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The human genome contains nearly 100 deubiquitinating enzymes (DUBs) responsible for removing ubiquitin moieties from a large variety of substrates. Which DUBs are responsible for targeting which substrates remain mostly unknown. Here we implement the bioUb approach to identify DUB substrates in a systematic manner, combining gene silencing and proteomics analyses. Silencing of individual DUB enzymes is used to reduce their ubiquitin deconjugating activity, leading to an increase of the ubiquitination of their substrates, which can then be isolated and identified. We report here quantitative proteomic data of the putative substrates of 5 human DUBs. Furthermore, we have built a novel interactive database of DUB substrates to provide easy access to our data and collect DUB proteome data from other groups as a reference resource in the DUB substrates research field. Authors of this paper are members of Proteored, PRB3 and are supported by grant PT17/0019 of the PE I+D+i 2013-2016, funded by Instituto de Salud Carlos III (ISCIII) and European Regional Development Fund (ERDF). This research was also funded by the Spanish Ministry of Economy, grant number SAF2016-76898-P and the University of the Basque Country (UPV/EHU), grant number US19/05. J.R was supported with a postdoctoral research fellowship from the UPV/EHU.

Angelman syndrome is a complex neurodevelopmental disorder caused by the lack of function in the brain of a single gene, UBE3A. The E3 ligase coded by this gene is known to build K48-linked ubiquitin chains, a modification historically considered to target substrates for degradation by the proteasome. However, a change in protein abundance is not proof that a candidate UBE3A substrate is indeed ubiquitinated by UBE3A. We have here used an unbiased ubiquitin proteomics approach, the bioUb strategy, to identify 79 proteins that appear more ubiquitinated in the Drosophila photoreceptor cells when Ube3a is over-expressed. We found a significantly high number of those proteins to be proteasomal subunits or proteasome-interacting proteins, suggesting a wide proteasomal perturbation in the brain of Angelman patients. We focused on validating the ubiquitination by Ube3a of Rngo, a proteasomal component conserved from yeast (Ddi1) to humans (DDI1 and DDI2), but yet scarcely characterized. Ube3a-mediated Rngo ubiquitination in fly neurons was confirmed by immunoblotting. Using human neuroblastoma SH-SY5Y cells in culture, we also observed that human DDI1 is ubiquitinated by UBE3A, without being targeted for degradation. The novel observation that DDI1 is expressed in the developing mice brain, with a significant peak at E16.5, strongly suggests that DDI1 has biological functions not yet described that could be of relevance for Angelman syndrome clinical research.

Both Parkin and UBE3A are E3 ubiquitin ligases whose mutations result in severe brain dysfunction. Several of their substrates have been identified using cell culture models in combination with proteasome inhibitors, but not in more physiological settings. We recently developed theUbbiostrategy to isolate ubiquitinated proteins in flies and have now identified by mass spectrometry analysis the neuronal proteins differentially ubiquitinated by those ligases. This is an example of how flies can be used to provide biological material in order to reveal steady state substrates of disease causing genes. Collectively our results provide new leads to the possible physiological functions of the activity of those two disease causing E3 ligases. Particularly, in the case of Parkin the novelty of our data originates from the experimental setup, which is not overtly biased by acute mitochondrial depolarisation. In the case of UBE3A, it is the first time that a nonbiased screen for its neuronal substrates has been reported.

The necrotrophic fungus Botrytis cinerea is a very damaging phytopathogen of wide host range and environmental persistence. It is difficult to control because of its genetic versatility, expressed in the many phenotypical differences among isolates. The genomes of the B. cinerea B05.10 and T4 strains have been recently sequenced, becoming a model system for necrotrophic pathogens, and thus opening new alternatives for functional genomics analysis. In this work, the mycelium and secreted proteome of six wild-type strains with different host range, and grown in liquid minimal medium, have been analyzed by using complementary gel-based (1-DE and 2-DE) and gel-free/label-free (nUPLC–MS E ) approaches. We found differences in the protein profiles among strains belonging to both the mycelium and the secretome. A total of 47 and 51 variable proteins were identified in the mycelium and the secretome, respectively. Some of them, such as malate dehydrogenase or peptidyl-prolyl cis–trans isomerase from the mycelium, and endopolygalacturonase, aspartic protease or cerato-platanin protein from the secretome have been reported as virulence factors, which are involved in host-tissue invasion, pathogenicity or fungal development. Biological significance The necrotrophic fungus Botrytis cinerea is an important phytopathogen of wide host range and environmental persistence, causing substantial economic losses worldwide. In this work, the mycelium and secreted proteome of six B. cinerea wild-type strains with different host range have been analyzed by using complementary gel-based and gel-free/label-free approaches. Fungal genetic versatility was confirmed at the proteome level for both mycelium proteome and secreted proteins. A high number of hypothetical proteins with conserved domains related to toxin compounds or to unknown functions were identified, having qualitative differences among strains. The identification of hypothetical proteins suggests that the B. cinerea strains differ mostly in processes involved in adaptation to a particular environment or a growth condition, rather than in essential metabolic reactions. Proteomics can help in the identification of variable proteins related to the infection and colonization of host plant tissues, as well as of virulence and aggressiveness factors among different B. cinerea wild-type strains. This article is part of a Special Issue entitled: Trends in Microbial Proteomics.

