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 .

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.

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.

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.

Cyr61 as mediator of Src signaling in triple negative breast cancer cells.

SFKs are involved in tumorigenesis and metastasis. Here we analyzed c-Src contribution to initial steps of metastasis by tetracycline-dependent expression of a specific shRNA-c-Src, which suppressed c-Src mRNA and protein levels in metastatic MDA-MB-231 cells. c-Src suppression did not alter cell proliferation or survival, but it significantly reduced anchorage-independent growth. Concomitantly with diminished tyrosine-phosphorylation/activation of Fak, caveolin-1, paxillin and p130CAS, c-Src depletion also inhibited cellular migration, invasion and transendothelial migration. Quantitative proteomic analyses of the secretome showed that Cyr61 levels, which were detected in the exosomal fraction, were diminished upon shRNA-c-Src expression. In contrast, Cyr61 expression was unaltered inside cells. Cyr61 partially colocalized with cis-Golgi gp74 marker and with exosomal marker CD63, but c-Src depletion did not alter their cellular distribution. In SUM159PT cells, transient c-Src suppression also reduced secreted exosomal Cyr61 levels. Furthermore, conditional expression of a c-Src dominant negative mutant (SrcDN, c-Src-K295M/Y527F) in MDA-MB-231 and in SUM159PT diminished secreted Cyr61 as well. Cyr61 transient suppression in MDA-MB-231 inhibited invasion and transendothelial migration. Finally, in both MDA-MB-231 and SUM159PT, a neutralizing Cyr61 antibody restrained migration. Collectively, these results suggest that c-Src regulates secreted proteins, including the exosomal Cyr61, which are involved in modulating the metastatic potential of triple negative breast cancer cells.

Development of data representation standards by the human proteome organization proteomics standards initiative.

OBJECTIVE: To describe the goals of the Proteomics Standards Initiative (PSI) of the Human Proteome Organization, the methods that the PSI has employed to create data standards, the resulting output of the PSI, lessons learned from the PSI's evolution, and future directions and synergies for the group. MATERIALS AND METHODS: The PSI has 5 categories of deliverables that have guided the group. These are minimum information guidelines, data formats, controlled vocabularies, resources and software tools, and dissemination activities. These deliverables are produced via the leadership and working group organization of the initiative, driven by frequent workshops and ongoing communication within the working groups. Official standards are subjected to a rigorous document process that includes several levels of peer review prior to release. RESULTS: We have produced and published minimum information guidelines describing what information should be provided when making data public, either via public repositories or other means. The PSI has produced a series of standard formats covering mass spectrometer input, mass spectrometer output, results of informatics analysis (both qualitative and quantitative analyses), reports of molecular interaction data, and gel electrophoresis analyses. We have produced controlled vocabularies that ensure that concepts are uniformly annotated in the formats and engaged in extensive software development and dissemination efforts so that the standards can efficiently be used by the community.Conclusion In its first dozen years of operation, the PSI has produced many standards that have accelerated the field of proteomics by facilitating data exchange and deposition to data repositories. We look to the future to continue developing standards for new proteomics technologies and workflows and mechanisms for integration with other omics data types. Our products facilitate the translation of genomics and proteomics findings to clinical and biological phenotypes. The PSI website can be accessed at http://www.psidev.info.

The Spanish biology/disease initiative within the human proteome project: Application to rheumatic diseases.

The Spanish Chromosome 16 consortium is integrated in the global initiative Human Proteome Project, which aims to develop an entire map of the proteins encoded following a gene-centric strategy (C-HPP) in order to make progress in the understanding of human biology in health and disease (B/D-HPP). Chromosome 16 contains many genes encoding proteins involved in the development of a broad range of diseases, which have a significant impact on the health care system. The Spanish HPP consortium has developed a B/D platform with five programs focused on selected medical areas: cancer, obesity, cardiovascular, infectious and rheumatic diseases. Each of these areas has a clinical leader associated to a proteomic investigator with the responsibility to get a comprehensive understanding of the proteins encoded by Chromosome 16 genes. Proteomics strategies have enabled great advances in the area of rheumatic diseases, particularly in osteoarthritis, with studies performed on joint cells, tissues and fluids. BIOLOGICAL SIGNIFICANCE: In this manuscript we describe how the Spanish HPP-16 consortium has developed a B/D platform with five programs focused on selected medical areas: cancer, obesity, cardiovascular, infectious and rheumatic diseases. Each of these areas has a clinical leader associated to a proteomic investigator with the responsibility to get a comprehensive understanding of the proteins encoded by Chromosome 16 genes. We show how the Proteomic strategy has enabled great advances in the area of rheumatic diseases, particularly in osteoarthritis, with studies performed on joint cells, tissues and fluids. This article is part of a Special Issue entitled: HUPO 2014.

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.

Response to copper of Acidithiobacillus ferrooxidans ATCC 23270 grown in elemental sulfur.

The response of Acidithiobacillus ferrooxidans ATCC 23270 to copper was analyzed in sulfur-grown cells by using quantitative proteomics. Forty-seven proteins showed altered levels in cells grown in the presence of 50 mM copper sulfate. Of these proteins, 24 were up-regulated and 23 down-regulated. As seen before in ferrous iron-grown cells, there was a notorious up-regulation of RND-type Cus systems and different RND-type efflux pumps, indicating that these proteins are very important in copper resistance. Copper also triggered the down-regulation of the major outer membrane porin of A. ferrooxidans in sulfur-grown bacteria, suggesting they respond to the metal by decreasing the influx of cations into the cell. On the contrary, copper in sulfur-grown cells caused an overexpression of putative TadA and TadB proteins known to be essential for biofilm formation in bacteria. Surprisingly, sulfur-grown microorganisms showed increased levels of proteins related with energy generation (rus and petII operons) in the presence of copper. Although rus operon is overexpressed mainly in cells grown in ferrous iron, the up-regulation of rusticyanin in sulfur indicates a possible role for this protein in copper resistance as well. Finally, copper response in A. ferrooxidans appears to be influenced by the substrate being oxidized by the microorganism.

A DERL3-associated defect in the degradation of SLC2A1 mediates the Warburg effect.

Cancer cells possess aberrant proteomes that can arise by the disruption of genes involved in physiological protein degradation. Here we demonstrate the presence of promoter CpG island hypermethylation-linked inactivation of DERL3 (Derlin-3), a key gene in the endoplasmic reticulum-associated protein degradation pathway, in human tumours. The restoration of in vitro and in vivo DERL3 activity highlights the tumour suppressor features of the gene. Using the stable isotopic labelling of amino acids in cell culture workflow for differential proteome analysis, we identify SLC2A1 (glucose transporter 1, GLUT1) as a downstream target of DERL3. Most importantly, SLC2A1 overexpression mediated by DERL3 epigenetic loss contributes to the Warburg effect in the studied cells and pinpoints a subset of human tumours with greater vulnerability to drugs targeting glycolysis.

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.

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.

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.

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.

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.