosis. Alterations in post-translational modifications (PTMs), including NEDDylation, result in abnormal protein dynamics, cell disturbances and disease. Herein, we investigate the role of NEDDylation in CCA development and progression. METHODS: Levels and functions of NEDDylation, together with response to pevonedistat (NEDDylation inhibitor) or CRISPR/Cas9 against NAE1 were evaluated invitro, invivo and/or in patients with CCA. The development of preneoplastic lesions in Nae1+/- mice was investigated using an oncogene-driven CCA model. The impact of NEDDylation in CCA cells on tumor-stroma crosstalk was assessed using CCA-derived cancer-associated fibroblasts (CAFs). Proteomic analyses were carried out by mass-spectrometry. RESULTS: The NEDDylation machinery was found overexpressed and overactivated in human CCA cells and tumors. Most NEDDylated proteins found upregulated in CCA cells, after NEDD8-immunoprecipitation and further proteomics, participate in the cell cycle, proliferation or survival. Genetic (CRISPR/Cas9-NAE1) and pharmacological (pevonedistat) inhibition of NEDDylation reduced CCA cell proliferation and impeded colony formation invitro. NEDDylation depletion (pevonedistat or Nae1+/- mice) halted tumorigenesis in subcutaneous, orthotopic, and oncogene-driven models of CCA invivo. Moreover, pevonedistat potentiated chemotherapy-induced cell death in CCA cells invitro. Mechanistically, impaired NEDDylation triggered the accumulation of both cullin RING ligase and NEDD8 substrates, inducing DNA damage and cell cycle arrest. Furthermore, impaired NEDDylation in CCA cells reduced the secretion of proteins involved in fibroblast activation, angiogenesis, and oncogenic pathways, ultimately hampering CAF proliferation and migration. CONCLUSION: Aberrant protein NEDDylation contributes to cholangiocarcinogenesis by promoting cell survival and proliferation. Moreover, NEDDylation impacts the CCA-stroma crosstalk. Inhibition of NEDDylation with pevonedistat may represent a potential therapeutic strategy for patients with CCA. LAY SUMMARY: Little is known about the role of post-translational modifications of proteins in cholangiocarcinoma development and progression. Herein, we show that protein NEDDylation is upregulated and hyperactivated in cholangiocarcinoma, promoting tumor growth. Pharmacological inhibition of NEDDylation halts cholangiocarcinogenesis and could be an effective therapeutic strategy to tackle these tumors. This article is based upon work from the COST Action CA18122 European Cholangiocarcinoma Network supported by COST (European Cooperation in Science and Technology: www.cost.eu).

Introduction: Borrelia burgdorferi sensu lato, the causative agents of Lyme borreliosis, are transmitted by Ixodes ticks. Tick saliva proteins are instrumental for survival of both the vector and spirochete and have been investigated as targets for vaccine targeting the vector. In Europe, the main vector for Lyme borreliosis is Ixodes ricinus, which predominantly transmits Borrelia afzelii. We here investigated the differential production of I. ricinus tick saliva proteins in response to feeding and B. afzelii infection. Method: Label-free Quantitative Proteomics and Progenesis QI software was used to identify, compare, and select tick salivary gland proteins differentially produced during tick feeding and in response to B. afzelii infection. Tick saliva proteins were selected for validation, recombinantly expressed and used in both mouse and guinea pig vaccination and tick-challenge studies. Results: We identified 870 I. ricinus proteins from which 68 were overrepresented upon 24-hours of feeding and B. afzelii infection. Selected tick proteins were successfully validated by confirming their expression at the RNA and native protein level in independent tick pools. When used in a recombinant vaccine formulation, these tick proteins significantly reduced the post-engorgement weights of I. ricinus nymphs in two experimental animal models. Despite the reduced ability of ticks to feed on vaccinated animals, we observed efficient transmission of B. afzelii to the murine host. Conclusion: Using quantitative proteomics, we identified differential protein production in I. ricinus salivary glands in response to B. afzelii infection and different feeding conditions. These results provide novel insights into the process of I. ricinus feeding and B. afzelii transmission and revealed novel candidates for an anti-tick vaccine.

The purpose of this study was to analyze the proteomic composition of plasma rich in growth factors eye drops (PRGF) in comparison to lyophilized PRGF eye drops (PRGF lyo). The differential protein expression of keratocyte (HK) cells after PRGF or PRGF lyo treatment was also determined. Blood from different donors was collected and processed to obtain PRGF and PRGF lyo eye drops. Then, HK cells were treated with both formulations. A proteomic analysis was performed to evaluate the differential proteomic profile between PRGF and PRGF lyo, and the differential protein expression by HK cells after treatment with both blood-derived products. About 280 proteins were detected between both blood-derived formulation, with only 8 of them reaching significant differences. Furthermore, 101 out of 3213 proteins showed statistically significant deregulation in HK cells after treatment with PRGF or PRGF lyo. Gene Ontology analysis showed that these significant deregulated proteins were involved in 30 functional processes. However, the Ingenuity Pathway Analysis showed that no significant differences were found in any of the identified processes. In summary, the present study show that no significant differences were found in the proteomic profile or in the signaling pathways activation in HK cells between PRGF and PRGF lyo.

[EN] Calcium ions are used in the development of biomaterials for the promotion of coagulation, bone regeneration, and implant osseointegration. Upon implantation, the time-dependent release of calcium ions from titanium implant surfaces modifies the physicochemical characteristics at the implant-tissue interface and thus, the biological responses. The aim of this study is to examine how the dynamics of protein adsorption on these surfaces change over time. Titanium discs with and without Ca were incubated with human serum for 2 min, 180 min, and 960 min. The layer of proteins attached to the surface was characterised using nLC-MS/MS. The adsorption kinetics was different between materials, revealing an increased adsorption of proteins associated with coagulation and immune responses prior to Ca release. Implant-blood contact experiments confirmed the strong coagulatory effect for Ca surfaces. We employed primary human alveolar osteoblasts and THP-1 monocytes to study the osteogenic and inflammatory responses. In agreement with the proteomic results, Ca-enriched surfaces showed a significant initial inflammation that disappeared once the calcium was released. The distinct protein adsorption/desorption dynamics found in this work demonstrated to be useful to explain the differential biological responses between the titanium and Ca-ion modified implant surfaces., This work was supported by MINECO [MAT2017-86043-R; RTC-2017-6147-1], Generalitat Valenciana [GRISOLIAP/2018/091; APOSTD/2020/036, PROMETEO/2020/069], Universitat Jaume I under [ UJI-B2017-37], the University of the Basque Country under [GIU18/189] and Basque Government under [PRE_2017_2_0044]. The authors would like to thank Raquel Oliver, Jose Ortega and Iraide Escobes for their valuable technical assistance.

Alcohol abuse has a high impact on the mortality and morbidity related to a great number of diseases and is responsible for the development of alcoholic liver disease (ALD). It remains challenging to detect and evaluate its severity, which is crucial for prognosis. In this work, we studied if urinary EVs (uEVs) could serve in diagnose and evaluate cirrhosis in ALD. To this purpose, uEVs characterization by cryo-electron microscopy (Cryo-EM), Nanoparticle Tracking Analysis (NTA) and Western blotting (WB) was performed in a cohort of 21 controls and 21 cirrhotic patients. Then, proteomics of uEVs was carried out in a second cohort of 6 controls and 8 patients in order to identify new putative biomarkers for cirrhosis in ALD. Interestingly, uEVs concentration, size and protein composition were altered in cirrhotic patients. From a total of 1304 proteins identified in uEVs, 90 of them were found to be altered in cirrhotic patients. The results suggest that uEVs could be considered as a tool and a supplier of new biomarkers for cirrhosis in ALD, whose application would be especially relevant in chronic patients. Yet, further research is necessary to obtain more relevant result in clinical terms.

