Sorting nexin 27 interactome in T-lymphocytes identifies zona occludens-2 dynamic redistribution at the immune synapse.

T Lymphocyte recognition of antigens leads to the formation of a highly organized structure termed immune synapse (IS) by analogy with the neuronals synapse. Sorting nexin 27 (SNX27) controls the endosomal traffic of PSD95, Dlg1, ZO-1 (PDZ) domain-interacting proteins, and its alteration is associated with impaired synaptic function and neurological diseases. In T-lymphocytes, SNX27-positive vesicles polarize to the IS, the identity of SNX27 interactors in these conditions nonetheless remains unknown. Here we used proteomics to analyze the SNX27 interactome purified from IS-forming T cells, and confirmed the conserved nature of the SNX27/WASH/retromer association in hematopoietic cells. Furthermore, our comparative interactome analysis of SNX27 wild-type and a mutant-deficient for PDZ cargo recognition identified the epithelial cell-cell junction protein zona occludens-2 (ZO-2) as an IS component. Biochemistry and microscopy approaches in T cells confirmed SNX27/ZO-2 PDZ-dependent interaction, and demonstrated its role controlling the dynamic localization of ZO-2 at the IS. This study broadens our knowledge of SNX27 function in T lymphocytes, and suggests that pathways that delimit polarized structures in nervous and epithelial systems also participate in IS regulation.

PACOM: A Versatile Tool for Integrating, Filtering, Visualizing, and Comparing Multiple Large Mass Spectrometry Proteomics Data Sets.

Mass-spectrometry-based proteomics has evolved into a high-throughput technology in which numerous large-scale data sets are generated from diverse analytical platforms. Furthermore, several scientific journals and funding agencies have emphasized the storage of proteomics data in public repositories to facilitate its evaluation, inspection, and reanalysis. (1) As a consequence, public proteomics data repositories are growing rapidly. However, tools are needed to integrate multiple proteomics data sets to compare different experimental features or to perform quality control analysis. Here, we present a new Java stand-alone tool, Proteomics Assay COMparator (PACOM), that is able to import, combine, and simultaneously compare numerous proteomics experiments to check the integrity of the proteomic data as well as verify data quality. With PACOM, the user can detect source of errors that may have been introduced in any step of a proteomics workflow and that influence the final results. Data sets can be easily compared and integrated, and data quality and reproducibility can be visually assessed through a rich set of graphical representations of proteomics data features as well as a wide variety of data filters. Its flexibility and easy-to-use interface make PACOM a unique tool for daily use in a proteomics laboratory. PACOM is available at https://github.com/smdb21/pacom .

Comparative Analysis of the Endogenous Peptidomes Displayed by HLA-B*27 and Mamu-B*08: Two MHC Class I Alleles Associated with Elite Control of HIV/SIV Infection.

Indian rhesus macaques are arguably the most reliable animal models in AIDS research. In this species the MHC class I allele Mamu-B*08, among others, is associated with elite control of SIV replication. A similar scenario is observed in humans where the expression of HLA-B*27 or HLA-B*57 has been linked to slow or no progression to AIDS after HIV infection. Despite having large differences in their primary structure, it has been reported that HLA-B*27 and Mamu-B*08 display peptides with sequence similarity. To fine-map the Mamu-B*08 binding motif and assess its similarities with that of HLA-B*27, we affinity purified the peptidomes bound to these MHC class I molecules and analyzed them by LC-MS, identifying several thousands of endogenous ligands. Sequence analysis of both sets of peptides revealed a degree of similarity in their binding motifs, especially at peptide position 2 (P2), where arginine was present in the vast majority of ligands of both allotypes. In addition, several differences emerged from this analysis: (i) ligands displayed by Mamu-B*08 tended to be shorter and to have lower molecular weight, (ii) Mamu-B*08 showed a higher preference for glutamine at P2 as a suboptimal binding motif, and (iii) the second major anchor position, found at PΩ, was much more restrictive in Mamu-B*08. In this regard, HLA-B*27 bound efficiently peptides with aliphatic, aromatic (including tyrosine), and basic C-terminal residues while Mamu-B*08 preferred peptides with leucine and phenylalanine in this position. Finally, in silico estimations of binding efficiency and competitive binding assays to Mamu-B*08 of several selected peptides revealed a good correlation between the characterized anchor motif and binding affinity. These results deepen our understanding of the molecular basis of the presentation of peptides by Mamu-B*08 and can contribute to the detection of novel SIV epitopes restricted by this allotype.

