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We analysed the cellular and molecular changes in the leaf growth zone of tolerant and sensitive rice varieties in response to suboptimal temperatures. Cold reduced the final leaf length by 35% and 51% in tolerant and sensitive varieties, respectively. Tolerant lines exhibited a smaller reduction of the leaf elongation rate and greater compensation by an increased duration of leaf growth. Kinematic analysis showed that cold reduced cell production in the meristem and the expansion rate in the elongation zone, but the latter was compensated for by a doubling of the duration of cell expansion. We performed iTRAQ proteome analysis on proliferating and expanding parts of the leaf growth zone. We identified 559 and 542 proteins, of which 163 and 210 were differentially expressed between zones, and 96 and 68 between treatments, in the tolerant and sensitive lines, respectively. The categories protein biosynthesis and redox homeostasis were significantly overrepresented in the up-regulated proteins. We therefore measured redox metabolites and enzyme activities in the leaf growth zone, demonstrating that tolerance of rice lines to suboptimal temperatures correlates with the ability to up-regulate enzymatic antioxidants in the meristem and non-enzymatic antioxidants in the elongation zone. Fil: Gázquez, Ayelén. Universiteit Antwerp; Bélgica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Abdelgawad, Hamada. Universiteit Antwerp; Bélgica Fil: Baggerman, Geert. Universiteit Antwerp; Bélgica Fil: Van Raemdonck, Geert. Universiteit Antwerp; Bélgica Fil: Asard, Han. Universiteit Antwerp; Bélgica Fil: Maiale, Santiago Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Rodriguez, Andres Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Beemster, Gerrit T. S.. Universiteit Antwerp; Bélgica

© 2019 Elsevier B.V. This study has focussed on the effect of low temperature storage on the postharvest ripening of green tomatoes; more specifically, on the ethylene signal transduction in relation to the ethylene biosynthesis pathway. To this end, the first elements of the ethylene signal transduction, ethylene receptors, CTR and EIN2 have been analysed in tomatoes stored at three different temperatures in terms of both their gene and protein expression levels. These results have been related to quality changes and enzymes and intermediates of the ethylene biosynthesis pathway to better understand the ethylene control of ripening under low temperatures. The first changes observed during low temperature storage took place at the ethylene biosynthesis level, which might enhance changes in the ethylene signal transduction. Although expression of most ethylene signalling genes decreased with chilling, none of the protein levels showed a significant decrease, pointing at a differential regulation at the gene and protein level. ispartof: POSTHARVEST BIOLOGY AND TECHNOLOGY vol:155 pages:1-10 status: published

Maternal lipolytic metabolic disorders result in a lipotoxic microenvironment in the ovarian follicular fluid (FF) which deteriorates oocyte quality. Although cellular stress response mechanisms are well defined in somatic cells, they remain largely unexplored in oocytes, which have distinct organelle structure and nuclear transcription patterns. Here we used shotgun proteomic analyses to study cellular responses of bovine oocytes and cumulus cells (CCs) after in vitro maturation under lipotoxic conditions; in the presence of pathophysiological palmitic acid (PA) concentration as a model. Differentially regulated proteins (DRPs) were mainly localized in the endoplasmic reticulum, mitochondria and nuclei of CCs and oocytes, however the DRPs and their direction of change were cell-type specific. Proteomic changes in PA-exposed CCs were predominantly pro-apoptotic unfolded protein responses (UPRs), mitochondrial and metabolic dysfunctions, and apoptotic pathways. This was also functionally confirmed. Interestingly, although the oocytes were enclosed by CCs during PA exposure, elevated cellular stress levels were also evident. However, pro-survival UPRs, redox regulatory and compensatory metabolic mechanisms were prominent despite evidence of mitochondrial dysfunction, oxidative stress, and reduced subsequent embryo development. The data provides a unique insight that enriches the understanding of the cellular stress responses in metabolically-compromised oocytes and forms a fundamental base to identify new targets for fertility treatments as discussed within.

