Rosana Navajas

Generalized Method for Probability-based Peptide and Protein Identification from Tandem Mass Spectrometry Data and Sequence Database Searching

Tandem mass spectrometry-based proteomics is currently in great demand of computational methods that facilitate the elimination of likely false positives in peptide and protein identification. In the last few years, a number of new peptide identification programs have been described, but scores or other significance measures reported by these programs cannot always be directly translated into an easy to interpret error rate measurement such as the false discovery rate. In this work we used generalized lambda distributions to model frequency distributions of database search scores computed by MASCOT, X!TANDEM with k-score plug-in, OMSSA, and InsPecT. From these distributions, we could successfully estimate p values and false discovery rates with high accuracy. From the set of peptide assignments reported by any of these engines, we also defined a generic protein scoring scheme that enabled accurate estimation of protein-level p values by simulation of random score distributions that was also found to yield good estimates of protein-level false discovery rate. The performance of these methods was evaluated by searching four freely available data sets ranging from 40,000 to 285,000 MS/MS spectra.

Guidelines for reporting quantitative mass spectrometry based experiments in proteomics

UNLABELLED Mass spectrometry is already a well-established protein identification tool and recent methodological and technological developments have also made possible the extraction of quantitative data of protein abundance in large-scale studies. Several strategies for absolute and relative quantitative proteomics and the statistical assessment of quantifications are possible, each having specific measurements and therefore, different data analysis workflows. The guidelines for Mass Spectrometry Quantification allow the description of a wide range of quantitative approaches, including labeled and label-free techniques and also targeted approaches such as Selected Reaction Monitoring (SRM). BIOLOGICAL SIGNIFICANCE The HUPO Proteomics Standards Initiative (HUPO-PSI) has invested considerable efforts to improve the standardization of proteomics data handling, representation and sharing through the development of data standards, reporting guidelines, controlled vocabularies and tooling. In this manuscript, we describe a key output from the HUPO-PSI-namely the MIAPE Quant guidelines, which have developed in parallel with the corresponding data exchange format mzQuantML [1]. The MIAPE Quant guidelines describe the HUPO-PSI proposal concerning the minimum information to be reported when a quantitative data set, derived from mass spectrometry (MS), is submitted to a database or as supplementary information to a journal. The guidelines have been developed with input from a broad spectrum of stakeholders in the proteomics field to represent a true consensus view of the most important data types and metadata, required for a quantitative experiment to be analyzed critically or a data analysis pipeline to be reproduced. It is anticipated that they will influence or be directly adopted as part of journal guidelines for publication and by public proteomics databases and thus may have an impact on proteomics laboratories across the world. This article is part of a Special Issue entitled: Standardization and Quality Control.

β‐arrestin‐1 mediates the TCR‐triggered re‐routing of distal receptors to the immunological synapse by a PKC‐mediated mechanism

T‐cell receptors (TCR) recognize their antigen ligand at the interface between T cells and antigen‐presenting cells, known as the immunological synapse (IS). The IS provides a means of sustaining the TCR signal which requires the continual supply of new TCRs. These are endocytosed and redirected from distal membrane locations to the IS. In our search for novel cytoplasmic effectors, we have identified β‐arrestin‐1 as a ligand of non‐phosphorylated resting TCRs. Using dominant‐negative and knockdown approaches we demonstrate that β‐arrestin‐1 is required for the internalization and downregulation of non‐engaged bystander TCRs. Furthermore, TCR triggering provokes the β‐arrestin‐1‐mediated downregulation of the G‐protein coupled chemokine receptor CXCR4, but not of other control receptors. We demonstrate that β‐arrestin‐1 recruitment to the TCR, and bystander TCR and CXCR4 downregulation, are mechanistically mediated by the TCR‐triggered PKC‐mediated phosphorylation of β‐arrestin‐1 at Ser163. This mechanism allows the first triggered TCRs to deliver a stop migration signal, and to promote the internalization of distal TCRs and CXCR4 and their translocation to the IS. This receptor crosstalk mechanism is critical to sustain the TCR signal.

The ProteoRed MIAPE web toolkit: A User-friendly Framework to Connect and Share Proteomics Standards

The development of the HUPO-PSIs (Proteomics Standards Initiative) standard data formats and MIAPE (Minimum Information About a Proteomics Experiment) guidelines should improve proteomics data sharing within the scientific community. Proteomics journals have encouraged the use of these standards and guidelines to improve the quality of experimental reporting and ease the evaluation and publication of manuscripts. However, there is an evident lack of bioinformatics tools specifically designed to create and edit standard file formats and reports, or embed them within proteomics workflows. In this article, we describe a new web-based software suite (The ProteoRed MIAPE web toolkit) that performs several complementary roles related to proteomic data standards. First, it can verify that the reports fulfill the minimum information requirements of the corresponding MIAPE modules, highlighting inconsistencies or missing information. Second, the toolkit can convert several XML-based data standards directly into human readable MIAPE reports stored within the ProteoRed MIAPE repository. Finally, it can also perform the reverse operation, allowing users to export from MIAPE reports into XML files for computational processing, data sharing, or public database submission. The toolkit is thus the first application capable of automatically linking the PSIs MIAPE modules with the corresponding XML data exchange standards, enabling bidirectional conversions. This toolkit is freely available at http://www.proteored.org/MIAPE/.