Sepsis is a syndrome without a standard validated diagnostic test. Early recognition is crucial. Serum proteome analysis in children with sepsis may identify new biomarkers. This study aimed to find suitable blood biomarkers for an early diagnosis of sepsis. An analytical observational case-control study was carried out in a single center. Children admitted to a Pediatric Intensive Care Unit with clinical diagnosed sepsis were eligible for study. A proteomic analysis conducted by mass spectrometry was performed. Forty patients with sepsis and 24 healthy donors were recruited. Proteomics results revealed 44 proteins differentially expressed between patients and healthy controls. Six proteins were selected to be validated: lactoferrin, serum amyloid-A1 (SAA-1), complement factor B, leucine-rich alpha-2 glycoprotein (LRG1), soluble interleukin-2 alpha chain receptor (sCD25) and soluble haptoglobin-hemoglobin receptor. Our results showed that sCD25, SAA-1, and LRG1 had high levels of specificity and sensitivity, as well as an excellent area under the ROC curve (>0.9). Our study provides a serum proteomic analysis that identifies new diagnostic biomarkers in sepsis. SAA-1, sCD25 and LRG1 were able to separate septic from healthy donor, so they could be used together with other clinical and analytical features to improve sepsis diagnosis in children. This work was funded by Research Projects from University of Basque Country (US10/02) and from the Basque Government (SAIO10-PE10BF02, SAIO12-PE12BF002, 2012111052, 2019111056). CICbioGUNE is supported by Basque Department of Industry, Tourism and Trade (Etortek and Elkartek programs), the Innovation Technology Department of the Bizkaia County, the ProteoRed-ISCIII (Grant PRB3 IPT17/0019), CIBERehd Network and Severo Ochoa Grant (SEV-2016-0644).

Contains fulltext : 225281.pdf (Publisher’s version ) (Closed access) Contains fulltext : 225281pos.pdf (Author’s version postprint ) (Open Access) BACKGROUND & AIMS: Polycystic liver diseases (PLDs) are genetic disorders characterized by progressive development of multiple biliary cysts. Recently, novel PLD-causative genes, encoding for endoplasmic reticulum (ER)-resident proteins involved in protein biogenesis and transport, were identified. We hypothesized that aberrant proteostasis contributes to PLD pathogenesis, representing a potential therapeutic target. METHODS: ER stress was analysed at transcriptional (qPCR), proteomic (mass spectrometry), morphological (transmission electron microscopy, TEM) and functional (proteasome activity) levels in different PLD models. The effect of ER stress inhibitors [4-phenylbutyric acid (4-PBA)] and/or activators [tunicamycin (TM)] was tested in polycystic (PCK) rats and cystic cholangiocytes in vitro. RESULTS: The expression levels of unfolded protein response (UPR) components were upregulated in liver tissue from PLD patients and PCK rats, as well as in primary cultures of human and rat cystic cholangiocytes, compared to normal controls. Cystic cholangiocytes showed altered proteomic profiles, mainly related to proteostasis (ie synthesis, folding, trafficking and degradation of proteins), marked enlargement of the ER lumen (by TEM) and hyperactivation of the proteasome. Notably, chronic treatment of PCK rats with 4-PBA decreased liver weight, as well as both liver and cystic volumes, of animals under baseline conditions or after TM administration compared to controls. In vitro, 4-PBA downregulated the expression (mRNA) of UPR effectors, normalized proteomic profiles related to protein synthesis, folding, trafficking and degradation and reduced the proteasome hyperactivity in cystic cholangiocytes, reducing their hyperproliferation and apoptosis. CONCLUSIONS: Restoration of proteostasis in cystic cholangiocytes with 4-PBA halts hepatic cystogenesis, emerging as a novel therapeutic strategy.

After over two decades of constant evolution, proteomics can be truly considered nowadays as a high-throughput technique. Latest advances performed in sample preparation, instrumentation, and data analysis tools enable proteome-wide detection and quantification of proteins in complex samples.Label-free quantification by nanoscale liquid chromatography coupled online to tandem mass spectrometry (nLC MS /MS ) is a straightforward procedure for relative protein quantification. This approach allows to get deeper insights of what molecular changes are involved in the biological system we want to study in an unbiased manner.This chapter describes methods for sample preparation prior to mass spectrometry analysis. Besides, we describe a standard acquisition method, and some common bioinformatics analyses that help extracting biologically relevant information out of the achieved data.

The proteomic profiling of serum samples supposes a challenge due to the large abundance of a few blood proteins in comparison with other circulating proteins coming from different tissues and cells. Although the sensitivity of protein detection has increased enormously in the last years, specific strategies are still required to enrich less abundant proteins and get rid of abundant proteins such as albumin, lipoproteins, and immunoglobulins. One of the alternatives that has become more promising is to characterize circulating extracellular vesicles from serum samples that have great interest in biomedicine. In the present work, we enriched the extracellular vesicles fraction from human serum by applying different techniques, including ultracentrifugation, size-exclusion chromatography, and two commercial precipitation methods based on different mechanisms of action. To improve the performance and efficacy of the techniques to promote purity of the preparations, we have employed a small volume of serum samples (&lt, 100 mL). The comparative proteomic profiling of the enriched preparations shows that ultracentrifugation procedure yielded a larger and completely different set of proteins than other techniques, including mitochondrial and ribosome related proteins. The results showed that size exclusion chromatography carries over lipoprotein associated proteins, while a polymer-based precipitation kit has more affinity for proteins associated with granules of platelets. The precipitation kit that targets glycosylation molecules enriches differentially protein harboring glycosylation sites, including immunoglobulins and proteins of the membrane attack complex.

Chaperone-mediated autophagy (CMA) is a homeostatic process essential for the lysosomal degradation of a selected subset of the proteome. CMA activity directly depends on the levels of LAMP2A, a critical receptor for CMA substrate proteins at the lysosomal membrane. In glioblastoma (GBM), the most common and aggressive brain cancer in adulthood, high levels of LAMP2A in the tumor and tumor-associated pericytes have been linked to temozolomide resistance and tumor progression. However, the role of LAMP2A, and hence CMA, in any cancer stem cell type or in glioblastoma stem cells (GSC) remains unknown. In this work, we show that LAMP2A expression is enriched in patient-derived GSCs, and its depletion diminishes GSC-mediated tumorigenic activities. Conversely, overexpression of LAMP2A facilitates the acquisition of GSC properties. Proteomic and transcriptomic analysis of LAMP2A-depleted GSCs revealed reduced extracellular matrix interaction effectors in both analyses. Moreover, pathways related to mitochondrial metabolism and the immune system were differentially deregulated at the proteome level. Furthermore, clinical samples of GBM tissue presented overexpression of LAMP2, which correlated with advanced glioma grade and poor overall survival. In conclusion, we identified a novel role of CMA in directly regulating GSCs activity via multiple pathways at the proteome and transcriptome levels. Significance: A receptor of chaperone-mediated autophagy regulates glioblastoma stem cells and may serve as a potential biomarker for advanced tumor grade and poor survival in this disease.

Embryo implantation is one of the most inefficient steps in assisted reproduction, so the identifying drugs with a potential clinical application to improve it has a strong interest. This work applies artificial intelligence and systems biology-based mathematical modeling strategies to unveil potential treatments by computationally analyzing and integrating available molecular and clinical data from patients. The mathematical models of embryo implantation computationally generated here simulate the molecular networks underneath this biological process. Once generated, these models were analyzed in order to identify potential repositioned drugs (drugs already used for other indications) able to improve embryo implantation by modulating the molecular pathways involved. Interestingly, the repositioning analysis has identified drugs considering two endpoints: (1) drugs able to modulate the activity of proteins whose role in embryo implantation is already bibliographically acknowledged, and (2) drugs that modulate key proteins in embryo implantation previously predicted through a mechanistic analysis of the mathematical models. This second approach increases the scope open for examination and potential novelty of the repositioning strategy. As a result, a list of 23 drug candidates to improve embryo implantation after IVF was identified by the mathematical models. This list includes many of the compounds already tested for this purpose, which reinforces the predictive capacity of our approach, together with novel repositioned candidates (e.g., Infliximab, Polaprezinc, and Amrinone). In conclusion, the present study exploits existing molecular and clinical information to offer new hypotheses regarding molecular mechanisms in embryo implantation and therapeutic candidates to improve it. This information will be very useful to guide future research. Abbreviations: IVF: in vitro fertilization; EI: Embryo implantation; TPMS: Therapeutic Performance Mapping System; MM: mathematical models; ANN: Artificial Neuronal Networks; TNFα: tumour necrosis factor factor-alpha; HSPs: heat shock proteins; VEGF: vascular endothelial growth factor; PPARA: peroxisome proliferator activated receptor-α PXR: pregnane X receptor; TTR: transthyretin; BED: Biological Effectors Database; MLP: multilayer perceptron

