Absolute quantification of dengue virus serotype 4 chimera vaccine candidate in Vero cell culture by targeted mass spectrometry.

Infection by dengue flavivirus is transmitted by mosquitoes and affects tens to hundreds of millions people around the world each year. Four serotypes have been described, all of which cause similar disease. Currently, there no approved vaccines or specific therapeutics for dengue, although several vaccine prototypes are in different stages of clinical development. Among them, a chimeric vaccine, built from the replication machinery of the yellow fever 17D virus, has shown promising results in phase III trials. Accurate quantitation of expressed viral particles in alive attenuated viral antigen vaccine is essential and determination of infectious titer is usually the method of choice. The current paper describes an alternative or orthogonal strategy, namely, a multiplexed and absolute assay of four proteins of the chimera yellow fever/dengue serotype 4 virus using targeted MS in SRM mode. Over 1 month, variability of the assay using a partially purified Vero cell extract was between 8 and 17%, and accuracy was between 80 and 120%. In addition, the assay was linear between 6.25 and 200 nmol/L and could therefore be used in the near future to quantify dengue virus type 4 during production and purification from Vero cells.

Absolute quantification of podocalyxin, a potential biomarker of glomerular injury in human urine, by liquid chromatography-mass spectrometry.

Podocalyxin is a protein present in specialized glomerulus cells called podocytes and may be released in the urine in case of kidney injury. In this context, its quantification could be of great interest in order to monitor glomerular injury. Liquid chromatography tandem mass spectrometry (LC-MS/MS), in selected reaction monitoring (SRM) mode, has been demonstrated as a powerful technique that can be applied to protein quantification. This paper describes the development of a quantification method of human podocalyxin in urine by LC-MS/MS in SRM mode by monitoring one proteotypic peptide with an isotope-dilution standardization strategy employing (13)C/(15)N labelled peptides. Inter/intra assay precisions and accuracies of the assay were below 10% and between 90% and 106.1%, respectively. In addition, the method was linear between 0.78 and 100 ng/mL and could therefore be used to quantify endogenous level of podocalyxin that was estimated between 15.2 and 44.2 ng/mL in urine samples from healthy donor.

NGF and ProNGF: Regulation of neuronal and neoplastic responses through receptor signaling.

Nerve growth factor (NGF) and its precursor (proNGF) are primarily considered as regulators of neuronal function that induce their responses via the tyrosine kinase receptor TrkA and the pan-neurotrophin receptor p75NTR. It has been generally held that NGF exerts its effects primarily through TrkA, inducing a cascade of tyrosine kinase-initiated responses, while proNGF binds more strongly to p75NTR. When this latter entity interacts with a third receptor, sortilin, apoptotic responses are induced in contrast to the survival/differentiation associated with the other two. Recent studies have outlined portions of the downstream phosphoproteome of TrkA in the neuronal PC12 cells and have clarified the contribution of individual docking sites in the TrkA endodomain. The patterns observed showed a similarity with the profile induced by the epidermal growth factor receptor, which is extensively associated with oncogenesis. Indeed, as with other neurotrophic factors, the distribution of TrkA and p75NTR is not limited to neuronal tissue, thus providing an array of targets outside the nervous systems. One such source is breast cancer cells, in which NGF and proNGF stimulate breast cancer cell survival/growth and enhance cell invasion, respectively. This latter activity is exerted via TrkA (as opposed to p75NTR) in conjunction with sortilin. Another tissue overexpressing proNGF is prostate cancer and here the ability of cancer cells to induce neuritogenesis has been implicated in cancer progression. These studies show that the non-neuronal functions of proNGF/NGF are likely integrated with their neuronal activities and point to the clinical utility of these growth factors and their receptors as biomarkers and therapeutic targets for metastasis and cancer pain.

Combination of a discovery LC-MS/MS analysis and a label-free quantification for the characterization of an epithelial-mesenchymal transition signature.