Increased diversity of the HLA-B40 ligandome by the presentation of peptides phosphorylated at their main anchor residue.

Human leukocyte antigen (HLA) class I molecules bind peptides derived from the intracellular degradation of endogenous proteins and present them to cytotoxic T lymphocytes, allowing the immune system to detect transformed or virally infected cells. It is known that HLA class I-associated peptides may harbor posttranslational modifications. In particular, phosphorylated ligands have raised much interest as potential targets for cancer immunotherapy. By combining affinity purification with high-resolution mass spectrometry, we identified more than 2000 unique ligands bound to HLA-B40. Sequence analysis revealed two major anchor motifs: aspartic or glutamic acid at peptide position 2 (P2) and methionine, phenylalanine, or aliphatic residues at the C terminus. The use of immobilized metal ion and TiO2 affinity chromatography allowed the characterization of 85 phosphorylated ligands. We further confirmed every sequence belonging to this subset by comparing its experimental MS2 spectrum with that obtained upon fragmentation of the corresponding synthetic peptide. Remarkably, three phospholigands lacked a canonical anchor residue at P2, containing phosphoserine instead. Binding assays showed that these peptides bound to HLA-B40 with high affinity. Together, our data demonstrate that the peptidome of a given HLA allotype can be broadened by the presentation of peptides with posttranslational modifications at major anchor positions. We suggest that ligands with phosphorylated residues at P2 might be optimal targets for T-cell-based cancer immunotherapy.

Proteogenomics Dashboard for the Human Proteome Project.

dasHPPboard is a novel proteomics-based dashboard that collects and reports the experiments produced by the Spanish Human Proteome Project consortium (SpHPP) and aims to help HPP to map the entire human proteome. We have followed the strategy of analog genomics projects like the Encyclopedia of DNA Elements (ENCODE), which provides a vast amount of data on human cell lines experiments. The dashboard includes results of shotgun and selected reaction monitoring proteomics experiments, post-translational modifications information, as well as proteogenomics studies. We have also processed the transcriptomics data from the ENCODE and Human Body Map (HBM) projects for the identification of specific gene expression patterns in different cell lines and tissues, taking special interest in those genes having little proteomic evidence available (missing proteins). Peptide databases have been built using single nucleotide variants and novel junctions derived from RNA-Seq data that can be used in search engines for sample-specific protein identifications on the same cell lines or tissues. The dasHPPboard has been designed as a tool that can be used to share and visualize a combination of proteomic and transcriptomic data, providing at the same time easy access to resources for proteogenomics analyses. The dasHPPboard can be freely accessed at: http://sphppdashboard.cnb.csic.es.

A novel CD44-binding peptide from the pro-matrix metalloproteinase-9 hemopexin domain impairs adhesion and migration of chronic lymphocytic leukemia (CLL) cells.