[EN] Magnesium is the fourth most abundant element in the human body with a wide battery of functions in the maintenance of normal cell homeostasis. In the bone, this element incorporates in the hydroxyapatite structure and it takes part in mineral metabolism and regulates osteoclast functions. In this study, sol-gel materials with increasing concentrations of MgCl2 (0.5, 1, and 1.5%) were synthesized and applied onto Ti surfaces as coatings. The materials were first physicochemically characterized. In vitro responses were examined using the MC3T3-E1 osteoblastic cells and RAW264.7 macrophages. Human serum protein adsorption was evaluated employing nLC-MS/MS. The incorporation of Mg did not affect the crosslinking of the sol-gel network, and a controlled release of Mg was observed; it was not cytotoxic at any of the tested concentrations. The cytoskeleton arrangement of MC3T3-E1 cells cultured on the Mg-doped materials changed in comparison with controls; the cells became more elongated, with protruded lamellipodia and increased cell surface. The expression of integrins (ITGA5 and ITGB1) was boosted by Mg-coatings. The ALP activity and expression of TGF-beta, OSX and RUNX2 genes were also increased. In RAW264.7 cells, TNF-alpha secretion was reduced, while TGF-beta and IL-4 expression rose. These changes correlated with the altered protein adsorption patterns. The Mg-doped coatings showed increased adsorption of anti-inflammatory (CLUS, IC1, CFAH, and VTNC), cell adhesion (DSG1, FILA2, and DESP) and tissue regeneration (VTNC and CYTA) proteins. This integrated approach to biomaterial characterization revealed the potential of Mg in bone tissue regeneration., This work was supported by MINECO [MAT2017-86043-R; RTC2017-6147-1], Generalitat Valenciana [GRISOLIAP/2018/091, APOSTD/2020/036, PROMETEO/2020/069], Universitat Jaume I under [UJI-B2017-37, Posdoc/2019/28], the University of the Basque Country under [GIU18/189] and Basque Government under [PRE_2017_2_0044]. The authors would like to thank Raquel Oliver, Jose Ortega, Jos ' e Miguel Pedra and Iraide Escob ' es for their valuable technical assistance and Antonio Coso (GMI-Ilerimplant) for producing the titanium discs.

[EN] Zinc is an essential element with an important role in stimulating the osteogenesis and mineralization and suppressing osteoclast differentiation. In this study, new bioactive ZnCl2-doped sol-gel materials were designed to be applied as coatings onto titanium. The biomaterials were physicochemically characterized and the cellular responses evaluated in vitro using MC3T3-E1 osteoblasts and RAW264.7 macrophages. The effect of Zn on the adsorption of human serum proteins onto the material surface was evaluated through nLC-MS/MS. The incorporation of Zn did not affect the crosslinking of the sol-gel network. A controlled Zn2+ release was obtained, reaching values below 10 ppm after 21 days. The materials were no cytotoxic and lead to increased gene expression of ALP, TGF-beta, and RUNX2 in the osteoblasts. In macrophages, an increase of IL-1 beta, TGF-beta, and IL-4 gene expression was accompanied by a reduced TNF-alpha liberation. Proteomic results showed changes in the adsorption patterns of proteins associated with immunological, coagulative, and regenerative functions, in a Zn dose-dependent manner. The variations in protein adsorption might lead to the downregulation of the NF-kappa B pathway, thus explain the observed biological effects of Zn incorporation into biomaterials. Overall, these coatings demonstrated their potential to promote bone tissue regeneration., This work was supported by MINECO [MAT2017-86043-R; RTC2017-6147-1], Generalitat Valenciana [GRISOLIAP/2018/091], Universitat Jaume I under [UJI-B2017-37, Posdoc/2019/28], the University of the Basque Country under [GIU18/189] and Basque Government under [PRE_2017_2_0044]. The authors would like to thank Raquel Oliver, Jose Ortega and Iraide Escobes for their valuable technical assistance, and Antonio Coso (GMI-Ilerimplant) for producing the titanium discs.

Incluye material complementario The Human Proteome Project (HPP) consortium aims to functionally characterize the dark proteome. On the basis of the relevance of olfaction in early neurodegeneration, we have analyzed the dark proteome using data mining in public resources and omics data sets derived from the human olfactory system. Multiple dark proteins localize at synaptic terminals and may be involved in amyloidopathies such as Alzheimer's disease (AD). We have characterized the dark PITH domain-containing protein 1 (PITHD1) in olfactory metabolism using bioinformatics, proteomics, in vitro and in vivo studies, and neuropathology. PITHD1-/- mice exhibit olfactory bulb (OB) proteome changes related to synaptic transmission, cognition, and memory. OB PITHD1 expression increases with age in wild-type (WT) mice and decreases in Tg2576 AD mice at late stages. The analysis across 6 neurological disorders reveals that olfactory tract (OT) PITHD1 is specifically upregulated in human AD. Stimulation of olfactory neuroepithelial (ON) cells with PITHD1 alters the ON phosphoproteome, modifies the proliferation rate, and induces a pro-inflammatory phenotype. This workflow applied by the Spanish C-HPP and Human Brain Proteome Project (HBPP) teams across the ON-OB-OT axis can be adapted as a guidance to decipher functional features of dark proteins. Data are available via ProteomeXchange with identifiers PXD018784 and PXD021634. This work was funded by grants from the Spanish Ministry of Science Innovation and Universities (ref. PID2019-110356RB-I00 to JFI and ES) and Department of Economic and Business Development from Government of Navarra (ref. 0011-1411-2020-000028) to ES and from MEXT-JSPS 17K08884 and Takeda Science Foundation to IO. The Proteomics Unit of Navarrabiomed is a member of Proteored (PRB3-ISCIII) and is supported by grant PT17/0019/009 to JFI, of the PE I+D+I 2013–2016 funded by ISCIII and FEDER. MLM was supported by a postdoctoral fellowship from the Public University of Navarra (UPNA).

Study question Is there any difference in the protein composition of the endometrial fluid aspirate (EFA) obtained the day of embryo transfer in in vitro fertilization (IVF) cycles achieving and not achieving pregnancy? Summary answer Comparative analysis identified a differential protein expression pattern in 'implantative' and 'non-implantative' IVF cycles. What is known already EFA allows non-invasive characterization of the endometrium, and may contain important information on its receptivity when performing (IVF) cycles. Endometrial side of implantation has usually been studied with endometrial biopsy in an IVF cycle prior to embryo transfer, focusing on 'receptive/non-receptive' endometria and with low-throughput proteomic techniques. Study design, size, duration We have compared the protein expression patterns in EFA from a total of 110 women undergoing IVF, corresponding to 50 implantative and 60 non-implantative IVF cycles. Discovery (38 patients) and Validation (42 patients) sample cohorts were analyzed using a high-throughput differential proteomic approach. Then, the differential expression of glycogen phosphorylase B (PYGB) was validated by western blotting in an additional cohort (30 patients). The study period was 18 months. Participants/materials, setting, methods The population under study consisted of 110 women aged 18-40 years old, undergoing their first or second IVF/ intracytoplasmic sperm injection cycle, with normal uterus and endometrium, and 1-2 good quality embryos, and embryo transfer being performed on Day 3. Endometrial fluid aspiration was performed immediately before the embryo transfer. Samples (80) were initially distributed in two independent cohorts and analyzed by liquid chromatography-mass spectrometry. The first cohort was used for the discovery and the second for the validation of the results. Filter-aided sample preparation was used for the in-solution tryptic digestion of the proteins present in the samples, followed by label-free mass spectrometry analysis. In order to unravel the molecular features of receptivity, the lists of differential proteins were thoroughly analyzed using different bioinformatic tools, including GSEA, IPA and GO analysis. Main results and the role of chance A false discovery rate-based correction of the t-test P-values was carried out in order to strengthen the reliability of the results. Functional analyses denoted the deregulation of important processes governing receptivity, such as antimicrobial response, cell-cell interaction, immune response and inflammatory signaling, among others. Overall eight proteins were commonly deregulated in both studied datasets and brain form glycogen phosphorylase (PYGB) was selected for confirmatory analysis. Limitations, reasons for caution Our results were obtained from patients with normal uterus and endometrium and with good quality embryos, who had fresh Day-3 embryo transfer, in stimulated cycles. Therefore, our observations may not be applicable to poor prognosis cases or non-stimulated cycles. Wider implications of the findings This work provides insights into the molecular features of implantative IVF cycles using non-invasive methods. It reveals that EFA may reflect an increased inflammatory state in non-implantative endometrium. Additionally, it proposes PYGB as a potential biomarker for endometrial receptivity or implantation success. This knowledge opens a new avenue for developing embryo transfer strategies, through the improvement of embryo culture media or modifying endometrial fluid composition to increase pregnancy rates. Study funding/competing interest(s) This study was partially funded by a Grant for Fertility Innovation (GFI, 2011) from Merck (Darmstadt, Germany). Authors declare no competing interests. Trial registration number Not applicable.