Disease phenotype reorganizations are the consequences of signaling pathway perturbations and protein abundance modulations. Characterizing the protein signature of a biological event allows the identification of new candidate biomarkers, new targets for treatments and selective patient therapy. The combination of discovery LC-MS/MS analyses and targeted mass spectrometry using selected reaction monitoring (SRM) mode has emerged as a powerful technology for biomarker identification and quantification owing to faster development time and multiplexing capability. The epithelial-mesenchymal transition (EMT) is a process that controls local invasion and metastasis generation by stimulating changes in adhesion and migration of cells but also in metabolic pathways. In this study, the non-transformed human breast epithelial cell line MCF10A, treated by TGFß or overexpressing mutant K-Ras(v12), two EMT inducers frequently involved in cancer progression, was used to characterize protein abundance changes during an EMT event. The LC-MS/MS analysis and label-free quantification revealed that TGFß and K-Ras(v12) induce a similar pattern of protein regulation and that besides the expected cytoskeletal changes, a strong increase in the anabolism and energy production machinery was observed. BIOLOGICAL SIGNIFICANCE: To our knowledge, this is the first proteomic analysis combining a label-free quantification with an SRM validation of proteins regulated by TGFß and K-Rasv12. This study reveals new insights in the characterization of the changes occurring during an epithelial-mesenchymal transition (EMT) event. Notably, a strong increase in the anabolism and energy production machinery was observed upon both EMT inducers.

Implementing visible 473 nm photodissociation in a Q-Exactive mass spectrometer: towards specific detection of cysteine-containing peptides.

Improvement of the fragmentation specificity may streamline data processing of bottom-up proteomic experiments by drastically reducing either the amount of MS/MS data to process in the discovery phase or the detection of interfering signals in targeted quantification. Photodissociation at appropriate wavelengths is a promising alternative technique to the non-discriminating conventional activation mode by collision. Here, we describe the implementation of visible LID at 473 nm in a Q-Exactive-Orbitrap mass spectrometer for the specific detection of cysteine-containing peptides tagged with a Dabcyl group. HCD cell DC offset and irradiation time were optimized to obtain high fragmentation yield and spectra free of contaminating CID product ions, while keeping the irradiation time scale compatible with chromatographic separation. With this optimized experimental set-up, the selective detection of cysteine-containing peptides in a whole tryptic hydrolysate of three combined proteins is demonstrated by comparing all ion fragmentation (AIF) spectra recorded online with and without laser irradiation.

Combined collision-induced dissociation and photo-selected reaction monitoring mass spectrometry modes for simultaneous analysis of coagulation factors and estrogens.

Oral estrogens are directly associated with changes in plasma levels of coagulation proteins. Thus, the detection of any variation in protein concentrations due to estrogen contraceptives, by a simultaneous analysis of both coagulation proteins and estrogens, would be a very informative tool. In the present study, the merit of photo-selected reaction monitoring (SRM), a new analytical tool, was evaluated towards estrogens detection in plasma. Then, SRM and photo-SRM detection modes were combined for the simultaneous analysis of estrogen molecules together with heparin co-factor and factor XIIa, two proteins involved in the coagulation cascade. This study shows that photo-SRM could open new multiplexed analytical routes.

Dissecting the roles of tyrosines 490 and 785 of TrkA protein in the induction of downstream protein phosphorylation using chimeric receptors.

Receptor tyrosine kinases generally act by forming phosphotyrosine-docking sites on their own endodomains that propagate signals through cascades of post-translational modifications driven by the binding of adaptor/effector proteins. The pathways that are stimulated in any given receptor tyrosine kinase are a function of the initial docking sites that are activated and the availability of downstream participants. In the case of the Trk receptors, which are activated by nerve growth factor, there are only two established phosphotyrosine-docking sites (Tyr-490 and Tyr-785 on TrkA) that are known to be directly involved in signal transduction. Taking advantage of this limited repertoire of docking sites and the availability of PC12 cell lines stably transfected with chimeric receptors composed of the extracellular domain of the PDGF receptor and the transmembrane and intracellular domains of TrkA, the downstream TrkA-induced phosphoproteome was assessed for the "native" receptor and mutants lacking Tyr-490 or both Tyr-490 and Tyr-785. Basal phosphorylation levels were compared with those formed after 20 min of stimulation with PDGF. Several thousand phosphopeptides were identified after TiO2 enrichment, and many were up- or down-regulated by receptor activation. The modified proteins in the native sample contained many of the well established participants in TrkA signaling. The results from the mutant receptors allowed grouping of these downstream targets by their dependence on the two characterized docking site(s). A clear subset that was not dependent on either Tyr-490 or Tyr-785 emerged, providing direct evidence that there are other sites on TrkA that are involved in downstream signaling.

