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p38α (encoded by MAPK14) is a protein kinase that regulates cellular responses to almost all types of environmental and intracellular stresses. Upon activation, p38α phosphorylates many substrates both in the cytoplasm and nucleus, allowing this pathway to regulate a wide variety of cellular processes. While the role of p38α in the stress response has been widely investigated, its implication in cell homeostasis is less understood. To investigate the signaling networks regulated by p38α in normally proliferating cancer cells, we performed quantitative proteomic and phosphoproteomic analyses in breast cancer cells in which this pathway had been either genetically targeted or chemically inhibited. Our study identified with high confidence 35 proteins and 82 phosphoproteins (114 phosphosites) that are modulated by p38α, and highlighted the implication of various protein kinases, including MK2 and mTOR, in the p38α-regulated signaling networks. Moreover, functional analyses revealed an important contribution of p38α to the regulation of cell adhesion, DNA replication and RNA metabolism. Indeed, we provide experimental evidence supporting that p38α negatively regulates cell adhesion, and showed that this p38α function is likely mediated by the modulation of the adaptor protein ArgBP2. Collectively, our results illustrate the complexity of the p38α regulated signaling networks, provide valuable information on p38α-dependent phosphorylation events in cancer cells, and document a mechanism by which p38α can regulate cell adhesion.

We describe furan as a triggerable 'warhead' for site-specific cross-linking using the actin and thymosin β4 (Tβ4)-complex as model of a weak and dynamic protein-protein interaction (PPI) with known 3D structure and with application potential in disease contexts. The identified cross-linked residues demonstrate that lysine is a target for the furan warhead. The presented in vitro validation of covalently acting 'furan-armed' Tβ4-variants provides initial proof to further exploit furan-technology for covalent drug design targeting lysines.

We report a quantitative proteomics data analysis pipeline which, coupled to protein-directed dynamic combinatorial chemistry (DDC) experiments, enables the rapid discovery and direct characterization of protein-protein interaction (PPI) modulators. A low-affinity PD-1 binder was incubated with a library of >100 D-peptides under thiol-exchange favoring conditions, in the presence of the target protein PD-1, and we determined the S-linked dimeric species that resulted amplified in the protein samples versus the controls. We chemically synthesized the target dimer candidates and validated them by thermophoresis binding and protein-protein interaction assays. The results provide a proof-of-concept for using this strategy in the high-throughput search of improved drug-like peptide binders that block therapeutically relevant protein-protein interactions.

Background Vertebrate CPEB proteins bind mRNAs at cytoplasmic polyadenylation elements (CPEs) in their 3′ UTRs, leading to cytoplasmic changes in their poly(A) tail lengths; this can promote translational repression or activation of the mRNA. However, neither the regulation nor the mechanisms of action of the CPEB family per se have been systematically addressed to date. Results Based on a comparative analysis of the four vertebrate CPEBs, we determine their differential regulation by phosphorylation, the composition and properties of their supramolecular assemblies, and their target mRNAs. We show that all four CPEBs are able to recruit the CCR4-NOT deadenylation complex to repress the translation. However, their regulation, mechanism of action, and target mRNAs define two subfamilies. Thus, CPEB1 forms ribonucleoprotein complexes that are remodeled upon a single phosphorylation event and are associated with mRNAs containing canonical CPEs. CPEB2–4 are regulated by multiple proline-directed phosphorylations that control their liquid–liquid phase separation. CPEB2–4 mRNA targets include CPEB1-bound transcripts, with canonical CPEs, but also a specific subset of mRNAs with non-canonical CPEs. Conclusions Altogether, these results show how, globally, the CPEB family of proteins is able to integrate cellular cues to generate a fine-tuned adaptive response in gene expression regulation through the coordinated actions of all four members.