To understand the growth response to drought, we performed a proteomics study in the leaf growth zone of maize (Zea mays L.) seedlings and functionally characterized the role of starch biosynthesis in the regulation of growth, photosynthesis and antioxidant capacity, using the shrunken-2 mutant (sh2), defective in ADP-glucose pyrophosphorylase. Drought altered the abundance of 284 proteins overrepresented for photosynthesis, amino acid, sugar and starch metabolism, and redox-regulation. Changes in protein levels correlated with enzyme activities (increased ATP synthase, cysteine synthase, starch synthase, RuBisCo, peroxiredoxin, glutaredoxin, thioredoxin and decreased triosephosphate isomerase, ferredoxin, cellulose synthase activities, respectively) and metabolite concentrations (increased ATP, cysteine, glycine, serine, starch, proline and decreased cellulose levels). The sh2 mutant showed a reduced increase of starch levels under drought conditions, leading to soluble sugar starvation at the end of the night and correlating with an inhibition of leaf growth rates. Increased RuBisCo activity and pigment concentrations observed in WT, in response to drought, were lacking in the mutant, which suffered more oxidative damage and recovered more slowly after re-watering. These results demonstrate that starch biosynthesis contributes to maintaining leaf growth under drought stress and facilitates enhanced carbon acquisition upon recovery. ispartof: PLANT CELL AND ENVIRONMENT vol:43 issue:9 pages:2254-2271 ispartof: location:United States status: published

As an alternative to the conventional packed bed nano LC columns that are frequently used in bottom-up proteomics research, a breakthrough in column manufacturing recently led to the introduction of silicon-based micromachined nano LC chip columns known as micro pillar array columns (μPAC™). These columns have an inherent high permeability and an unprecedented low ‘on-column’ dispersion due to the perfect order of the separation bed .Due to the perfect order, the peak dispersion originating from heterogeneous flow paths in the separation bed is eliminated (no A-term contributions). Sample components remain therefore much more concentrated during separation resulting in unprecedented separation performance. The freestanding nature of the pillars also leads to much lower backpressure allowing a high operational flow rate flexibility with exceptional peak capacities. In addition, the monolithic nature of the beds makes them extremely rugged and guarantees an unprecedented longevity of the columns. Moreover, it also guarantees run-to-run time repeatability that is about an order of magnitude better than packed bed columns, thus facilitating and accelerating protein identification. After the introduction of a 200 cm long column, which is ideally suited to perform comprehensive proteome research, a 50 cm long μPAC™ column is now available which can be used in a more routine research setting. With an internal volume of 3 μL, this column is perfectly suited to perform high throughput analyses with shorter gradient solvent times (30, 60 and 90 minute gradients) and it can be used over a wide range of flow rates, between 100 and 2000 nL/min. Recently performed experiments with 500 ng of HeLa cell digest indicate that an increase in protein identifications up to 50% and a gain of 70% in peptide identifications can be achieved when comparing the 50 cm μPAC™ column to the current state-of-the-art packed bed columns. In the recent months, we have also been developing a column that is deeper and wider to meet the requirements of capillary LC flow rate. New data on these columns will be presented at the conference.

Aim: A diagnostic test that could detect Treponema pallidum antigens in urine would facilitate the prompt diagnosis of syphilis. Materials & methods: Urine from 54 individuals with various clinical stages of syphilis and 6 controls were pooled according to disease stage and interrogated with complementary mass spectrometry techniques to uncover potential syphilis biomarkers. Results & conclusion: In total, 26 unique peptides were uncovered corresponding to four unique T. pallidum proteins that have low genetic sequence similarity to other prokaryotes and human proteins. This is the first account of direct T. pallidum protein detection in human clinical samples using mass spectrometry. The implications of these findings for future diagnostic test development is discussed. Data are available via ProteomeXchange with identifier PXD009707.