(pro)MMP-9 binds to CLL cells through the PEX9 domain and contributes to CLL progression. To biochemically characterize this interaction and identify potential therapeutic targets, we prepared GST-PEX9 forms containing structural blades B1B2 or B3B4. We recently described a sequence in blade B4 (P3 sequence) that bound α4ß1 integrin and partially impaired cell adhesion and migration. We have now studied the possible contribution of the B1B2 region to cell interaction with PEX9. CLL cells bound to GST-B1B2 and CD44 was the primary receptor. GST-B1B2 inhibited CLL cell migration as effectively as GST-B3B4. Overlapping synthetic peptides spanning the B1B2 region identified the sequence FDAIAEIGNQLYLFKDGKYW, present in B1 and contained in peptide P6, as the most effective site. P6 inhibited cell adhesion to PEX9 in a dose-dependent manner and with an IC50 value of 90 µM. P6 also inhibited cell adhesion to hyaluronan but had no effect on adhesion to VCAM-1 (α4ß1 integrin ligand), confirming its specific interaction with CD44. Spatial localization analyses mapped P6 to the central cavity of PEX9, in close proximity to the previously identified P3 sequence. Both P6 and P3 equally impaired cell adhesion to (pro)MMP-9. Moreover, P6 synergistically cooperated with P3, resulting in complete inhibition of CLL cell binding to PEX9, chemotaxis, and transendothelial migration. Thus, P6 is a novel sequence in PEX9 involved in cell-PEX9/(pro)MMP-9 binding by interacting with CD44. Targeting both sites, P6 and P3, should efficiently prevent (pro)MMP-9 binding to CLL cells and its pathological consequences.

Transient PKCα shuttling to the immunological synapse is governed by DGKζ and regulates L-selectin shedding.

Considerable evidence indicates that diacylglycerol (DAG) generation at the immunological synapse (IS) determines T cell functions by regulating the duration and amplitude of Ras/ERK signals. The exact mechanism by which DAG regulates Ras/ERK activation downstream of the T cell receptor (TCR) nonetheless remains poorly understood. Here we characterize PKCα as a previously unrecognized component of the machinery that translates cell receptor occupancy into Ras/ERK-propagated signals. We show transient translocation of PKCα to the IS, mediated by DAG generation at the contact area. Diacylglycerol kinase (DGK)ζ negatively regulated PKCα translocation kinetics, whereas PKCα activity limited its own persistence at the IS. Coordinated activation of DGKζ and PKCα in response to antigen recognition regulated the amplitude and duration of Ras/ERK activation; this in turn mediated early processes of T cell surface proteolysis such as L-selectin shedding. Analysis of DGKζ-deficient mice further showed that increased DAG signaling is translated to downstream elements of this pathway, as reflected by enhanced PKCα-dependent L-selectin shedding. We propose that early activation of a DAG-PKCα axis contributes to the mechanisms by which antigen affinity translates into TCR biological responses.

The mzQuantML data standard for mass spectrometry-based quantitative studies in proteomics.

The range of heterogeneous approaches available for quantifying protein abundance via mass spectrometry (MS)(1) leads to considerable challenges in modeling, archiving, exchanging, or submitting experimental data sets as supplemental material to journals. To date, there has been no widely accepted format for capturing the evidence trail of how quantitative analysis has been performed by software, for transferring data between software packages, or for submitting to public databases. In the context of the Proteomics Standards Initiative, we have developed the mzQuantML data standard. The standard can represent quantitative data about regions in two-dimensional retention time versus mass/charge space (called features), peptides, and proteins and protein groups (where there is ambiguity regarding peptide-to-protein inference), and it offers limited support for small molecule (metabolomic) data. The format has structures for representing replicate MS runs, grouping of replicates (for example, as study variables), and capturing the parameters used by software packages to arrive at these values. The format has the capability to reference other standards such as mzML and mzIdentML, and thus the evidence trail for the MS workflow as a whole can now be described. Several software implementations are available, and we encourage other bioinformatics groups to use mzQuantML as an input, internal, or output format for quantitative software and for structuring local repositories. All project resources are available in the public domain from the HUPO Proteomics Standards Initiative http://www.psidev.info/mzquantml.

The chromosome-centric human proteome project at FEBS Congress.