Alzheimer’s disease (AD) is the most common form of dementia. Over fifty years of intense research have revealed many key elements of the biology of this neurodegenerative disorder. However, our understanding of the molecular bases of the disease is still incomplete, and the medical treatments available for AD are mainly symptomatic and hardly effective. Indeed, the robustness of biological systems has revealed that the modulation of a single target is unlikely to yield the desired outcome and we should therefore move from gene-centric to systemic therapeutic strategies. Here we present the complete characterization of three murine models of AD at different stages of the disease (i.e. onset, progression and advanced). To identify genotype-to-phenotype relationships, we combine the cognitive assessment of these mice with histological analyses and full transcriptional and protein quantification profiling of the hippocampus. Comparison of the gene and protein expression trends observed in AD progression and physiological aging revealed certain commonalities, such as the upregulation of microglial and inflammation markers. However, although AD models show accelerated aging, other factors specifically associated with Aβ pathology are involved. Despite the clear correlation between mRNA and protein levels of the dysregulated genes, we discovered a few proteins whose abundance increases with AD progression, while the corresponding transcript levels remain stable. Indeed, we show that at least two of these proteins, namely lfit3 and Syt11, co-localize with Aβ plaques in the brain. Finally, we derived specific Aβ-related molecular AD signatures and looked for drugs able to globally revert them. We found two NSAIDs (dexketoprofen and etodolac) and two anti-hypertensives (penbutolol and bendroflumethiazide) that overturn the cognitive impairment in AD mice while reducing Aβ plaques in the hippocampus and partially restoring the physiological levels of AD signature genes to wild-type levels.TeaserThe comprehensive characterization of three AD mouse models reveals disease signatures that we used to identify approved drugs able to modify the etiology of the pathology and overturn cognitive impairment.

The Tousled-like kinases 1 and 2 (TLK1 and TLK2) are involved in many fundamental processes, including DNA replication, cell cycle checkpoint recovery and chromatin remodelling. Mutations inWe re-evaluate whole exome sequencing and array-CGH data from a large cohort of patients affected by neurodevelopmental disorders. Using spatial proteomics (BioID) and single-cell gel electrophoresis, we investigated the proximity interaction landscape ofWe identified three new unrelated MRD57 families. Two were sporadic and caused by a missense change (c.1652AG; p.(Asp551Gly)) or a 39 kb deletion encompassingOur study identified novel

IntroductionThe Tousled-like kinases 1 and 2 (TLK1 and TLK2) are involved in many fundamental processes, including DNA replication, cell cycle checkpoint recovery and chromatin remodelling. Mutations in TLK2 were recently associated with ‘Mental Retardation Autosomal Dominant 57’ (MRD57, MIM# 618050), a neurodevelopmental disorder characterised by a highly variable phenotype, including mild-to-moderate intellectual disability, behavioural abnormalities, facial dysmorphisms, microcephaly, epilepsy and skeletal anomalies.MethodsWe re-evaluate whole exome sequencing and array-CGH data from a large cohort of patients affected by neurodevelopmental disorders. Using spatial proteomics (BioID) and single-cell gel electrophoresis, we investigated the proximity interaction landscape of TLK2 and analysed the effects of p.(Asp551Gly) and a previously reported missense variant (c.1850C>T; p.(Ser617Leu)) on TLK2 interactions, localisation and activity.ResultsWe identified three new unrelated MRD57 families. Two were sporadic and caused by a missense change (c.1652A>G; p.(Asp551Gly)) or a 39 kb deletion encompassing TLK2, and one was familial with three affected siblings who inherited a nonsense change from an affected mother (c.1423G>T; p.(Glu475Ter)). The clinical phenotypes were consistent with those of previously reported cases. The tested mutations strongly impaired TLK2 kinase activity. Proximal interactions between TLK2 and other factors implicated in neurological disorders, including CHD7, CHD8, BRD4 and NACC1, were identified. Finally, we demonstrated a more relaxed chromatin state in lymphoblastoid cells harbouring the p.(Asp551Gly) variant compared with control cells, conferring susceptibility to DNA damage.ConclusionOur study identified novel TLK2 pathogenic variants, confirming and further expanding the MRD57-related phenotype. The molecular characterisation of missense variants increases our knowledge about TLK2 function and provides new insights into its role in neurodevelopmental disorders.