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

Anti-cancer immunotherapies commonly rely on the use of interleukin-2 (IL-2) to promote the expansion of T lymphocytes. IL-2- dependent proliferation is the culmination of a complex network of phosphorylation-driven signaling events that impact on gene transcription through mechanisms that are not clearly understood. To study the role of IL-2 in the regulation of nuclear protein function we have performed an unbiased mass spectrometry-based study of the nuclear phosphoproteome of resting and IL-2-treated CD4(+) T lymphocytes. We detected 8521distinct phosphosites including many that are not yet reported in curated phosphorylation databases. Although most phosphorylation sites remained unaffected upon IL-2 treatment, 391 sites corresponding to 288 gene products showed robust IL-2-dependent regulation. Importantly, we show that ATP-citrate lyase (ACLY) is a key phosphoprotein effector of IL-2-mediated T-cell responses. ACLY becomes phosphorylated on serine 455 in T lymphocytes upon IL-2-driven activation of AKT, and depletion or inactivation of ACLY compromises IL-2-promoted T-cell growth. Mechanistically, we demonstrate that ACLY is required for enhancing histone acetylation levels and inducing the expression of cell cycle regulating genes in response to IL-2. Thus, the metabolic enzyme ACLY emerges as a bridge between cytokine signaling and proliferation of T lymphocytes, and may be an attractive candidate target for the development of more efficient anti-cancer immunotherapies.

Currently the bottom up approach is the most popular for characterizing protein samples by mass spectrometry. This is mainly attributed to the fact that the bottom up approach has been successfully optimized for high throughput studies. However, the bottom up approach is associated with a number of challenges such as loss of linkage information between peptides. Previous publications have addressed some of these problems which are commonly referred to as protein inference. Nevertheless, all previous publications on the subject are oversimplified and do not represent the full complexity of the proteins identified. To this end we present here SIR (spectra based isoform resolver) that uses a novel transparent and systematic approach for organizing and presenting identified proteins based on peptide spectra assignments. The algorithm groups peptides and proteins into five evidence groups and calculates sixteen parameters for each identified protein that are useful for cases where deterministic protein inference is the goal. The novel approach has been incorporated into SIR which is a user-friendly tool only concerned with protein inference based on imports of Mascot search results. SIR has in addition two visualization tools that facilitate further exploration of the protein inference problem.

Interleukin-2 (IL-2) is major cytokine involved in T cell proliferation, differentiation and apoptosis. Association between IL-2 and its receptor (IL-2R), triggers activation of complex signaling cascade governed by tyrosine phosphorylation that culminates in transcription of genes involved in modulation of the immune response. The complete characterization of the IL-2 pathway is essential to understand how aberrant IL-2 signaling results in several diseases such as cancer or autoimmunity and also how IL-2 treatments affect cancer patients. To gain insights into the downstream machinery activated by IL-2, we aimed to define the global tyrosine-phosphoproteome of IL-2 pathway in human T cell line Kit225 using high resolution mass spectrometry combined with phosphotyrosine immunoprecipitation and SILAC. The molecular snapshot at 5min of IL-2 stimulation resulted in identification of 172 proteins among which 79 were found with increased abundance in the tyrosine-phosphorylated complexes, including several previously not reported IL-2 downstream effectors. Combinatorial site-specific phosphoproteomic analysis resulted in identification of 99 phosphorylated sites mapping to the identified proteins with increased abundance in the tyrosine-phosphorylated complexes, of which 34 were not previously described. In addition, chemical inhibition of the identified IL-2-mediated JAK, PI3K and MAPK signaling pathways, resulted in distinct alteration on the IL-2 dependent proliferation.

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.

The phytopathogenic fungus Botrytis cinerea infects more than different 200 plant species and causes substantial losses in numerous crops. The B05.10 and T4 wild-type strain genomes have been recently sequenced, becoming a model system for necrotrophic pathogens, as well as opening up new alternatives in functional genomics, such as proteomics. We analyzed B. cinerea mycelium from these two wild-type strains, introducing label-free shotgun nUPLC-MS(E) methodology to complement the 2-DE-MS-based approach. We assessed the label-free nUPLC-MS(E) methodology for protein identification and quantification using five mycelium protein dilutions. A total of 225 and 170 protein species were identified by nUPLC-MS(E) in the B05.10 and T4 strains, respectively. Moreover, 129 protein species were quantified in both strains. Significant differences in protein abundance were found in 15 more abundant and 16 less abundant protein species in the B05.10 strain compared to the T4 strain. Twenty-nine qualitative and 15 significant quantitative differences were found using 2-DE. The label-free nUPLC-MS(E) was a reliable, reproducible and sensitive method for protein identification and quantification to study the B. cinerea mycelial proteome. Results obtained by gel-based and gel-free complementary approaches allow a deeper characterization of this fungus, as well as the identification of potential virulence factors.