In the summer of 2013, distinguished global representatives of proteome science gathered to discuss the futuristic visions of the chromosome-centric human proteome project (C-HPP) (Cochairs: Y. K. Paik, G. Omenn; hosted by A. Archakov, Institute of Biomedical Chemistry, Russia) that was broadcast to the annual Federation of European Biochemical Societies Congress (St. Petersburg, Russia, July 10-11, 2013). Technology breakthroughs presented included a new ultra-sensitive Tribrid mass-spectrometer from Thermo and SOMAmers-Slow Off-rate Modified Aptamers (SOMAlogic, USA), a new type of protein capture reagents. Professor Archakov's group introduced the "rectangle" concept of proteome size as a product of proteome width and depth. The discussion on proteome width culminated with the introduction of digital biomarkers-low-copied aberrant proteins that differ from their typical forms by PTMs, alternative splicing, or single amino acid polymorphisms. The aberrant proteoforms, a complement to whole-genome proteomic surveys, were presented as an ultimate goal for the proteomic community.

Meeting new challenges: The 2014 HUPO-PSI/COSMOS Workshop: 13-15 April 2014, Frankfurt, Germany.

The Annual 2014 Spring Workshop of the Proteomics Standards Initiative (PSI) of the Human Proteome Organization (HUPO) was held this year jointly with the metabolomics COordination of Standards in MetabOlomicS (COSMOS) group. The range of existing MS standards (mzML, mzIdentML, mzQuantML, mzTab, TraML) was reviewed and updated in the light of new methodologies and advances in technologies. Adaptations to meet the needs of the metabolomics community were incorporated and a new data format for NMR, nmrML, was presented. The molecular interactions workgroup began work on a new version of the existing XML data interchange format. PSI-MI XML3.0 will enable the capture of more abstract data types such as protein complex topology derived from experimental data, allosteric binding, and dynamic interactions. Further information about the work of the HUPO-PSI can be found at http://www.psidev.info.

New copper resistance determinants in the extremophile acidithiobacillus ferrooxidans: a quantitative proteomic analysis.

Acidithiobacillus ferrooxidans is an extremophilic bacterium used in biomining processes to recover metals. The presence in A. ferrooxidans ATCC 23270 of canonical copper resistance determinants does not entirely explain the extremely high copper concentrations this microorganism is able to stand, suggesting the existence of other efficient copper resistance mechanisms. New possible copper resistance determinants were searched by using 2D-PAGE, real time PCR (qRT-PCR) and quantitative proteomics with isotope-coded protein labeling (ICPL). A total of 594 proteins were identified of which 120 had altered levels in cells grown in the presence of copper. Of this group of proteins, 76 were up-regulated and 44 down-regulated. The up-regulation of RND-type Cus systems and different RND-type efflux pumps was observed in response to copper, suggesting that these proteins may be involved in copper resistance. An overexpression of most of the genes involved in histidine synthesis and several of those annotated as encoding for cysteine production was observed in the presence of copper, suggesting a possible direct role for these metal-binding amino acids in detoxification. Furthermore, the up-regulation of putative periplasmic disulfide isomerases was also seen in the presence of copper, suggesting that they restore copper-damaged disulfide bonds to allow cell survival. Finally, the down-regulation of the major outer membrane porin and some ionic transporters was seen in A. ferrooxidans grown in the presence of copper, indicating a general decrease in the influx of the metal and other cations into the cell. Thus, A. ferrooxidans most likely uses additional copper resistance strategies in which cell envelope proteins are key components. This knowledge will not only help to understand the mechanism of copper resistance in this extreme acidophile but may help also to select the best fit members of the biomining community to attain more efficient industrial metal leaching processes.

Surfing transcriptomic landscapes. A step beyond the annotation of chromosome 16 proteome.

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.

Proteomic analysis reveals heat shock protein 70 has a key role in polycythemia Vera.