Purpose Despite improvement in vaccines against human papilloma virus (HPV), the causative agent of cervical cancer, screening women for cervical precancer will remain indispensable in the coming 3040 years. A simple test that could be performed at home or at a doctors practice and that informs the woman whether she is at risk would significantly help make a broader group of patients who aware that they need medical treatment. Cervical vaginal fluid (CVF) is a body fluid that is very well suited for such a test. Methods Narrative review of cervical (pre)cancer candidate biomarkers from cervicovaginal fluid, is based on a detailed review of the literature. We will also discuss the possibilities that these biomarkers create for the development of a self-test or point-of-care test for cervical (pre)cancer. Results Several DNA, DNA methylation, miRNA, and protein biomarkers were identified in the cervical vaginal fluid; however, not all of these biomarkers are suited for development of a simple diagnostic assay. Conclusions Proteins, especially alpha-actinin-4, are most suited for development of a simple assay for cervical (pre)cancer. Accuracy of the test could further be improved by combination of several proteins or by combination with a new type of biomarker, e.g., originating from the cervicovaginal microbiome or metabolome.

Capitalizing on the massive increase in sample concentrations which are produced by extremely low elution volumes, nano-LC-ESI-MS/MS is currently one of the most sensitive analytical technologies for the comprehensive characterization of complex protein samples.However, despite tremendous technological improvements made in the production and the packing of monodisperse spherical particles for nano-flow HPLC, current state-of-the-art systems still suffer from limits in operation at the maximum potential of the technology.With the recent introduction of the µPAC system, which provides perfectly ordered micro-pillar array based chromatographic support materials, completely new chromatographic concepts for optimization towards the needs of ultra-sensitive proteomics become available.Here we report on a series of benchmarking experiments comparing the performance of a commercially available 50 cm micro-pillar array column to a widely used nano-flow HPLC column for the proteomics analysis of 10 ng tryptic HeLa cell digest.Comparative analysis of LC-MS/MS-data corroborated that micro-pillar array cartridges provide outstanding chromatographic performance, excellent retention time stability, increase sensitivity in the analysis of low-input proteomics samples, and thus repeatedly yielded almost twice as many unique peptide and unique protein group identifications when compared to conventional nano-flow HPLC columns.

G protein-coupled receptors (GPCRs) constitute the largest family of transmembrane protein receptors. These receptors regulate a wide array of physiological processes in both healthy and diseased conditions via G protein-dependent and –independent signaling pathways. Remarkably, a subfamily of class A GPCRs, Mas-related G protein-coupled receptors (MRGPRs), is predominantly expressed in sensory neurons of the dorsal root and trigeminal ganglia, as well as on mast cells. Interestingly, we recently discovered that Mas-related G protein-coupled receptor F (MRGPRF), which is known to be expressed by brain, spinal cord and enteric neurons, is also expressed by enteroendocrine cells in the gastrointestinal tract. MRGPRF expression was predominantly found at basal and basolateral sites, indicating a novel role in endocrine regulation. However, due to the orphan nature of MRGPRF, little is known about its role in the pathophysiology of intestinal inflammation. Additionally, increasing evidence suggests a prominent role of peptides and proteases, predominantly secreted by macrophages, microglia and enteroendocrine cells, in inflammatory conditions of the gut, but the therapeutic perspectives are still restricted because the underlying molecular mechanisms are unknown. Here, we report for the first time on the activation of human MRGPRF by the cysteine protease Cathepsin S (Cat-S). To this end, we first performed in-silico analysis of the N-terminal sequence of MRGPRF, which revealed consensus motif sites of cleavage by Cat-S. Furthermore, the N-terminal peptide of MRGPRF was synthesized, incubated with Cat-S and analyzed for cleaved sites using mass spectroscopy. The results clearly showed cleavage sites and as such corroborated our in-silico analysis. Moreover, luciferase tagged at the N-terminus of MRGPRF was expressed in HeLa cells incubated with Cat-S and receptor N-terminal cleavage was assessed by measuring luminescence activity in the supernatant. The high luciferase activity in the supernatant compared to its control (no Cat-S treatment) indicated cleavage of the N-terminus of MRGPRF. We next sought to determine receptor activation upon Cat-S treatment using live cell calcium imaging with Fluo-4. HeLa cells transiently transfected with MRGPRF and incubated with Cat-S, gave rise to protease-based receptor activation, as evident by a fluorescence trace when compared to controls (non-transfected HeLa cells). These results pave the way for deorphanization of MRGPRF and call for further pathological investigations related to MRGPRF, in particular with regard to its involvement in neuro-immune communication within the gut.