Macrophages are cells of the innate immune system with the ability to phagocytose and induce a global pattern of responses that depend on several signaling pathways. We have determined the biosignature of murine bone marrow-derived macrophages and human blood monocytes using transcriptomic and proteomic approaches. We identified a common pattern of genes that are transcriptionally regulated and overall indicate that the response to B. burgdorferi involves the interaction of spirochetal antigens with several inflammatory pathways corresponding to primary (triggered by pattern-recognition receptors) and secondary (induced by proinflammatory cytokines) responses. We also show that the Toll-like receptor family member CD180 is downregulated by the stimulation of macrophages, but not monocytes, with the spirochete. Silencing Cd180 results in increased phagocytosis while tempering the production of the proinflammatory cytokine TNF. Cd180-silenced cells produce increased levels of Itgam and surface CD11b, suggesting that the regulation of CD180 by the spirochete initiates a cascade that increases CR3-mediated phagocytosis of the bacterium while repressing the consequent inflammatory response.

Tears are a complex biological mixture containing electrolytes, metabolites, lipids, mucins, some small organic molecules, and proteins. The tear film has various roles in the lubrication, protection from the external environment, and nutrition of the cornea; it is also involved in the modulation of the optical properties of the eye. Tear composition reflects the physiological condition of the underlying tissues. Therefore, the tear fluid is useful in the evaluation of health and disease states and it is a valuable source of biomarkers for objective analysis of ocular and systemic diseases. The relatively high protein concentration of this fluid and the ease of noninvasive sample collection make it suitable for diagnostic and prognostic purposes. Efforts in proteomics research have positively affected to the field of ophthalmology, and the knowledge on the tear proteome has expanded considerably in the last few years. Nevertheless, despite a large amount of available data and the many biomarkers proposed for several eye and systemic diseases, the extent of translation to well-characterized and clinically useful tools has been largely insufficient. As for most of other biofluids, the road from discovery to clinical application is still long and full of pitfalls. In this review, we discuss the proteomic approaches used in the characterization of tear protein and peptide content, recapitulating the main studies and the progress done. We also present a brief summary of the path from discovery to clinical application of tear protein markers, with some representative examples of translation from the bench to the bedside. Significance In this review we cover the most relevant proteomic approaches used in the characterization of the tear proteome, and for the first time we also focus in advances performed in the nowadays emerging peptide content characterization. In this context, we recapitulate on the main studies and the progresses done in this field. We also present a concise overview of the course that may be happen from discovery to clinical application for tear protein markers. Finally we include some representative examples of translation from the bench to the bedside.

Macrophages mediate the elimination of pathogens by phagocytosis resulting in the activation of specific signaling pathways that lead to the production of cytokines, chemokines and other factors. Borrelia burgdorferi, the causative agent of Lyme disease, causes a wide variety of pro-inflammatory symptoms. The proinflammatory capacity of macrophages is intimately related to the internalization of the spirochete. However, most receptors mediating this process are largely unknown. We have applied a multiomic approach, including the proteomic analysis of B. burgdorferi-containing phagosome-enriched fractions, to identify surface receptors that are involved in the phagocytic capacity of macrophages as well as their inflammatory output. Sucrose gradient protein fractions of human monocyte-derived macrophages exposed to B. burgdorferi contained the phagocytic receptor, CR3/CD14 highlighting the major role played by these proteins in spirochetal phagocytosis. Other proteins identified in these fractions include C-type lectins, scavenger receptors or Siglecs, of which some are directly involved in the interaction with the spirochete. We also identified the Fc gamma receptor pathway, including the binding receptor, CD64, as involved both in the phagocytosis of, and TNF induction in response to B. burgdorferi in the absence of antibodies. The common gamma chain, FcγR, mediates the phagocytosis of the spirochete, likely through Fc receptors and C-type lectins, in a process that involves Syk activation. Overall, these findings highlight the complex array of receptors involved in the phagocytic response of macrophages to B. burgdorferi., Author summary Macrophages eliminate infecting microorganisms through the concerted action of surface receptors and signaling molecules. As a consequence, these cells produce a series of soluble factors that participate in the inflammatory response during infections. The composition of the full complement of receptors that participate in the recognition and internalization of the causative agent of Lyme disease, Borrelia burgdorferi, is largely unknown. We have analyzed the protein composition of phagosomes containing B. burgdorferi from human macrophages and identified a series of surface proteins that may be involved in the process. Through the use of gene silencing techniques, we have determined the participation of several of these receptors both in the internalization of the bacterium and the subsequent inflammatory response. Among these, we have identified the Fc gamma receptor pathway as involved in this process in the absence of antibodies. We have also identified receptors that are directly involved in the attachment of B. burgdorferi, while others seem to have an accessory role in the internalization and/or induction of proinflammatory cytokines in response to the spirochete. These data clarify the complex array of interactions between macrophages and B. burgdorferi and shed light on the overall response to this infectious agent.

International audience; Post-translational modification is a key mechanism regulating protein homeostasis and function in eukaryotic cells. Among all ubiquitin-like proteins in liver cancer, the modification by SUMO (Small Ubiquitin MOdifier) has been given the most attention. Isolation of endogenous SUMOylated proteins in vivo is challenging due to the presence of active SUMO-specific proteases. Initial studies of SUMOylation in vivo were based on the molecular detection of specific SUMOylated proteins (e.g., by western blot). However, in many cases, antibodies, generally made with non-modified recombinant protein, did not immunoprecipitate SUMOylated forms of the protein of interest. Nickel chromatography has been the other approach to study SUMOylation by capturing histidine-tagged versions of SUMO molecules. This approach is mainly used in cells stably expressing or transiently transfected with His-SUMO molecules. To overcome these limitations, SUMO-binding entities (SUBEs) were developed to isolate endogenous SUMOylated proteins. Herein, we describe all the steps required for the enrichment, isolation, and identification of SUMOylated substrates from human hepatoma cells and hepatic tissues from a liver cancer mouse model by using SUBEs. Firstly, we describe methods involved in the preparation and lysis of the human hepatoma cells and liver tumor tissue samples. Then, a thorough explanation of the preparation of SUBEs and controls is detailed along with the protocol for the protein pull-down assays. Finally, some examples are provided regarding the options available for the identification and characterization of the SUMOylated proteome, namely the use of western-blot analysis for the detection of a specific SUMOylated substrate from liver tumors or the use of proteomics by mass spectrometry for high-throughput characterization of the SUMOylated proteome and interactome in hepatoma cells.

[EN] Calcium is an element widely used in the development of biomaterials for bone tissue engineering as it plays important roles in bone metabolism and blood coagulation. The Ca ions can condition the microenvironment at the tissue-material interface, affecting the protein deposition process and cell responses. The aim of this study was to analyze the changes in the patterns of protein adsorption on the silica hybrid biomaterials supplemented with different amounts of CaCl2, which can function as release vehicles. This characterization was carried out by incubating the Ca-biomaterials with human serum. LC-MS/MS analysis was used to characterize the adsorbed protein layers and compile a list of proteins whose affinity for the surfaces might depend on the CaCl2 content. The attachment of pro- and anti-clotting proteins, such as THRB, ANT3, and PROC, increased significantly on the Ca-materials. Similarly, VTNC and APOE, proteins directly involved on osteogenic processes, attached preferentially to these surfaces. To assess correlations with the proteomic data, these formulations were tested in vitro regarding their osteogenic and inflammatory potential, employing MC3T3-E1 and RAW 264.7 cell lines, respectively. The results confirmed a Ca dose-dependent osteogenic and inflammatory behavior of the materials employed, in accordance with the protein attachment patterns., This work was supported by MINECO [MAT2017-86043-R]; Universitat Jaume I [Grant numbers Predoc/2014/25, UJI-B2017-37]; Basque Government [Grant numbers IT611-13, Predoc/2016/1/0141]; University of the Basque Country [Grant number UFI11/56]. CIC bioGUNE is supported by Basque Department of Industry, Tourism and Trade (Etortek and Elkartek programs), the Innovation Technology Department of the Bizkaia County; The ProteoRed-ISCIII (Grant PRB3 IPT17/0019); CIBERehd Network, and Severo Ochoa Grant (SEV-2016-0644). Authors would like to thank Antonio Coso and Jaime Franco (GMI-Ilerimplant) for their inestimable contribution to this study, and Raquel Oliver, Jose Ortega (UJI) and Iraide Escobes (CIC bioGUNE) for their valuable technical assistance.