Protamine 1 (P1) and protamine 2 (P2) family are extremely basic, sperm-specific proteins, packing 85-95% of the paternal DNA. P1 is synthesized as a mature form, whereas P2 components (HP2, HP3, and HP4) arise from the proteolysis of the precursor (pre-P2). Due to the particular protamine physical-chemical properties, their identification by standardized bottom-up mass spectrometry (MS) strategies is not straightforward. Therefore, the aim of this study was to identify the sperm protamine proteoforms profile, including their post-translational modifications, in normozoospermic individuals using two complementary strategies, a top-down MS approach and a proteinase-K-digestion-based bottom-up MS approach. By top-down MS, described and novel truncated P1 and pre-P2 proteoforms were identified. Intact P1, pre-P2, and P2 mature proteoforms and their phosphorylation pattern were also detected. Additionally, a +61 Da modification in different proteoforms was observed. By the bottom-up MS approach, phosphorylated residues for pre-P2, as well as the new P2 isoform 2, which is not annotated in the UniProtKB database, were revealed. Implementing these strategies in comparative studies of different infertile phenotypes, together with the evaluation of P1/P2 and pre-P2/P2 MS-derived ratios, would permit determining specific alterations in the protamine proteoforms and elucidate the role of phosphorylation/dephosphorylation dynamics in male fertility.

Cerebrospinal fluid (CSF) is the fluid of choice to study pathologies and disorders of the central nervous system (CNS). Its composition, especially its proteins and peptides, holds the promise that it may reflect the pathological state of an individual. Traditionally, proteins and peptides in CSF have been analyzed using bottom-up proteomics technologies in the search of high proteome coverage. However, the limited protein sequence coverage of this technology means that information regarding post-translational modifications (PTMs) and alternative splice variants is lost. As an alternative technology, top-down proteomics offers low to medium proteome coverage, but high protein coverage enabling almost a full characterization of the proteins' primary structure. This allows us to precisely identify distinct molecular forms of proteins (proteoforms) as well as naturally occurring bioactive peptide fragments, which could be of critical biological relevance and would otherwise remain undetected with a classical proteomics approach.Here, we describe various strategies including sample preparation protocols, off-line intact protein prefractionation, and LC-MS/MS methods together with data analysis pipelines to analyze cerebrospinal fluid (CSF) by top-down proteomics. However, there is not a unique or standardized method and the selection of the top-down strategy will depend on the exact goal of the study. Here, we describe various top-down proteomics methods that enable rapid protein characterization and may be an excellent companion analytical workflow in the search for new protein biomarkers in neurodegenerative diseases.

Drosophila l(3)mbt malignant brain tumors present sexual dimorphism., The notable male predominance across many human cancer types remains unexplained. Here, we show that Drosophila l(3)mbt brain tumors are more invasive and develop as malignant neoplasms more often in males than in females. By quantitative proteomics, we have identified a signature of proteins that are differentially expressed between male and female tumor samples. Prominent among them is the conserved chromatin reader PHD finger protein 7 (Phf7). We show that Phf7 depletion reduces sex-dependent differences in gene expression and suppresses the enhanced malignant traits of male tumors. Our results identify potential regulators of sex-linked tumor dimorphism and show that these genes may serve as targets to suppress sex-linked malignant traits.