Neuroglobin is a heme protein of which increased levels provide neuroprotection against amyloid proteinopathy and hemorrhagic damage. These cellular stressors involve the promotion of ferroptosis&mdash, an iron-dependent, lipid peroxide-accreting form of cell death. Hence, we questioned whether neuroglobin could oppose ferroptosis initiation. We detected human neuroglobin (hNgb)-EGFP-expressing SH-SY5Y cells to be significantly more resistant to ferroptosis induction, identifying 0.68-fold less cell death. To elucidate the underlying pathways, this study investigated hNgb-protein interactions with a Co-IP-MS/MS approach both under a physiological and a ferroptotic condition. hNgb binds to proteins of the cellular iron metabolism (e.g., RPL15 and PCBP3) in an unstressed condition and shows an elevated binding ratio towards cell death-linked proteins, such as HNRNPA3, FAM120A, and ABRAXAS2, under ferroptotic stress. Our data also reveal a constitutive interaction between hNgb and the longevity-associated heterodimer XRCC5/XRCC6. Disentangling the involvement of hNgb and its binding partners in cellular processes, using Ingenuity Pathway Analysis, resulted in the integration of hNgb in the ubiquitination pathway, mTOR signaling, 14-3-3-mediated signaling, and the glycolysis cascade. We also detected a previously unknown strong link with motor neuropathies. Hence, this study contributes to the elucidation of neuroglobin&rsquo, s involvement in cellular mechanisms that provide neuroprotection and the upkeep of homeostasis.

We report on a comparative study of the basic separation kinetics of commercial packed bed columns and a micro-pillar array column (mu PAC) working in the 1-10 mu L/min flow rate range, i.e., operating in the area of capillary flow LC. This is done using a basic test mixture of 8 alkylphenones under both isocratic and gradient separation conditions. Care was taken the mu PAC and the packed bed columns have similar volumes (around 10 mu L) and hence also similar t 0-times when compared at the same flow rate. In addition, the isocratic mobile phase composition and gradient programs were selected such to have similar elution windows (in absolute times) for all 4 column types. It was found that the mu PAC produces significantly more theoretical plates (up to 3 times) in the 1-4 mu L/min range, while, the packed bed columns perform better at the higher flow rates because of the relatively large inter-pillar distance in the mu PAC. Under gradient conditions, the mu PAC produces a clearly higher peak capacity than any of the three packed bed columns over the entire range of investigated flow rates, albeit that this is also partly to be owed to the steeper gradient that needed to be used in the mu PAC in order to maintain a similar elution window on all columns. (c) 2020ElsevierB.V. Allrightsreserved.

Ethylene, the plant ripening hormone of climacteric fruit, is perceived by ethylene receptors which is the first step in the complex ethylene signal transduction pathway. Much progress has been made in elucidating the mechanism of this pathway, but there is still a lot to be done in the proteomic quantification of the main proteins involved, particularly during fruit ripening. This work focuses on the mass spectrometry based identification and quantification of the ethylene receptors (ETRs) and the downstream components of the pathway, CTR-like proteins (CTRs) and ETHYLENE INSENSITIVE 2 (EIN2). We used tomato as a model fruit to study changes in protein abundance involved in the ethylene signal transduction during fruit ripening. In order to detect and quantify these low abundant proteins located in the membrane of the endoplasmic reticulum, we developed a workflow comprising sample fractionation and MS analysis using parallel reaction monitoring. This work shows the feasibility of the identification and absolute quantification of all seven ethylene receptors, three out of four CTRs and EIN2 in four ripening stages of tomato. In parallel, gene expression was analyzed through real-time qPCR. Correlation between transcriptomic and proteomic profiles during ripening was only observed for three of the studied proteins, suggesting that the other signaling proteins are likely post-transcriptionally regulated. Based on our quantification results we were able to show that the protein levels of SlETR3 and SlETR4 increased during ripening, probably to control ethylene sensitivity. The other receptors and CTRs showed either stable levels that could sustain, or decreasing levels that could promote fruit ripening. ispartof: FRONTIERS IN PLANT SCIENCE vol:9 ispartof: location:Switzerland status: published