JAK-STAT signaling through the JAK2V617F mutation is central to the pathogenesis of myeloproliferative neoplasms (MPN). However, other events could precede the JAK2 mutation. The aim of this study is to analyze the phenotypic divergence between polycytemia vera (PV) and essential thrombocytemia (ET) to find novel therapeutics targets by a proteomic and functional approach to identify alternative routes to JAK2 activation. Through 2D-DIGE and mass spectrometry of granulocyte protein from 20 MPN samples, showed differential expression of HSP70 in PV and ET besides other 60 proteins. Immunohistochemistry of 46 MPN bone marrow samples confirmed HSP70 expression. The median of positive granulocytes was 80% in PV (SD 35%) vs. 23% in ET (SD 34.25%). In an ex vivo model KNK437 was used as an inhibition model assay of HSP70, showed dose-dependent inhibition of cell growth and burst formation unit erythroid (BFU-E) in PV and ET, increased apoptosis in the erythroid lineage, and decreased pJAK2 signaling, as well as a specific siRNA for HSP70. These data suggest a key role for HSP70 in proliferation and survival of the erythroid lineage in PV, and may represent a potential therapeutic target in MPN, especially in PV.

Quantitative proteomics: A strategic ally to map protein interaction networks.

Many physiological processes are regulated by dynamic protein interaction networks whose characterization provides valuable information on cell biology. Several strategies can be used to analyze protein-protein interactions. Among them, affinity purification combined with mass spectrometry (AP-MS) is arguably the most widely employed technique, not only owing to its high throughput and sensitivity but also because it can answer critical questions such as where, when, and how protein-protein interactions occur. In AP-MS workflows, both the target protein and its interacting partners are isolated before being identified by MS. The main challenge of this approach is to distinguish bona fide binders from background contaminants. This review focuses on the different strategies designed to circumvent this limitation. In this regard, the combination of quantitative proteomics and affinity purification emerges as one of the most powerful, yet relatively simple, strategies to characterize protein-protein interactions.

Five years later: the current status of the use of proteomics and transcriptomics in EMF research.

The World Health Organization's and Radiation and Nuclear Safety Authority's "Workshop on Application of Proteomics and Transcriptomics in Electromagnetic Fields Research" was held in Helsinki in the October/November 2005. As a consequence of this meeting, Proteomics journal published in 2006 a special issue "Application of Proteomics and Transcriptomics in EMF Research" (Vol. 6 No. 17; Guest Editor: D. Leszczynski). This Proteomics issue presented the status of research, of the effects of electromagnetic fields (EMF) using proteomics and transcriptomics methods, present in 2005. The current overview/opinion article presents the status of research in this area by reviewing all studies that were published by the end of 2010. The review work was a part of the European Cooperation in the Field of Scientific and Technical Research (COST) Action BM0704 that created a structure in which researchers in the field of EMF and health shared knowledge and information. The review was prepared by the members of the COST Action BM0704 task group on the high-throughput screening techniques and electromagnetic fields (TG-HTST-EMF).

From proteomics data representation to public data flow: a report on the HUPO-PSI workshop September 2011, Geneva, Switzerland.

The plenary session of the Proteomics Standards Initiative (PSI) of the Human Proteome Organization at the Tenth annual HUPO World Congress updated the delegates on the ongoing activities of this group. The Molecular Interactions workgroup described the success of the PSICQUIC web service, which enables users to access multiple interaction resources with a single query. One user instance is the IMEx Consortium, which uses the service to enable users to access a non-redundant set of protein-protein interaction records. The mass spectrometry data formats, mzML for mass spectrometer output files and mzIdentML for the output of search engines, are now successfully established with increasing numbers of implementations. A format for the output of quantitative proteomics data, mzQuantML, and also TraML, for SRM/MRM transition lists, are both currently nearing completion. The corresponding MIAPE documents are being updated in line with advances in the field, as is the shared controlled vocabulary PSI-MS. In addition, the mzTab format was introduced, as a simpler way to report MS proteomics and metabolomics results. Finally, the ProteomeXchange Consortium, which will supply a single entry point for the submission of MS proteomics data to multiple data resources including PRIDE and PeptideAtlas, is currently being established.

PRIDEViewer: a novel user-friendly interface to visualize PRIDE XML files.

Current standardization initiatives have greatly contributed to share the information derived by proteomics experiments. One of these initiatives is the XML-based repository PRIDE (PRoteomics IDEntification database), although an XML-based document does not appear to present a user-friendly view at the first glance. PRIDEViewer is a novel Java-based application that presents the information available in a PRIDE XML file in a user-friendly manner, facilitating the interaction among end users as well as the understanding and evaluation of the compiled information. PRIDEViewer is freely available at: http://proteo.cnb.csic.es/prideviewer/.