Cerebrospinal fluid (CSF) is the fluid of choice to study pathologies and disorders of the central nervous system (CNS). Its composition, especially its proteins and peptides, holds the promise that it may reflect the pathological state of an individual. Traditionally, proteins and peptides in CSF have been analyzed using bottom-up proteomics technologies in the search of high proteome coverage. However, the limited protein sequence coverage of this technology means that information regarding post-translational modifications (PTMs) and alternative splice variants is lost. As an alternative technology, top-down proteomics offers low to medium proteome coverage, but high protein coverage enabling almost a full characterization of the proteins' primary structure. This allows us to precisely identify distinct molecular forms of proteins (proteoforms) as well as naturally occurring bioactive peptide fragments, which could be of critical biological relevance and would otherwise remain undetected with a classical proteomics approach.Here, we describe various strategies including sample preparation protocols, off-line intact protein prefractionation, and LC-MS/MS methods together with data analysis pipelines to analyze cerebrospinal fluid (CSF) by top-down proteomics. However, there is not a unique or standardized method and the selection of the top-down strategy will depend on the exact goal of the study. Here, we describe various top-down proteomics methods that enable rapid protein characterization and may be an excellent companion analytical workflow in the search for new protein biomarkers in neurodegenerative diseases.

Brain-derived amyloid-β (Aβ) dimers are associated with Alzheimer's disease (AD). However, their covalent nature remains controversial. This feature is relevant, as a covalent cross-link has been proposed to make brain-derived dimers (brain dimers) more synaptotoxic than Aβ monomers and would also make them suitable candidates for biomarker development. To resolve this controversy, we here present a three-step approach. First, we validated a type of synthetic cross-linked Aβ (CL Aβ) dimers, obtained by means of the photoinduced cross-linking of unmodified proteins (PICUP) reaction, as well-defined mimics of putative brain CL Aβ dimers. Second, we used these PICUP CL Aβ dimers as standards to improve the isolation of brain Aβ dimers and to develop state-of-the-art mass spectrometry (MS) strategies to allow their characterization. Third, we applied these MS methods to the analysis of brain Aβ dimer samples allowing the detection of the CL [Aβ(6-16)]2 peptide comprising a dityrosine cross-link. This result demonstrates the presence of CL Aβ dimers in the brains of patients with AD and opens up avenues for establishing new therapeutic targets and developing novel biomarkers for this disease.

A selected library of nine novel platinum(II) complexes having differently functionalized 1,2-bis(aminomethyl)cyclohexane carrier ligands with a 1,4-diamino framework and iodides as labile ligands have been synthesized and evaluated in vitro for their tumor cell growth inhibitory activity, in front of one pair of human carcinoma cell lines A2780 and A2780cisR. These cell lines were chosen based on studying all the known main mechanisms of resistance of cisplatin. A2780cisR cells are resistant through a combination of reduced drug transport enhanced DNA repair/tolerance and elevated glutathione (GSH) levels with respect to the parental A2780 cells. Most platinum complexes evaluated showed a very low resistant factor, up to 16 times lower than that of cisplatin, which indicates their ability to overcome the cisplatin resistance in ovarian cancer A2780cisR cells. Structure-activity studies have been performed in order to know the influence of the several organic functionalities (C=C double bond, free OH group, MeO group, etc.) and the stereochemistry on the cytotoxic activity. Moreover, studies of interaction with DNA of these complexes were performed via three techniques: circular dichroism (CD), electrophoresis on agarose gel (EF) and atomic force microscopy (AFM) in order to evaluate the modifications of secondary and tertiary structure of DNA, induced by platinum complexes. These studies allowed us to correlate the IC50 values of complexes and the intensity of interaction to DNA, the main target for these compounds.

© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. We present several bioinformatics applications for the identification and quantification of phosphoproteome components by MS. These applications include a front-end graphical user interface that combines several Thermo RAW formats to MASCOT™ Generic Format extractors (EasierMgf), two graphical user interfaces for search engines OMSSA and SEQUEST (OmssaGui and SequestGui), and three applications, one for the management of databases in FASTA format (FastaTools), another for the integration of search results from up to three search engines (Integrator), and another one for the visualization of mass spectra and their corresponding database search results (JsonVisor). These applications were developed to solve some of the common problems found in proteomic and phosphoproteomic data analysis and were integrated in the workflow for data processing and feeding on our LymPHOS database. Applications were designed modularly and can be used standalone. These tools are written in Perl and Python programming languages and are supported on Windows platforms. They are all released under an Open Source Software license and can be freely downloaded from our software repository hosted at GoogleCode., This work was supported by grants BIO2009-11735 and BIO2013-46492 from the Spanish Ministerio de Ciencia e Innovación. The CSIC/UAB Proteomics Facility of IIBB-CSIC belongs to ProteoRed, PRB2-ISCIII, supported by grant PT13/0001