[EN] One of the events occurring when a biomaterial is implanted in an host is the protein deposition onto its surface, which might regulate cell responses. When a biomaterial displays a compromised biocompatibility, distinct complement pathways can be activated to produce a foreign body reaction. In this article, we have designed different types of biomaterial surfaces to study the inflammation process. Here, we used different concentrations of (3-glycidoxypropyl)-trimethoxysilane (GPTMS), an organically-modified alkoxysilane as a precursor for the synthesis of various types of sol-gel materials functionalizing coatings for titanium implants to regulate biological responses. Our results showed that greater GPTMS surface concentrations induced greater secretion of TNF-alpha and IL-10 on RAW 264.7 macrophages. When implanted into rabbit tibia, osseointegration decreased with higher GPTMS concentrations. Interestingly, higher deposition of complement-related proteins C-reactive protein (CRP) and ficolin-2 (FCN2), two main activators of distinct complement pathways, was observed. Taking all together, inflammatory potential increase seems to be GPTMS concentration-dependent. Our results show that a greater adsorption of complement proteins can condition macrophage polarization., This work was supported by MINECO [MAT2017-86043-R]; Universitat Jaume I [Predoc/2014/25, UJI-B2017-37]; Basque Government [IT611-13, Predoc/2016/1/0141]; University of the Basque Country [UFI11/56]; CIC bioGUNE is supported by Basque Department of Industry, Tourism and Trade (Etortek and Elkartek programs), ProteoRed-ISCIII [PRB3 IPT17/0019]; CIBERehd Network and Severo Ochoa Grant [SEV-2016-0644]. Authors would like to thank Antonio Coso and Jaime Franco (GMI-Ilerimplant) for their inestimable contribution to this study, and Raquel Oliver, Jose Ortega (UJI), René van Rheden, Vicent Cuijpers (Radboudumc) and Iraide Escobes (CIC bioGUNE) for their valuable technical assistance.

[EN] It is known strontium can both inhibit the osteoclast formation and stimulate the osteoblast maturation, so biomaterials containing this element can favour bone structure stabilisation. The addition of Sr to biomaterials could affect their interactions with proteins and cells. Here, a silica-hybrid sol-gel network doped with different amounts of SrCl2 and applied as coatings on titanium discs was examined. in vitro analysis was performed to determine the potential effect of Sr in the coatings, showing enhanced gene expression of osteogenic markers (alkaline phosphatase and transforming growth factor-beta) in MC3T3-E1 incubated with Sr-doped biomaterials. The examination of inflammatory markers (tumour necrosis factor-alpha and interleukin 10) in RAW 264.7 macrophages revealed an anti-inflammatory potential of these materials. Proteins adsorbed onto the coatings incubated with human serum (3 h at 37 degrees C) were also analysed; mass spectrometry was used to characterise the proteins adhering to materials with different Sr content. Adding Sr to the coatings increased their affinity to APOE and VTNC proteins (associated with anti-inflammatory and osteogenic functions). Moreover, the proteins involved in coagulation processes, such as prothrombin, were more abundant on the coatings containing Sr than on the base sol-gel surfaces. Correlations between gene expression and proteomic results were also examined., This work was supported by MINECO (MAT2017-86043-R); Universitat Jaume I (grant numbers Predoc/2014/25, UJI-B2017-37); Basque Government (grant numbers IT611-13, Predoc/2016/1/0141), and University of the Basque Country (UFI11/56). Authors would like to thank Antonio Coso and Jaime Franco (GMI-Ilerimplant) for their inestimable contribution to this study, and Raquel Oliver, Jose Ortega (UJI), and Iraide Escobes (CIC bioGUNE) for their valuable technical assistance.

Questions concerning longitudinal data quality and reproducibility of proteomic laboratories spurred the Protein Research Group of the Association of Biomolecular Resource Facilities (ABRF-PRG) to design a study to systematically assess the reproducibility of proteomic laboratories over an extended period of time. Developed as an open study, initially 64 participants were recruited from the broader mass spectrometry community to analyze provided aliquots of a six bovine protein tryptic digest mixture every month for a period of nine months. Data were uploaded to a central repository, and the operators answered an accompanying survey. Ultimately, 45 laboratories submitted a minimum of eight LC-MSMS raw data files collected in data-dependent acquisition (DDA) mode. No standard operating procedures were enforced; rather the participants were encouraged to analyze the samples according to usual practices in the laboratory. Unlike previous studies, this investigation was not designed to compare laboratories or instrument configuration, but rather to assess the temporal intralaboratory reproducibility. The outcome of the study was reassuring with 80% of the participating laboratories performing analyses at a medium to high level of reproducibility and quality over the 9-month period. For the groups that had one or more outlying experiments, the major contributing factor that correlated to the survey data was the performance of preventative maintenance prior to the LC-MSMS analyses. Thus, the Protein Research Group of the Association of Biomolecular Resource Facilities recommends that laboratories closely scrutinize the quality control data following such events. Additionally, improved quality control recording is imperative. This longitudinal study provides evidence that mass spectrometry-based proteomics is reproducible. When quality control measures are strictly adhered to, such reproducibility is comparable among many disparate groups. Data from the study are available via ProteomeXchange under the accession code PXD002114.

The composition of endometrial fluid reflects the status of the endometrium; it is a good atraumatic source of information on embryo implantation processes and possible pathological conditions. Although some attempts have been made to characterise its proteome, the catalogue of its proteins remains incomplete and little has been done to analyse the natural peptides it contains. Here, we present a comprehensive analysis of the proteins and natural peptides of the endometrial fluid. The protein content of samples from 11 individuals was analysed using the novel timsTOF Pro mass spectrometer. We identified 4694 proteins with at least one peptide with FDR 1%, of which 2261 were found in50% of the samples. A pooled endometrial fluid sample was used for isolation and analysis of the natural peptides. Mass spectrometry analysis identified 3899 naturally occurring peptides from 238 different proteins. Among these, there were some putative natural antibacterial peptides. Antimicrobial activity of peptides derived from elafin and Cu/Zn superoxide dismutase was confirmed using microbiological assays. Our results substantially expand the catalogue of known endometrial fluid proteins and provide extensive new information on the natural peptide content of this fluid. SIGNIFICANCE: The endometrial fluid contains many proteins whose clinical relevance is still unknown. Some might be merely markers of endometrial function, but others might play a role in embryo nutrition and/or implantation. Human endometrial fluid analysis might open the door to new developments in embryo transfer strategies in in-vitro fertilisation programmes and lead to improvements in the composition of embryo culture media. Here, we report, for the first time, antimicrobial activity of endometrial fluid peptides. Such peptides could play an important role in the balance of the recently described uterine microbiota.

Hepatocellular carcinoma (HCC) is recognized as the fifth most common neoplasm and currently represents the second leading form of cancer-related death worldwide. Despite great progress has been done in the understanding of its pathogenesis, HCC represents a heavy societal and economic burden as most patients are still diagnosed at advanced stages and the 5-year survival rate remain below 20%. Early detection and revolutionary therapies that rely on the discovery of new molecular biomarkers and therapeutic targets are therefore urgently needed to develop precision medicine strategies for a more efficient management of patients. Areas covered: This review intends to comprehensively analyse the proteomics-based research conducted in the last few years to address some of the principal still open riddles in HCC biology, based on the identification of molecular drivers of tumor progression and metastasis. Expert commentary: The technical advances in mass spectrometry experienced in the last decade have significantly improved the analytical capacity of proteome wide studies. Large-scale protein and protein variant (post-translational modifications) identification and quantification have allowed detailed dissections of molecular mechanisms underlying HCC progression and are already paving the way for the identification of clinically relevant proteins and the development of their use on patient care.

Background: Sepsis is a life-threatening illness with a challenging diagnosis. Current serum biomarkers are not sensitive enough for diagnosis. With the aim of finding proteins associated with sepsis, serum protein profile was compared between patients and healthy donors and serum classical inflammatory proteins were analyzed in both groups. Methods: Serum protein profiles were characterized by two-dimensional electrophoresis (2DE). Identification of the proteins was carried out by mass spectrophotometry and their validation was performed by Enzyme-Linked-ImmunoSorbent Assay (ELISA) in a cohort of 85 patients and 67 healthy donors. Seven classical inflammatory proteins were analyzed in the same cohort by ELISA: interleukin-2 receptor α-chain (sCD25), scavenger receptor cysteine-rich-type-1 (sCD163), tumor-necrosis factor receptor superfamily-member-6 (sFas), hemeoxigenase-1 decycling (HO-1), interleukin-6 (IL-6), interleukin-18 (IL-18) and intercellular adhesion-molecule-1 (sICAM-1). Results: After 2DE, 20 significantly differently expressed spots were identified by mass spectrometry analysis, revealing deregulation of six different proteins upon sepsis and 50% were validated by ELISA: Antithrombin-III (AT-III), Clusterin (CLUS) and Serum amyloid A-1 (SAA-1). Serum concentration of AT-III and CLUS was significantly lower in patients′ serum, whereas SAA-1 showed higher values in septic patients. Serum concentration of the seven inflammatory proteins was significantly increased in septic patients. Functional analysis of the ten deregulated proteins revealed an enrichment of proteins related mainly to the activation of the immune response. Conclusion: We have identified a panel of ten potential sepsis marker proteins biologically connected and validated in a large number of patients, whose analysis could be considered as a complementary tool for the diagnosis of sepsis. Keywords: ELISA, Mass spectrometry analysis, Proteome, Sepsis, Septic shock, Serum markers

Peptidomics is an emerging field focused in the analysis of endogenous peptides. Naturally occurring peptides are often endogenously produced protein fragments. Cleavage of precursor proteins by proteases generates peptides that may gain specialized functions not ascribed to their precursors, and which could reflect the state of a cell under certain physiological conditions or pathological processes.Since peptides are found in complex matrices (e.g., serum, tear, urine, cerebrospinal fluid), they need to be isolated from the matrix and concentrated before they can be analyzed on mass spectrometry. This chapter describes methods for sample preparation prior to mass spectrometry analysis. In addition, different peptide fragmentation techniques are described which are complementary when analyzing naturally occurring peptides by liquid chromatography coupled online to tandem mass spectrometry.