Enabling BioSharing - a report on the Annual Spring Workshop of the HUPO-PSI April 11-13, 2011, EMBL-Heidelberg, Germany.

The Annual Spring workshop of the HUPO-PSI was this year held at the EMBL International Centre for Advanced Training (EICAT) in Heidelberg, Germany. Delegates briefly reviewed the successes of the group to date. These include the wide spread implementation of the molecular interaction data exchange formats, PSI-MI XML2.5 and MITAB, and also of mzML, the standard output format for mass spectrometer output data. These successes have resulted in enhanced accessibility to published data, for example the development of the PSICQUIC common query interface for interaction data and the development of databases such as PRIDE to act as public repositories for proteomics data and increased biosharing, through the development of consortia, for example IMEx and ProteomeXchange which will both share the burden of curating the increasing amounts of data being published and work together to make this more accessible to the bench scientist. Work then started over the three days of the workshop, with a focus on advancing the draft format for handling quantitative mass spectrometry data (mzQuantML) and further developing TraML, a standardized format for the exchange and transmission of transition lists for SRM experiments.

Quantitative proteome and acidic subproteome profiling of Candida albicans yeast-to-hypha transition.

Candida albicans yeast-to-hypha morphological transition is involved in the virulence strategy of this opportunistic fungal pathogen. Changes in relative abundance of the Candida proteome related to this process were analyzed using different two-dimensional differential in-gel electrophoresis (2D-DIGE)-based approaches. First, a comparative analysis of yeast and hyphal cytoplasmic proteins allowed the detection of 106 protein spots with significant variation in abundance. Sixty-one of them, corresponding to 46 proteins, were identified. As most of the differentially abundant proteins had an acidic isoelectric point, a large-scale prefractionation approach to analyze the acidic C. albicans subproteome was carried out. Ninety acidic C. albicans proteins were identified by either gel-based or nongel-based approaches. Additionally, different workflows combining preparative isoelectric focusing, Cy labeling, and narrow pH gradient 2-DE gels were tested to analyze the differences in relative protein abundance between yeast and hyphal acidic subproteomes. It was possible to identify 21 differentially abundant acidic proteins; 10 of them were not identified in the previous 2D-DIGE gels. Functional and network interaction analyses of the 56 differentially abundant proteins identified by both approaches rendered an integrated view of metabolic and cellular process reorganization during the yeast-to-hypha transition. With these results, we propose a model of metabolic reorganization.

Inverse regulation in the metabolic genes pckA and metE revealed by proteomic analysis of the Salmonella RcsCDB regulon.

The RcsC, RcsD, and RcsB proteins compose a system used by enteric bacteria to sense envelope stress. Signal transmission occurs from the sensor RcsC to the transcriptional regulator RcsB. Accessory proteins, such as IgaA, are known to adjust the response level. In a previous transcriptomic study, we uncovered 85 genes differentially expressed in Salmonella enterica serovar Typhimurium igaA mutants. Here, we extended these observations to proteomics by performing differential isotope-coded protein labeling (ICPL) followed by liquid chromatography-electrospray ionization tandem mass spectrometry. Five-hundred five proteins were identified and quantified, with 75 of them displaying significant changes in response to alterations in the RcsCDB system. Divergent expression at the RNA and protein level was observed for the metabolic genes pckA and metE, involved in gluconeogenesis and methionine synthesis, respectively. When analyzed in diverse environmental conditions, including the intracellular niche of eukaryotic cells, inverse regulation was more evident for metE and in bacteria growing in defined minimal medium or to stationary phase. The RcsCDB system was also shown to repress the synthesis of the small RNA FnrS, previously reported to modulate metE expression. Collectively, these findings provide new insights into post-transcriptional regulatory mechanisms involving the RcsCDB system and its control over metabolic functions.