This article belongs to a special issue Special Issue: Standardization and Quality Control in Proteomics: Technical note.-- et al., 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).

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

Here we demonstrate the potential of nano-UPLC-LTQ-FT-MS and the Byonic™ proteomic search engine for the separation, detection, and identification of N- and O-glycopeptide glycoforms in standard glycoproteins. The use of a BEH C18 nanoACQUITY column allowed the separation of the glycopeptides present in the glycoprotein digest and a baseline-resolution of the glycoforms of the same glycopeptide on the basis of the number of sialic acids. Moreover, we evaluated several acquisition strategies in order to improve the detection and characterization of glycopeptide glycoforms with the maximum number of identification percentages. The proposed strategy is simple to set up with the technology platforms commonly used in proteomic labs. The method allows the straightforward and rapid obtention of a general glycosylated map of a given protein, including glycosites and their corresponding glycosylated structures. The MS strategy selected in this work, based on a gas phase fractionation approach, led to 136 unique peptides from four standard proteins, which represented 78% of the total number of peptides identified. Moreover, the method does not require an extra glycopeptide enrichment step, thus preventing the bias that this step could cause towards certain glycopeptide species. Data are available via ProteomeXchange with identifier PXD003578.We propose a simple and high-throughput glycoproteomics-based methodology that allows the separation of glycopeptide glycoforms on the basis of the number of sialic acids, and their automatic and rapid identification without prior knowledge of protein glycosites or type and structure of the glycans.

Phosphorylation is a reversible post-translational modification, playing a vital role in protein function. In T cells, protein phosphorylation is the key mechanism regulating T cell receptor-driven signaling pathways. In order to gain insights into the phosphoproteome evolution of T cell activation, we performed a large-scale quantitative phosphoproteomics study of Jurkat E6.1 (wild type) and Jurkat gamma1 (Phospholipase gamma1 null) cell clones upon costimulation with anti-CD3 and anti-CD28 antibodies at times ranging from 15 min to as long as 120 min. In total, we identified 5585 phosphopeptides belonging to 2008 phosphoproteins from both cell clones. We detected 130 and 114 novel phosphopeptides in Jurkat E6.1 and Jurkat gamma1 clones, respectively. A significantly lower number of proteins containing regulated phosphorylation sites were identified in Jurkat gamma1 in comparison to Jurkat E6.1, reflecting the vital role of Phospholipase gamma1 in T cell signaling. Several new phosphorylation sites from lymphocyte-specific protein tyrosine kinase (Lck) were identified. Of these, serine-121 showed significant changes in JE6.1 while only small changes in the Jgamma1 clone. Our data may contribute to the current human T cell phosphoproteome and provide a better understanding on T cell receptor signaling. Data are available via ProteomeXchange with identifier PXD002871, This work was supported by project BIO2013-46492-R from MINECO and VAST-CSIC bilateral action (2012VN0003). The CSIC/UAB Proteomics Laboratory of IIBB-CSIC is a member of Proteored, PRB2-ISCIII and is supported by grant PT13/0001, of the PE I+ D+i 2013-2016, funded by ISCIII and FEDER.