The prime objectives in the development of biomaterials for dental applications are to improve the quality of osseointegration and to short the time needed to achieve it. Design of implants nowadays involves changes in the surface characteristics to obtain a good cellular response. Incorporating osteoinductive elements is one way to achieve the best regeneration possible post-implantation. This study examined the osteointegrative potential of two distinct biomaterials: sandblasted acid-etched titanium and a silica sol–gel hybrid coating, 70% MTMOS-30% TEOS. In vitro, in vivo, and proteomic characterisations of the two materials were conducted. Enhanced expression levels of ALP and IL-6 in the MC3T3-E1 cells cultured with coated discs, suggest that growing cells on such surfaces may increase mineralisation levels. 70M30T-coated implants showed improved bone growth in vivo compared to uncoated titanium. Complete osseointegration was achieved on both. However, coated implants displayed osteoinductive properties, while uncoated implants demonstrated osteoconductive characteristics. Coagulation-related proteins attached predominantly to SAE-Ti surface. Surface properties of the material might drive the regenerative process of the affected tissue. Analysis of the proteins on the coated dental implant showed that few proteins specifically attached to its surface, possibly indicating that its osteoinductive properties depend on the silicon delivery from the implant.

There is an ever-increasing need to develop dental implants with ideal characteristics to achieve specific and desired biological response in the scope of improve the healing process post-implantation. Following that premise, enhancing and optimizing titanium implants through superficial treatments, like silica sol-gel hybrid coatings, are regarded as a route of future research in this area. These coatings change the physicochemical properties of the implant, ultimately affecting its biological characteristics. Sandblasted acid-etched titanium (SAE-Ti) and a silica hybrid sol-gel coating (35M35G30T) applied onto the Ti substrate were examined. The results of in vitro and in vivo tests and the analysis of the protein layer adsorbed to each surface were compared and discussed. In vitro analysis with MC3T3-E1 osteoblastic cells, showed that the sol-gel coating raised the osteogenic activity potential of the implants (the expression of osteogenic markers, the alkaline phosphatase (ALP) and IL-6 mRNAs, increased). In the in vivo experiments using as model rabbit tibiae, both types of surfaces promoted osseointegration. However, the coated implants demonstrated a clear increase in the inflammatory activity in comparison with SAE-Ti. Mass spectrometry (LC–MS/MS) analysis showed differences in the composition of protein layers formed on the two tested surfaces. Large quantities of apolipoproteins were found attached predominantly to SAE-Ti. The 35M35G30T coating adsorbed a significant quantity of complement proteins, which might be related to the material intrinsic bioactivity, following an associated, natural and controlled immune response. The correlation between the proteomic data and the in vitro and in vivo outcomes is discussed on this experimental work.

Peptidomics is an emerging field focused in the analysis of endogenous peptides. Naturally occurring peptides are often endogenously produced protein fragments. Cleavage of precursor proteins by proteases generates peptides that may gain specialized functions not ascribed to their precursors, and which could reflect the state of a cell under certain physiological conditions or pathological processes.Since peptides are found in complex matrices (e.g., serum, tear, urine, cerebrospinal fluid), they need to be isolated from the matrix and concentrated before they can be analyzed on mass spectrometry. This chapter describes methods for sample preparation prior to mass spectrometry analysis. In addition, different peptide fragmentation techniques are described which are complementary when analyzing naturally occurring peptides by liquid chromatography coupled online to tandem mass spectrometry.

To assess whether there are proteins in endometrial fluid aspirate (EFA) that predict implantation. The population under study consisted of 285 women undergoing embryo transfer (ET). Endometrial fluid aspiration was performed immediately before ET. Results of proteomic analysis of EFA were compared between 33 cases who achieved pregnancy and 33 who did not. Samples were analysed by 2D electrophoresis and mass spectrometry. Blood samples were studied by ELISA Pregnancy rates and maternal complications were compared to those in women refusing aspiration. We found 23 proteins differentially expressed in the EFA in conception cycles: 4 up-regulated proteins and 19 down-regulated (FC = 0.31 0.78) (among others, arginase-1, actin B, PARK-7, cofilin-1, stathmin, annexin-2 and CAPZB). Among the five studied proteins that were differentially expressed in EFA, none was differentially expressed in serum. The aspiration procedure had no impact on pregnancy rate. No maternal complications were reported. We found a very different protein profile in implantative cycles, the majority of proteins being down-regulated. This probably reflects a different endometrial functional status, more favourable to implantation. EFA proteomic analysis could be a useful tool in the planning ET strategies.

Osseointegration, including the foreign body reaction to biomaterials, is an immune‐modulated, multifactorial, and complex healing process in which various cells and mediators are involved. The buildup of the osseointegration process is immunological and inflammation‐driven, often triggered by the adsorption of proteins on the surfaces of the biomaterials and complement activation. New strategies for improving osseointegration use coatings as vehicles for osteogenic biomolecules delivery from implants. Natural polymers, such as gelatin, can mimic Collagen I and enhance the biocompatibility of a material. In this experimental study, two different base sol–gel formulations and their combination with gelatin were applied as coatings on sandblasted, acid‐etched titanium substrates, and their biological potential as osteogenic biomaterials was tested. We examined the proteins adsorbed onto each surface and their in vitro and in vivo effects. In vitro results showed an improvement in cell proliferation and mineralization in gelatin‐containing samples. In vivo testing showed the presence of a looser connective tissue layer in those coatings with substantially more complement activation proteins adsorbed, especially those containing gelatin. Vitronectin and FETUA, proteins associated with mineralization process, were significantly more adsorbed in gelatin coatings.

© The Author(s) 2017. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/, Lung cancer configures as one of the deadliest types of cancer. The future implementation of early screening methods such as exhaled breath condensate analysis and low dose computed tomography (CT) as an alternative to current chest imaging based screening will lead to an increased burden on bronchoscopy units. New approaches for improvement of diagnosis in bronchoscopy units, regarding patient management, are likely to have clinical impact in the future. Diagnostic approaches to address mortality of lung cancer include improved early detection and stratification of the cancers according to its prognosis and further response to drug treatment. In this study, we performed a detailed mass spectrometry based proteome analysis of acellular bronchoalveolar lavage (BAL) fluid samples on an observational prospective cohort consisting of 90 suspected lung cancer cases which were followed during two years. The thirteen new lung cancer cases diagnosed during the follow up time period clustered, based on liquid chromatography-mass spectrometry (LC-MS) data, with lung cancer cases at the time of BAL collection. Hundred and thirty-tree potential biomarkers were identified showing significantly differential expression when comparing lung cancer versus non-lung cancer. The regulated biomarkers showed a large overlap with biomarkers detected in tissue samples., All experiments including MS analysis were supported by Fundação para a Ciência e Tecnologia project EXPL/DTP-PIC/0616/2013. RM is supported by FCT investigator program 2012 (IF/01002/2012). ASC is supported by grant SFRH/BPD/85569/2012 funded by Fundação para a Ciência e Tecnologia.