Incomplete tyrosine 1680 sulphation in recombinant FVIII concentrates

condition of obligate carrier of haemophilia A. When we performed the test with usual combinations of oligonucleotides, 9Fint1h-2F -9cR (reaction 1) and 9Fint1h-2F int1h-2R (reaction 2) for analysis of family members, we found that the proband s mother and daughter were carriers of the same genetic pattern as the proband while the proband s sister did not show Inv1 (Fig. 1). To our knowledge, this is the first report describing this aberrant band pattern. Sukarova et al. reported two cases with an abnormal Inv1 pattern in Macedônia which, however, was different from the one reported here [5]. Preliminary experiments suggest that the defect reported here is due to the presence of an extra int1h copy, mediating the recombination event. In conclusion, we present the case of a patient with severe HA with an aberrant pattern of Inv1 not reported to date. This case confirms the importance of detecting the Inv1 and illustrates the complex molecular mechanisms associated with HA.

hCLE/C14orf166 Associates with DDX1-HSPC117-FAM98B in a Novel Transcription-Dependent Shuttling RNA-Transporting Complex

hCLE/C14orf166 is a nuclear and cytoplasmic protein that interacts with the RNAP II, modulates nuclear RNA metabolism and is present in cytoplasmic RNA granules involved in localized translation. Here we have studied whether hCLE shares common interactors in the nucleus and the cytosol, which could shed light on its participation in the sequential phases of RNA metabolism. Nuclear and cytoplasmic purified hCLE-associated factors were identified and proteins involved in mRNA metabolism, motor-related proteins, cytoskeletal and translation-related factors were found. Purified hCLE complexes also contain RNAs and as expected some hCLE-interacting proteins (DDX1, HSPC117, FAM98B) were found both in the nucleus and the cytoplasm. Moreover, endogenous hCLE fractionates in protein complexes together with DDX1, HSPC117 and FAM98B and silencing of hCLE down-regulates their nuclear and cytosolic accumulation levels. Using a photoactivatable hCLE-GFP protein, nuclear import and export of hCLE was observed indicating that hCLE is a shuttling protein. Interestingly, hCLE nuclear import required active transcription, as did the import of DDX1, HSPC117 and FAM98B proteins. The data indicate that hCLE probably as a complex with DDX1, HSPC117 and FAM98B shuttles between the nucleus and the cytoplasm transporting RNAs suggesting that this complex has a prominent role on nuclear and cytoplasmic RNA fate.

Immobilized Metal Affinity Chromatography/Reversed-Phase Enrichment of Phosphopeptides and Analysis by CID/ETD Tandem Mass Spectrometry

Major difficulties in phosphoprotein analysis relate to the presence of a huge number of nonphosphorylated proteins and to the wide concentration dynamic range among them. In order to overcome the analysis complexity, specific clean-up and highly efficient enrichment procedures are mandatory prior to the -chromatographic separation and identification by tandem mass spectrometry. In this chapter, a procedure based on immobilized metal affinity chromatography (IMAC)/reversed-phase phosphopeptide purification and analysis by nanoHPLC-ESI-MS/MS with ion trap is described in detail. CID (collision-induced -dissociation) and ETD (electron-transfer dissociation) fragmentation techniques are used in combination to specifically determine phosphorylation sites inside the peptide sequences, through the analysis of MS/MS spectra.

Arabidopsis Responds to Alternaria alternata Volatiles by Triggering Plastid Phosphoglucose Isomerase-Independent Mechanisms

Cytokinin-mediated responses of Arabidopsis to volatile compounds emitted by pathogenic microorganisms involve the activation of plastidic phosphoglucose isomerase-independent mechanisms. Volatile compounds (VCs) emitted by phylogenetically diverse microorganisms (including plant pathogens and microbes that do not normally interact mutualistically with plants) promote photosynthesis, growth, and the accumulation of high levels of starch in leaves through cytokinin (CK)-regulated processes. In Arabidopsis (Arabidopsis thaliana) plants not exposed to VCs, plastidic phosphoglucose isomerase (pPGI) acts as an important determinant of photosynthesis and growth, likely as a consequence of its involvement in the synthesis of plastidic CKs in roots. Moreover, this enzyme plays an important role in connecting the Calvin-Benson cycle with the starch biosynthetic pathway in leaves. To elucidate the mechanisms involved in the responses of plants to microbial VCs and to investigate the extent of pPGI involvement, we characterized pPGI-null pgi1-2 Arabidopsis plants cultured in the presence or absence of VCs emitted by Alternaria alternata. We found that volatile emissions from this fungal phytopathogen promote growth, photosynthesis, and the accumulation of plastidic CKs in pgi1-2 leaves. Notably, the mesophyll cells of pgi1-2 leaves accumulated exceptionally high levels of starch following VC exposure. Proteomic analyses revealed that VCs promote global changes in the expression of proteins involved in photosynthesis, starch metabolism, and growth that can account for the observed responses in pgi1-2 plants. The overall data show that Arabidopsis plants can respond to VCs emitted by phytopathogenic microorganisms by triggering pPGI-independent mechanisms.

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.

Translocase and major signal peptidase malfunctions affect aerial mycelium formation in Streptomyces lividans

Deficiency in the translocase complex (SecG mutant strain) or in the major type I signal peptidase (SipY mutant strain) function in Streptomyces lividans resulted, as expected, in a drastic reduction of secretory protein production and in a bald phenotype. The transcriptional profiling of both strains showed that the expression of a set of genes involved in the morphological differentiation process was down regulated in both mutant strains (bldG, bldN and bldM), whereas bldA and bldH were only down-regulated in the SipY mutant strain. Consistently, low temperature scanning electron microscopy revealed that the disruption of sipY had a more noticeable effect in the growth/morphological aspect of the mycelium than that of secG, suggesting that in the sipY mutant, the blockage of the export process might have more severe consequences than in the secG mutant. In both cases, the likely degradation of the proteins that cannot be secreted might provide nutrients that might be responsible for the lack of induction of the bald cascade, which is thought to be triggered under conditions of nutritional limitation.