1 art. (21 pags., 4 Figs.) + Supplemental Information (13 Pags., 3 Tabls, 6 Figs.). The definitive version is available at: http://www.pnas.org/, Globins constitute a superfamily of proteins widespread in all kingdoms of life, where they fulfill multiple functions, such as efficient O2 transport and modulation of nitric oxide bioactivity. In plants, the most abundant Hbs are the symbiotic leghemoglobins (Lbs) that scavenge O2 and facilitate its diffusion to the N2-fixing bacteroids in nodules. The biosynthesis of Lbs during nodule formation has been studied in detail, whereas little is known about the green derivatives of Lbs generated during nodule senescence. Here we characterize modified forms of Lbs, termed Lbam, Lbcm, and Lbdm, of soybean nodules. These green Lbs have identical globins to the parent red Lbs but their hemes are nitrated. By combining UV-visible, MS, NMR, and resonance Raman spectroscopies with reconstitution experiments of the apoprotein with protoheme or mesoheme, we show that the nitro group is on the 4-vinyl. In vitro nitration of Lba with excess nitrite produced several isomers of nitrated heme, one of which is identical to those found in vivo. The use of antioxidants, metal chelators, and heme ligands reveals that nitration is contingent upon the binding of nitrite to heme Fe, and that the reactive nitrogen species involved derives from nitrous acid and is most probably the nitronium cation. The identification of these green Lbs provides conclusive evidence that highly oxidizing and nitrating species are produced in nodules leading to nitrosative stress. These findings are consistent with a previous report showing that the modified Lbs are more abundant in senescing nodules and have aberrant O2 binding., This work was funded by Ministerio de Ciencia e Innovación-Fondo Europeo de Desarrollo Regional Grants AGL2008-01298 and AGL2011-24524; Gobierno de Aragón-Fondo Social Europeo Group A53; and Junta para la Ampliación de Estudios-Doc contracts from the Consejo Superior de Investigaciones Científicas (to J.N. and M.G.).

El pdf del artículo es la versión pre-print.-- et al., Senescence-accelerated prone (SAMP) strain 8 mice suffer an earlier development of cognitive age-related pathologies and a shorter life span than conventional mice. Protein alterations in astrocytes, in addition to those in neurons, may contribute to neurodegenerative damage. We applied proteomics techniques to study cell-specific early markers of brain aging-related degeneration in SAMP8. The two-dimensional protein expression patterns of the SAMP8 neuron and astrocyte cultures were compared with those obtained from senescence-accelerated resistant mouse strain 1 cultures. Differentially expressed spots were identified by matrix-assisted laser desorption/ionization–time of flight peptide map fingerprinting and database search. Proteins belonged to cell pathways of energy metabolism, biosynthesis, cell transduction and signaling, stress response, and the maintenance of cytoskeletal functions. Most of the changes were cell type specific. However, there was a general increase in cell transduction, signaling, and stress-related proteins and a decrease in cytoskeletal proteins. In addition, neurons showed an increased expression of proteins involved in biosynthetic pathways. A number of the protein alterations have been previously reported in the brain tissue proteome of SAMP8, aged brain or Alzheimer’s disease brain. Alterations in neuron and astrocyte proteoma indicated that both cell types are involved in the brain degenerative changes of SAMP8 mice. However, network analysis suggests that neuronal changes are more complex and have a greater influence., This study was supported by grants SAF2006-13092-C02-02 from the Spanish Ministerio de Educación y Ciencia, RD06/0013/1004 (RETICEF) from Instituto de Salud Carlos III and DURSI 2005/SGR/00826 of the Generalitat de Catalunya. S. García-Matas received a Generalitat de Catalunya fellowship.

T lymphocytes mediate cellular and humoral defense against foreign bodies or autoantigens. An understanding of T-cell information processing furthers studies of the immunological response. We describe a large-scale phosphorylation analysis of primary T cells using a multidimensional separation strategy, involving preparative SDS-PAGE for prefractionation, in-gel digestion and sequential phosphopeptide enrichment using IMAC and TiO2. A total of 281 phosphorylation sites (197 of high confidence, Ascore > 15), mapping to 204 human gene sequences, were identified by LC-MS(n) analysis in an LTQ linear ion trap. Subsequently, we created the LymPHOS database (http://lymphos.org), which links mass spectrometric peptide information to phosphorylation sites and phosphoprotein sequences., This work was supported by grant BIO2004-01788 from the Ministerio de Ciencia y Tecnología.