Titanium dental implants are commonly used due to their biocompatibility and biochemical properties; blasted acid-etched Ti is used more frequently than smooth Ti surfaces. In this study, physico-chemical characterisation revealed important differences in roughness, chemical composition and hydrophilicity, but no differences were found in cellular in vitro studies (proliferation and mineralization). However, the deposition of proteins onto the implant surface might affect in vivo osseointegration. To test that hypothesis, protein layers formed on discs of both surface type after incubation with human serum were analysed. Using mass spectrometry (LC/MS/MS), 218 proteins were identified, 30 of which were associated with bone metabolism. Interestingly, Apo E, antithrombin and protein C adsorbed mostly onto blasted and acid-etched Ti, whereas the proteins of the complement system (C3) were found predominantly on smooth Ti surfaces. These results suggest that physico-chemical characteristics could be responsible for the differences observed in the adsorbed protein layer. This work was supported by Ministerio de Economía y Competitividad (MINECO) [MAT 2014-51918-C2-2-R], Universidad de Castellón [P11B2014-19], Plan de Promoción de la Investigación de la Universidad Jaume I under grant [Predoc/2014/25] and Generalitat Valenciana under grant [Grisolia/2014/016]. The authors would like to thank Antonio Coso and Jaime Franco (GMI-Ilerimplant) for their inestimable contribution to this study, and Iraida Escobes (CIC bioGUNE) for her valuable technical assistance.

The interactions of implanted biomaterials with the host organism determine the success or failure of an implantation. Normally, their biocompatibility is assessed using in vitro tests. Unfortunately, in vitro and in vivo results are not always concordant; new, effective methods of biomaterial characterisation are urgently needed to predict the in vivo outcome. As the first layer of proteins adsorbed onto the biomaterial surfaces might condition the host response, mass spectrometry analysis was performed to characterise these proteins. Four distinct hybrid sol-gel biomaterials were tested. The in vitro results were similar for all the materials examined here. However, in vivo, the materials behaved differently. Six of the 171 adsorbed proteins were significantly more abundant on the materials with weak biocompatibility; these proteins are associated with the complement pathway. Thus, protein analysis might be a suitable tool to predict the in vivo outcomes of implantations using newly formulated biomaterials.

Extracellular vesicles have created great interest as possible source of biomarkers for different biological processes and diseases. Although the biological function of these vesicles is not fully understood, it is clear that they participate in the removal of unnecessary cellular material and act as carriers of various macromolecules and signals between the cells. In this report, we analyzed the proteome of extracellular vesicles secreted by primary hepatocytes. We used one- and two-dimensional liquid chromatography combined with data-independent mass spectrometry. Employing label-free quantitative proteomics, we detected significant changes in vesicle protein expression levels in this in vitro model after exposure to well-known liver toxins (galactosamine and Escherichia coli-derived lipopolysaccharide). The results allowed us to identify candidate markers for liver injury. We validated a number of these markers in vivo, providing the basis for the development of novel methods to evaluate drug toxicity. This report strongly supports the application of proteomics in the study of extracellular vesicles released by well-controlled in vitro cellular systems. Analysis of such systems should help to identify specific markers for various biological processes and pathological conditions. Biological significance Identification of low invasive candidate marker for hepatotoxicity. Support to apply proteomics in the study of extracellular vesicles released by well-controlled in vitro cellular systems to identify low invasive markers for diseases.

The success of a dental implant depends on its osseointegration, an important feature of the implant biocompatibility. In this study, two distinct sol-gel hybrid coating formulations [50% methyltrimethoxysilane: 50% 3-glycidoxypropyl-trimethoxysilane (50M50G) and 70% methyltrimethoxysilane with 30% tetraethyl orthosilicate (70M30T)] were applied onto titanium implants. To evaluate their osseointegration, in vitro and in vivo assays were performed. Cell proliferation and differentiation in vitro did not show any differences between the coatings. However, four and eight weeks after in vivo implantation, the fibrous capsule area surrounding 50M50G-implant was 10 and 4 times, respectively, bigger than the area of connective tissue surrounding the 70M30T treated implant. Thus, the in vitro results gave no prediction or explanation for the 50M50G-implant failure in vivo. We hypothesized that the first protein layer adhered to the surface may have direct implication in implant osseointegration, and perhaps correlate with the in vivo outcome. Human serum was used for adsorption analysis on the biomaterials, the first layer of serum proteins adhered to the implant surface was analyzed by proteomic analysis, using mass spectrometry (LC-MS/MS). From the 171 proteins identified; 30 proteins were significantly enriched on the 50M50G implant surface. This group comprised numerous proteins of the immune complement system, including several subcomponents of the C1 complement, complement factor H, C4b-binding protein alpha chain, complement C5 and C-reactive protein. This result suggests that these proteins enriched in 50M50G surface might trigger the cascade leading to the formation of the fibrous capsule observed. The implications of these results could open up future possibilities to predict the biocompatibility problems in vivo. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1477-1485, 2018.

Plasma rich in growth factors (PRGF®-Endoret®) is an autologous technology that contains a set of proteins specifically addressed to wound healing and tissue regeneration. The scaffold formed by using this technology is a clot mainly composed of fibrin protein, forming a three-dimensional (3D) macroscopic network. This biomaterial is easily obtained by biotechnological means from blood and can be used in a range of situations to help wound healing and tissue regeneration. Although the main constituent of this clot is the fibrin scaffold, little is known about other proteins interacting in this clot that may act as adjuvants in the healing process. The aim of this study was to characterize the proteins enclosed by PRGF–Endoret scaffold, using a double-proteomic approach that combines 1D-SDS–PAGE approach followed by LC–MS/MS, and 2-DE followed by MALDI–TOF/TOF. The results presented here provide a description of the catalogue of key proteins in close contact with the fibrin scaffold. The obtained lists of proteins were grouped into families and networks according to gene ontology. Taken together, an enrichment of both proteins and protein families specifically involved in tissue regeneration and wound healing has been found. Copyright © 2013 John Wiley & Sons, Ltd.

Despite the maturity reached by targeted proteomic strategies, reliable and standardized protocols are urgently needed to enhance reproducibility among different laboratories and analytical platforms, facilitating a more widespread use in biomedical research. To achieve this goal, the use of dimensionless relative retention times (iRT), defined on the basis of peptide standard retention times (RT), has lately emerged as a powerful tool. The robustness, reproducibility and utility of this strategy were examined for the first time in a multicentric setting, involving 28 laboratories that included 24 of the Spanish network of proteomics laboratories (ProteoRed-ISCIII). According to the results obtained in this study, dimensionless retention time values (iRTs) demonstrated to be a useful tool for transferring and sharing peptide retention times across different chromatographic set-ups both intra- and inter-laboratories. iRT values also showed very low variability over long time periods. Furthermore, parallel quantitative analyses showed a high reproducibility despite the variety of experimental strategies used, either MRM (multiple reaction monitoring) or pseudoMRM, and the diversity of analytical platforms employed. [Biological significance]: From the very beginning of proteomics as an analytical science there has been a growing interest in developing standardized methods and experimental procedures in order to ensure the highest quality and reproducibility of the results. In this regard, the recent (2012) introduction of the dimensionless retention time concept has been a significant advance. In our multicentric (28 laboratories) study we explore the usefulness of this concept in the context of a targeted proteomics experiment, demonstrating that dimensionless retention time values is a useful tool for transferring and sharing peptide retention times across different chromatographic set-ups., All laboratories from Spain are members of ProteoRed (Plataforma de Recursos Biomoleculares y Bioinformáticos) and are supported bygrant PT13/0001 funded by Instituto de Salud Carlos III (ISCIII) andFEDER

The successful use of proteasome inhibitors in clinical trials revealed the potential of the Ubiquitin Proteasome System for drug development. Protein remodeling through ubiquitylation is known to regulate the stability and activity of essential cellular factors through largely uncharacterized mechanisms. Here, we used Tandem repeated Ubiquitin Binding Entities (TUBEs) under non-denaturing conditions followed by mass spectrometry analysis to study global ubiquitylation events that may lead to the identification of potential drug targets. Using this approach we identified 643 proteins including known and unknown ubiquitin targets from human breast adenocarcinoma MCF7 cells treated with Adriamycin. Coherent with a global cellular response to this genotoxic insult, cellular factors identified are involved in protein synthesis, cellular transport, RNA post-transcriptional modification and signaling pathways regulating early stress responses. This includes components of large macromolecular complexes such as subunits and regulators of the proteasome, supporting the use of this method to characterize networks of molecular interactions coordinated by ubiquitylation. Further in vitro and in silico analysis confirmed that 84% of the total proteins identified here, are ubiquitylated. More importantly the enrichment of known biomarkers and targets for drug development, underlined the potential of this approach for the identification of this clinically relevant information. This article is part of a Special Issue entitled: Proteomics: The clinical link.