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.

11 páginas, 6 figuras.-- et al., The Chromosome 16 Consortium forms part of the Human Proteome Project that aims to develop an entire map of the proteins encoded by the human genome following a chromosome-centric strategy (C-HPP) to make progress in the understanding of human biology in health and disease (B/D-HPP). A Spanish consortium of 16 laboratories was organized into five working groups: Protein/Antibody microarrays, protein expression and Peptide Standard, S/MRM, Protein Sequencing, Bioinformatics and Clinical healthcare, and Biobanking. The project is conceived on a multicenter configuration, assuming the standards and integration procedures already available in ProteoRed-ISCIII, which is encompassed within HUPO initiatives. The products of the 870 protein coding genes in chromosome 16 were analyzed in Jurkat T lymphocyte cells, MCF-7 epithelial cells, and the CCD18 fibroblast cell line as it is theoretically expected that most chromosome 16 protein coding genes are expressed in at least one of these. The transcriptome and proteome of these cell lines was studied using gene expression microarray and shotgun proteomics approaches, indicating an ample coverage of chromosome 16. With regard to the B/D section, the main research areas have been adopted and a biobanking initiative has been designed to optimize methods for sample collection, management, and storage under normalized conditions and to define QC standards. The general strategy of the Chr-16 HPP and the current state of the different initiatives are discussed., 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 F.J.C. and EU FP7 grant ProteomeXchange (grant number 260558); BBVA Foundation for its support to HUPO initiatives.

Background Protein inference from peptide identifications in shotgun proteomics must deal with ambiguities that arise due to the presence of peptides shared between different proteins, which is common in higher eukaryotes. Recently data independent acquisition (DIA) approaches have emerged as an alternative to the traditional data dependent acquisition (DDA) in shotgun proteomics experiments. MSE is the term used to name one of the DIA approaches used in QTOF instruments. MSE data require specialized software to process acquired spectra and to perform peptide and protein identifications. However the software available at the moment does not group the identified proteins in a transparent way by taking into account peptide evidence categories. Furthermore the inspection, comparison and report of the obtained results require tedious manual intervention. Here we report a software tool to address these limitations for MSE data. Results In this paper we present PAnalyzer, a software tool focused on the protein inference process of shotgun proteomics. Our approach considers all the identified proteins and groups them when necessary indicating their confidence using different evidence categories. PAnalyzer can read protein identification files in the XML output format of the ProteinLynx Global Server (PLGS) software provided by Waters Corporation for their MSE data, and also in the mzIdentML format recently standardized by HUPO-PSI. Multiple files can also be read simultaneously and are considered as technical replicates. Results are saved to CSV, HTML and mzIdentML (in the case of a single mzIdentML input file) files. An MSE analysis of a real sample is presented to compare the results of PAnalyzer and ProteinLynx Global Server. Conclusions We present a software tool to deal with the ambiguities that arise in the protein inference process. Key contributions are support for MSE data analysis by ProteinLynx Global Server and technical replicates integration. PAnalyzer is an easy to use multiplatform and free software tool. Proteomics Core Facility-SGIKER is member of ProteoRed-ISCIII and is supported by UPV/EHU, MCINN, GV/EJ, ERDF and ESF. JMA is supported by the Department of Industry of the Basque Government (ETORTEK grant IE09-256).RM is supported by Fundação para a Ciência e a Tecnologia (FCT) Ciência 2007 and by European Social Fund. IPATIMUP is an Associate Laboratory of the Portuguese Ministry of Science, Technology and Higher Education and is partially supported by FCT. RM is further supported by FCT grants (PTDC/QUI-BIQ/099457/2008 and PTDC/EIA-EIA/099458/2008).

E2F transcription factors (E2F1-8) are best known for their role in cell proliferation, although it is clear that they regulate many other biological processes through the transcriptional modulation of distinct target genes. However, the specific set of genes regulated by each E2F remains to be characterized. To gain insight into the molecular pathways regulated by E2F2, we have analyzed the proteome of antigen receptor-activated T cells lacking E2F2. We report that loss of E2F2 results in a deregulated Aryl-hydrocarbon-receptor pathway. Proliferating E2F2(-/-) T lymphocytes expressed significantly higher levels of Aip, Ahr, and Arnt relative to wild-type (WT)(1) controls. The mechanism for increased levels of Aip appears straightforward, involving direct regulation of the Aip gene promoter by E2F2. Although the Ahr and Arnt promoters also bind E2F2, their regulation appears to be more complex. Nevertheless, exposure to the environmental xenobiotic 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a well-known exogenous ligand of the Ahr pathway, led to overexpression of the Ahr target gene Cyp1a1, and to increased sensitivity to TCDD-triggered apoptosis in E2F2(-/-) T cells compared with WT controls. These results suggest that E2F2 modulates cellular sensitivity to xenobiotic signals through the negative regulation of the Ahr pathway.