A selected chemical library of six platinum(II) complexes having 1,2-bis(aminomethyl)carbobicyclic ligands were synthesized after a rational design in order to evaluate their antiproliferative activity and the structure-activity relationships. The cytotoxicity studies were performed using cancer cell lines sensitive (A2780) and resistant (A2780R) to cisplatin. Excellent cytotoxicity was observed for most of complexes, which presented better resistance factors than cisplatin against the A2780R cell line. The interaction of these complexes with DNA, as the target biomolecule, was evaluated by several methods: DNA-platinum binding kinetics, changes in the DNA melting temperature, evaluation of the unwinding angle of supercoiled DNA, evaluation of the interstrand cross-links, and replication mapping. The kinetics of the interaction with glutathione was also investigated to better understand the resistant factors observed for the new complexes.

The studies of interaction with DNA and the cytotoxic activity of a new organometallic platinum(II) compound are presented. The ability of this new platinum complex to modify secondary DNA structure was explored by circular dichroism (CD). Electrophoretic mobility showed changes in tertiary DNA structure, and atomic force microscopy (AFM) revealed morphological changes of plasmid DNA (pBR322). This compound breaks the traditional structure-activity rules for cis-platinum compounds, but it could be of interest because of its different kinetics. An organometallic bond normally shows a trans-effect higher than that of an amine ligand, and that fact, a priori, could contribute to a higher DNA binding rate. Several ovarian cancer cell lines, resistant and non-resistant to cisplatin, were exposed to increasing concentrations of cisplatin and complex 5 for 24 h, after which time the cell number/viability was determined by the colorimetric MTT assay. A lower cytotoxicity but also a lower resistant factor was observed for organometallic compound 5 than for cisplatin, against A2780 and A2780cisR cell lines. This result is consistent with the DNA interaction degree observed by the aforementioned techniques.

The interaction with DNA and the cytotoxic activity of a new organometallic platinum(II) compound were studied. Different techniques were used to evaluate changes in secondary and tertiary DNA structures, and to obtain images of DNA morphological changes. The ability of platinum complex to modify secondary DNA structure was explored by circular dichroism (CD). Electrophoretic mobility showed changes in tertiary DNA structure. Finally, Atomic Force Microscopy (AFM) revealed morphological changes of plasmid DNA (pBR322). This compound breaks the traditional structure-activity rules for cis-platinum compounds, but it could be of interest because of its different kinetics. An organometallic bond normally shows a higher trans-effect than an amine ligand, and that fact, a priori, could contribute to a higher DNA binding rate. Human A431 and Capan-1 cells (vulvae carcinoma and pancreatic carcinoma, respectively) were exposed to increasing concentrations of cisplatin and complex 6 for 24 h, after which time the cell number/viability was determined by the colorimetric MTT assay. A low cytotoxicity of organometallic compound 6 against A431 and Capan-1 cancer cell lines was observed and this result is consistent with the low interaction with DNA observed in previous studies.

Two trichloroplatinum π-complexes and a diammonium tetrachloroplatinate (II) salt have been unexpectedly isolated during the attempt to synthesize the corresponding cisplatinum square-planar complexes of tertiary cyclohexane 1,4-diamines. All three compounds have been physically and spectroscopically characterized, including X-ray diffraction analysis in two of the three compounds. The formation of the π-complexes instead of the diamino cis-platinum complexes could be explained as due to the steric hindrance exerted by methyl groups on the tertiary amines. So, the π-electrons of the C C double bond are more available than the electron lone pair of nitrogen atom from the amino groups. When the π-electrons of the C C from the molecule of ligand were made unavailable by transforming the cyclohexene subunit into a benzene aromatic ring, no cis-diamino dichloro platinum(II) complex nor trichloroplatinum-complexes were isolated but the corresponding tetrachloroplatinate(II) diammonium salt of the 1,4-diamino ligand.