The 4-plex iTRAQ platform was utilized to analyze the protein profiles in four stages of grapevine berry skin ripening, from pre-veraison to fully ripening. Mass spectrometric data were acquired from three replicated analyses using a parallel acquisition method in an Orbitrap instrument by combining collision-induced dissociation (CID) and higher energy collision-induced dissociation (HCD) peptide ion fragmentations. As a result, the number of spectra suitable for peptide identification (either from CID or HCD) increased 5-fold in relation to those suitable for quantification (from HCD). Spectra were searched against an NCBInr protein database subset containing all the Vitis sequences, including those derived from whole genome sequencing. In general, 695 unique proteins were identified with more than one single peptide, and 513 of them were quantified. The sequence annotation and GO term enrichment analysis assisted by the automatic annotation tool Blast2GO permitted a pathway analysis which resulted in finding that biological processes and metabolic pathways de-regulated throughout ripening. A detailed analysis of the function-related proteins profiles helped discover a set of proteins of known Vitis gene origin as the potential candidates to play key roles in grapevine berry quality, growth regulation and disease resistance.

A frequent goal of MS-based proteomics experiments nowadays is to quantify changes in the abundance of proteins across several biological samples. The iTRAQ labeling method is a powerful technique; when combined with LC coupled to MS/MS it allows relative quantitation of up to eight different samples simultaneously. Despite the usefulness of iTRAQ current software solutions have limited functionality and require the combined use of several software programs for analysis of the data from different MS vendors. We developed an integrated tool, now available in the virtual expert mass spectrometrist (VEMS) program, for database-dependent search of MS/MS spectra, quantitation and database storage for iTRAQ-labeled samples. VEMS also provides useful alternative report types for large-scale quantitative experiments. The implemented statistical algorithms build on quantitative algorithms previously used in proposed iTRAQ tools as described in detail herein. We propose a new algorithm, which provides more accurate peptide ratios for data that show an intensity-dependent saturation. The accuracy of the proposed iTRAQ algorithm and the performance of VEMS are demonstrated by comparing results from VEMS, MASCOT and PEAKS Q obtained by analyzing data from a reference mixture of six proteins. Users can download VEMS and test data from "http://www.portugene.com/software.html".

Non-alcoholic fatty liver disease (NAFLD) is an important cause of chronic liver injury that has gained concern in clinical hepatology. The principal aim of this study was to find differences in protein expression between patients with NAFLD and healthy controls. Changes in protein expression of liver samples from each of the three groups of subjects, controls, non-alcoholic steatosis, and non-alcoholic steatohepatitis (NASH), were analyzed by two dimensional differential in gel electrophoresis (DIGE) combined with MALDI TOF/TOF analysis, a proteomic approach that allows to compare hundreds of proteins simultaneously. Forty-three proteins exhibiting significant changes (ratio ≥ 1.5, p

Genes that encode glycosylphosphatidylinositol anchored proteins (GPI-APs) constitute an estimated 1-2% of eukaryote genomes. Current computational methods for the prediction of GPI-APs are sensitive and specific; however, the analysis of the processing site (omega- or omega-site) of GPI-APs is still challenging. Only 10% of the proteins that are annotated as GPI-APs have the omega-site experimentally verified. We describe an integrated computational and experimental proteomics approach for the identification and characterization of GPI-APs that provides the means to identify GPI-APs and the derived GPI-anchored peptides in LC-MS/MS data sets. The method takes advantage of sequence features of GPI-APs and the known core structure of the GPI-anchor. The first stage of the analysis encompasses LC-MS/MS based protein identification. The second stage involves prediction of the processing sites of the identified GPI-APs and prediction of the corresponding terminal tryptic peptides. The third stage calculates possible GPI structures on the peptides from stage two. The fourth stage calculates the scores by comparing the theoretical spectra of the predicted GPI-peptides against the observed MS/MS spectra. Automated identification of C-terminal GPI-peptides from porcine membrane dipeptidase, folate receptor and CD59 in complex LC-MS/MS data sets demonstrates the sensitivity and specificity of this integrated computational and experimental approach.

Alexandre Campos et al., Proteomic technologies based on mass spectrometry (MS) have greatly evolved in the past years, and nowadays it is possible to routinely identify thousands of peptides from complex biological samples in a single LC-MS/MS experiment. Despite the advancements in proteomic technologies, the scientific community still faces important challenges in terms of depth and reproducibility of proteomics analyses. Here, we present a multicenter study designed to evaluate long-term performance of LC-MS/MS platforms within the Spanish Proteomics Facilities Network (ProteoRed-ISCIII). The study was performed under well-established standard operating procedures, and demonstrated that it is possible to attain qualitative and quantitative reproducibility over time. Our study highlights the importance of deploying quality assessment metrics routinely in individual laboratories and in multi-laboratory studies. The mass spectrometry data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD000205.This article is part of a Special Issue entitled: HUPO 2014., This work was supported by the Instituto de Salud Carlos III (ProteoRed grant #2005X747).

Plasma membrane proteins are displayed through diverse mechanisms, including anchoring in the extracellular leaflet via glycosylphosphatidylinositol (GPI) molecules. GPI-anchored membrane proteins (GPI-APs) are a functionally and structurally diverse protein family, and their importance is well-recognized as they are candidate cell surface biomarker molecules with potential diagnostic and therapeutic applications in molecular medicine. GPI-APs have also attracted interest in plant biotechnology because of their role in root development and cell remodeling. Using a shave-and-conquer concept, we demonstrate that phospholipase D (PLD) treatment of human and plant plasma membrane fractions leads to the release of GPI-anchored proteins that were identified and characterized by capillary liquid chromatography and tandem mass spectrometry. In contrast to phospholipase C, the PLD enzyme is not affected by structural heterogeneity of the GPI moiety, making PLD a generally useful reagent for proteomic investigations of GPI-anchored proteins in a variety of cells, tissues, and organisms. A total of 11 human GPI-APs and 35 Arabidopsis thaliana GPI-APs were identified, representing a significant addition to the number of experimentally detected GPI-APs in both species. Computational GPI-AP sequence analysis tools were investigated for the characterization of the identified GPI-APs, and these demonstrated that there is some discrepancy in their efficiency in classification of GPI-APs and the exact assignment of omega-sites. This study highlights the efficiency of an integrative proteomics approach that combines experimental and computational methods to provide the selectivity, specificity, and sensitivity required for characterization of post-translationally modified membrane proteins.

Breast cancer is a leading cause of cancer-associated death worldwide. One of the most important prognostic factors for survival is the early detection of the disease. Recent studies indicate that extracellular vesicles may provide diagnostic information for cancer management. We demonstrate the secretion of extracellular vesicles by primary breast epithelial cells enriched for stem/progenitor cells cultured as mammospheres, in non-adherent conditions. Using a proteomic approach we identified proteins contained in these vesicles whose expression is affected by hormonal changes in the cellular environment. In addition, we showed that these vesicles are capable of promoting changes in expression levels of genes involved in epithelial-mesenchymal transition and stem cell markers. Our findings suggest that secreted extracellular vesicles could represent potential diagnostic and/or prognostic markers for breast cancer and support a role for extracellular vesicles in cancer progression.

All participating laboratories are members of ProteoRed-ISCIII.-- et al., The Spanish team of the Human Proteome Project (SpHPP) marked the annotation of Chr16 and data analysis as one of its priorities. Precise annotation of Chromosome 16 proteins according to C-HPP criteria is presented. Moreover, Human Body Map 2.0 RNA-Seq and Encyclopedia of DNA Elements (ENCODE) data sets were used to obtain further information relative to cell/tissue specific chromosome 16 coding gene expression patterns and to infer the presence of missing proteins. Twenty-four shotgun 2D-LC–MS/MS and gel/LC–MS/MS MIAPE compliant experiments, representing 41% coverage of chromosome 16 proteins, were performed. Furthermore, mapping of large-scale multicenter mass spectrometry data sets from CCD18, MCF7, Jurkat, and Ramos cell lines into RNA-Seq data allowed further insights relative to correlation of chromosome 16 transcripts and proteins. Detection and quantification of chromosome 16 proteins in biological matrices by SRM procedures are also primary goals of the SpHPP. Two strategies were undertaken: one focused on known proteins, taking advantage of MS data already available, and the second, aimed at the detection of the missing proteins, is based on the expression of recombinant proteins to gather MS information and optimize SRM methods that will be used in real biological samples. SRM methods for 49 known proteins and for recombinant forms of 24 missing proteins are reported in this study., This work was supported by: ProteoRed and the Carlos III National Health Institute Agreement, ProteoRed-ISCIII; the agreement between FIMA and the “UTE project CIMA”; grants SAF2011-29312 from Ministerio de Ciencia e Innovación and ISCIII-RETIC RD06/0020 to FJC and EU FP7 grant ProteomeXchange [grant number 260558]. APM and DTM have been funded by Spanish grants from Ministerio de Ciencia e Innovación BIO2010-17527 and the Government of Madrid (P2010/BMD-2305). BBVA Foundation for its support to HUPO initiatives.