Evaluation of hydrophilic interaction chromatography (HILIC) versus C18 reversed-phase chromatography for targeted quantification of peptides by mass spectrometry.

Hydrophilic-interaction liquid chromatography (HILIC) is a widely used technique for small polar molecule analysis and offers the advantage of improved sensitivity in mass spectrometry. Although HILIC is today frequently employed as an orthogonal fractionation method for peptides during the proteomic discovery phase, it is still seldom considered for quantification. In this study, the performances in terms of peak capacity and sensitivity of 3 HILIC columns were compared to traditional reversed phase liquid C(18) column in the context of targeted quantification of proteotypic peptides using selected reaction monitoring mode (SRM). The results showed that the maximum sensitivity in HILIC chromatography was achieved by using an amide column without salt buffer and that the signal increased compared to classic reversed phase chromatography. However, the intensity improvement is quite low compared to the one obtained for small molecules. This is due on one hand to a higher matrix effect in HILIC and on the other hand to a change of charge states of peptides in organic solvent (doubly charged to monocharged). The doubly charged ions can be more readily dissociated than singly charged ions, making them ideal for SRM peptide quantification. As a result "supercharging" reagents are added to the mobile phase to shift from predominant singly charged ions to the more favorable doubly charged species. Using such optimized conditions, peptide signal is improved by a factor of between two and ten for 88% of the peptides of the 81 peptides investigated.

The induction of serine/threonine protein phosphorylations by a PDGFR/TrkA chimera in stably transfected PC12 cells.

Stably transfected PC12 cells expressing a chimeric receptor composed of the extracellular domain of the platelet-derived growth factor receptor BB and the transmembrane and intracellular domains of TrkA, the nerve growth factor receptor, were stimulated for 20 min with platelet-derived growth factor and the resulting phosphoproteome was determined from affinity purified tryptic peptides identified by tandem MS (MS/MS) analyses. The changes in the levels of individual phosphorylation sites in stimulated cells versus control were ascertained by the stable isotope labeling of amino acids in cell culture technique. A total of 2035 peptides (806 proteins) were indentified and quantified in both data sets. Of these, 424 phosphopeptides on 259 proteins were found to be up-regulated and 392 sites on 206 proteins were down-regulated (1.8-fold or more). Protein kinases and phosphatases, as well as sites in many proteins involved in G-protein signaling, were prominently represented in the up-regulated group and more than half of the kinase up-regulated phosphosites could be clustered into three sequence motifs; a similar distribution was also found for the down-regulated sites. A comparison of the up-regulated motif profile observed to that calculated from a previous study of the EGFR-induced phosphoproteome in human HeLa cells at the same time point showed a considerable amount of similarity, supporting the view that RTK signal transduction pathways and downstream modifications are likely to be extensively overlapping.

Receptor tyrosine kinase signaling--a proteomic perspective.