An unusual reactivity of cis-1,2-bis[(N,N-dimethylamino)methyl] cyclohexane with PtCl(2) was observed, resulting in the formation of a platinum(II) pi-olefin complex instead of the conventional square-planar cis Pt(II) coordination complex with the diamino ligand. This behavior was interpreted on the basis of the steric hindrance of the dimethylamino groups whose electron lone pairs are barely accessible to a platinum atom, which can make it difficult for both dimethylamino groups to bind platinum at the same time. This complex has been physically and spectroscopically characterized and its structure has been confirmed by using X-ray diffraction analyses on single crystals.

Intrinsically disordered regions (IDRs) are preferred sites for post-translational modifications essential for regulating protein function. The enhanced local mobility of IDRs facilitates their observation by NMR spectroscopy in vivo. Phosphorylation events can occur at multiple sites and respond dynamically to changes in kinase-phosphatase networks. Here we used real-time NMR spectroscopy to study the effect of kinases and phosphatases present in Xenopus oocytes and egg extracts on the phosphorylation state of the "unique domain" of c-Src. We followed the phosphorylation of S17 in oocytes, and of S17, S69, and S75 in egg extracts by NMR spectroscopy, MS, and western blotting. Addition of specific kinase inhibitors showed that S75 and S69 are phosphorylated by CDKs (cyclin-dependent kinases) differently from Cdk1. Moreover, although PKA (cAMP-dependent protein kinase) can phosphorylate S17 in vitro, this was not the major S17 kinase in egg extracts. Changes in PKA activity affected the phosphorylation levels of CDK-dependent sites, thus suggesting indirect effects of kinase-phosphatase networks. This study provides a proof-of-concept of the use of real-time in vivo NMR spectroscopy to characterize kinase/phosphatase effects on intrinsically disordered regulatory domains.

Major plasma protein families play different roles in blood physiology and hemostasis and in immunodefense. Other proteins in plasma can be involved in signaling as chemical messengers or constitute biological markers of the status of distant tissues. In this respect, the plasma phosphoproteome holds potentially relevant information on the mechanisms modulating these processes through the regulation of protein activity. In this work we describe for the first time a collection of phosphopeptides identified in human plasma using immunoaffinity separation of the seven major serum protein families from other plasma proteins, SCX fractionation, and TiO2 purification prior to LC-MS/ MS analysis. One-hundred and twenty-seven phosphosites in 138 phosphopeptides mapping 70 phosphoproteins were identified with FDR < 1%. A high-confidence collection of phosphosites was obtained using a combined search with the OMSSA, SEQUEST, and Phenyx search engines. © 2010 American Chemical Society., This work was supported by grant BIO2008-03369 from the Spanish Ministerio de Ciencia e Innovación.

HSA solutions account for 14% of the world market for plasma products. Albumin is indicated for reestablishing and maintaining circulatory volume in situations resulting from traumatic shock, surgery, or blood loss. Albumin is also used in extracorporeal liver support devices that perform blood dialysis against this protein. However, the protein composition of therapeutic albumin is only partially known. We performed an exhaustive analysis of albumin composition using a proteomic approach. Low abundance proteins and peptides in these samples were concentrated using a strong anion exchange resin. The absorbed material was eluted with a stepwise gradient of ammonium trifluoroacetate and the protein fraction was digested and analyzed by multidimensional liquid chromatography coupled to ESI-MS/MS using a linear ion trap. A total of 1219 peptides corresponding to 141 proteins different from albumin were identified with a false discovery rate, This work was partially funded by GAMBRO AB (Hechingen, Germany), a grant from the Ministerio de Educacion (SAF2005-03649), Spain, and the Centro de Investigaciones Biomedicas en Red de Enfermedades Hepaticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Spain.