The RTKs are one of the most important families mediating transmembrane signaling and they participate and are instrumental in regulating a broad range of physiological activities. Indeed, tyrosine kinases in general, and the processes that they control and/or stimulate, provide a rich source of drug targets, particularly in growth related disorders such as cancer (Zwick et al., 2002; Krause and Van Etten, 2005). However, there remain many questions regarding their activation and downstream signaling and the application of proteomic analyses promises to answer many of them. There have been relatively few detailed studies of this type to date and it will require considerably more of them to better define the pathways with respect to both the major and minor PTMs that, along with the protein-protein interactions, are the means to direct the flow of the signals generated. It will take such approaches to define the specificity that characterize the individual families, even appreciating that to some degree all are capable of activating many, if not all, of the principal pathways. It will also be necessary to understand, in the highly complex networks of intracellular phosphorylation (that contain thousands of sites of modification and clearly have not yet been fully determined in any paradigm), exactly which kinases modify which substrates, and to work out the inter-relationships with other modifications such as O-GlcNAcylation and acetylation. Only then will it be possible to determine which modifications are physiologically significant and which are simply background. Along theway, these studies should continue to provide potential drug targets and perhaps improve the current lackluster biomarker discovery track record.

Arabidopsis L10 ribosomal proteins in UV-B responses.

Ribosomal protein L10 (RPL10) is an ubiquitous protein that participates in joining the 40S and 60S ribosomal subunits into a functional 80S ribosome; however, increasing evidences indicate that RPL10 from various organisms has multiple extra ribosomal functions, besides being a constituent of ribosome and its role in translation. Arabidopsis thaliana contains in its genome three genes encoding RPL10, named RPL10A, RPL10B and RPL10C. Previously, we found that in maize and in A. thaliana, UV-B induces a reduction in protein biosynthesis, probably as a consequence of ribosomal damage; however, cellular recovery occurs in the absence of UV-B. Here, we show that RPL10s are differentially regulated by UV-B in a dosage and time dependent manner: RPL10C is induced, RPL10B is down regulated at high UV-B intensity, and RPL10A is not UV-B regulated. In addition, by coimmunoprecipitation studies using RPL10 antibodies and proteins from control and UV-B irradiated Arabidopsis plants, we demonstrate that RPL10 associates with different proteins under the two different conditions, including nuclear proteins, suggesting that at least one isoform may have extra-ribosomal roles.

Plant L10 ribosomal proteins have different roles during development and translation under ultraviolet-B stress.

Ribosomal protein L10 (RPL10) proteins are ubiquitous in the plant kingdom. Arabidopsis (Arabidopsis thaliana) has three RPL10 genes encoding RPL10A to RPL10C proteins, while two genes are present in the maize (Zea mays) genome (rpl10-1 and rpl10-2). Maize and Arabidopsis RPL10s are tissue-specific and developmentally regulated, showing high levels of expression in tissues with active cell division. Coimmunoprecipitation experiments indicate that RPL10s in Arabidopsis associate with translation proteins, demonstrating that it is a component of the 80S ribosome. Previously, ultraviolet-B (UV-B) exposure was shown to increase the expression of a number of maize ribosomal protein genes, including rpl10. In this work, we demonstrate that maize rpl10 genes are induced by UV-B while Arabidopsis RPL10s are differentially regulated by this radiation: RPL10A is not UV-B regulated, RPL10B is down-regulated, while RPL10C is up-regulated by UV-B in all organs studied. Characterization of Arabidopsis T-DNA insertional mutants indicates that RPL10 genes are not functionally equivalent. rpl10A and rpl10B mutant plants show different phenotypes: knockout rpl10A mutants are lethal, rpl10A heterozygous plants are deficient in translation under UV-B conditions, and knockdown homozygous rpl10B mutants show abnormal growth. Based on the results described here, RPL10 genes are not redundant and participate in development and translation under UV-B stress.

Nucleobindin co-localizes and associates with cyclooxygenase (COX)-2 in human neutrophils.

The inducible cyclooxygenase isoform (COX-2) is associated with inflammation, tumorigenesis, as well as with physiological events. Despite efforts deployed in order to understand the biology of this multi-faceted enzyme, much remains to be understood. Nucleobindin (Nuc), a ubiquitous Ca(2+)-binding protein, possesses a putative COX-binding domain. In this study, we investigated its expression and subcellular localization in human neutrophils, its affinity for COX-2 as well as its possible impact on PGE(2) biosynthesis. Complementary subcellular localization approaches including nitrogen cavitation coupled to Percoll fractionation, immunofluorescence, confocal and electron microscopy collectively placed Nuc, COX-2, and all of the main enzymes involved in prostanoid synthesis, in the Golgi apparatus and endoplasmic reticulum of human neutrophils. Immunoprecipitation experiments indicated a high affinity between Nuc and COX-2. Addition of human recombinant (hr) Nuc to purified hrCOX-2 dose-dependently caused an increase in PGE(2) biosynthesis in response to arachidonic acid. Co-incubation of Nuc with COX-2-expressing neutrophil lysates also increased their capacity to produce PGE(2). Moreover, neutrophil transfection with hrNuc specifically enhanced PGE(2) biosynthesis. Together, these results identify a COX-2-associated protein which may have an impact in prostanoid biosynthesis.

Characterization of prostaglandin E2 generation through the cyclooxygenase (COX)-2 pathway in human neutrophils.

In the present study, we characterized the generation of prostaglandin (PG)E2 in human neutrophils. We found that the Ca2+-dependent type IV cytosolic phospholipase A2 (cPLA2) was pivotally involved in the COX-2-mediated generation of PGE2 in response to a calcium ionophore, as determined by the use of selected PLA2 inhibitors. PGE2 biosynthesis elicited by bacterial-derived peptides or by phagocytic stimuli acting on cell surface receptors also showed to be dependent on cPLA2 activity. We then assessed metabolism of unesterified arachidonic acid (AA), and observed that PGE2 production becomes favored over that of LTB4 with higher AA concentrations. Withdrawal of calcium prevented the generation of PGE2 in response to a calcium ionophore but did not affect the up-regulation of COX-2 or its capacity to convert AA, thus limiting its implication at the level of cPLA2 activation. Of the main eicosanoids produced by neutrophils, only LTB4 was able to up-regulate COX-2 expression. Finally, the only PGE synthase isoform found in neutrophils is microsomal PGE synthase-1; it co-localized with COX-2 and its expression appeared mainly constitutive. These results highlight key differences in regulatory processes of the 5-LO and COX pathways, and enhance our knowledge at several levels in the PGE2 biosynthesis in neutrophils.

Carcinogenic properties of proteins with pro-inflammatory activity from Streptococcus infantarius (formerly S.bovis).

Several studies reported linkage between bacterial infections and carcinogenesis. Streptococcus bovis was traditionally considered as a lower grade pathogen frequently involved in bacteremia and endocarditis. This bacterium became important in human health as it was shown that 25-80% of patients who presented a S.bovis bacteremia had also a colorectal tumor. Moreover, in previous experiments, we demonstrated that S.bovis or S.bovis wall extracted antigens (WEA) were able to promote carcinogenesis in rats. The aim of the present study was: (i) to identify the S.bovis proteins responsible for in vitro pro-inflammatory properties; (ii) to purify them; (iii) to examine their ability to stimulate in vitro IL-8 and COX-2 expression by human colon cancer cells; and (iv) to assess in vivo their pro-carcinogenic potential in a rat model of colon carcinogenesis. The purified S300 fraction, as determined by proteomic analysis, contained 72 protein spots in two-dimensional gel electrophoresis representing 12 different proteins able to trigger human epithelial colonic Caco-2 cells and rat colonic mucosa to release CXC chemokines (human IL-8 or rat CINC/GRO) and prostaglandins E2, correlated with an in vitro over-expression of COX-2. Moreover, these proteins were highly effective in the promotion of pre-neoplastic lesions in azoxymethane-treated rats. In the presence of these proteins, Caco-2 cells exhibited enhanced phosphorylation of the three classes of MAP kinases. Our results show a relationship between the pro-inflammatory potential of S.bovis proteins and their pro-carcinogenic properties, confirming the linkage between inflammation and colon carcinogenesis. These data support the hypothesis that colonic bacteria can contribute to cancer development particularly in chronic infection/inflammation diseases where bacterial components may interfere with cell function.