To expand the knowledge on the porcine salivary proteome, secretions from the three major salivary glands were collected from anaesthetised piglets. Pilocarpine and isoproterenol were simultaneously injected intraperitoneally to increase the volume and protein concentration of the saliva, respectively. The protein composition and relative protein-specific abundance of saliva secreted by the parotid gland and by the mandibular and monostomatic sublingual gland, were determined using iTRAQ. When combining two detection methods, MALDI-TOF/TOF MS and Q-Exactive orbitrap MS/MS, a total of 122 porcine salivary proteins and 6 mammalian salivary proteins with a predicted porcine homolog were identified. Only a quantitative and not a qualitative difference was observed between both ductal secretions. The 128 proteins were detected in both secretions, however, at different levels. Twenty-four proteins (20 porcine and 4 mammalian with a predicted porcine homolog) were predominantly secreted by the parotid gland, such as carbonic anhydrase VI and alpha-amylase. Twenty-nine proteins (all porcine) were predominantly secreted by the mandibular and sublingual glands, for example salivary lipocalin and submaxillary apomucin protein. Data are available via ProteomeXchange with identifier PXD008853. SIGNIFICANCE: In humans, more than 3000 salivary proteins have been identified. To our knowledge, previous studies on porcine saliva only identified a total of 34 proteins. This research increased the total number of identified proteins in porcine saliva to 143. This insight into the porcine salivary proteome will facilitate the search for potential biomarkers that may help in the early detection of pathologies and follow-up of animal welfare. Moreover, it can also endorse the value of a porcine animal model and contribute to a better understanding of the animal's physiology. Additionally, this was the first study to collect and analyse gland specific saliva of pigs. The obtained relative-quantitative knowledge of the identified proteins is valuable when comparing data of stimulated (chewing on a device) vs. unstimulated (passive) saliva collection in the future, since salivary stimulation changes the relative contribution of the major salivary glands to the whole saliva in the oral cavity. For example, carbonic anhydrase VI, which is present in higher concentrations in parotid saliva, has a higher concentration in stimulated whole saliva because of the larger contribution of the parotid gland after stimulation by chewing.

Despite large gaps in our knowledge on the intracellular mechanism leading to cervical cancer, the pathways induced by oncogenic high-risk Human Papilloma Virus (HPV) and those finally causing cervical cancer are increasingly being unraveled. Assuming that precancerous tissue is recognized and lysed by the immune systemwhich is in many cases incomplete because of the counteraction by the HPV viruswe hypothesize that several intracellular factors, involved in induction and development of precancerous lesions and/or cervical cancer are being released into the cervicovaginal fluid (CVF). These factors can then be seen as markers for the precancerous state, and when they persist they are indicative for an increased risk for cervical carcinoma. In a previous study, we analyzed the proteomic profiles of six CVF samples from women with different stages of precancerous lesions and compared these with the CVF proteomes from healthy women. Here, we extend these observations by investigating these proteomes by Ingenuity Pathway Analysis (IPA). We show that proteins in CVF from precancerous women are clearly more involved in pathways that make up the hallmarks of cancer, as compared to CVF proteins from healthy persons. Moreover, after literature search, proteins classified by IPA in the cancer category, were more correlated with cervical cancer when they originated from CVF from precancerous women. Many of these proteins formed a network with angiotensin II as central mediator. The search for network biomarkers, rather than single biomarkers, could drastically increase specificity, sensitivity and prognostic value of cervical cancer diagnosis, making use of an easy to handle fluid, the CVF.

Background The spirochete bacterium Treponema pallidum ssp. pallidum is the etiological agent of syphilis, a chronic multistage disease. Little is known about the global T. pallidum proteome, therefore mass spectrometry studies are needed to bring insights into pathogenicity and protein expression profiles during infection. Methodology/Principal Findings To better understand the T. pallidum proteome profile during infection, we studied T. pallidum ssp. pallidum DAL-1 strain bacteria isolated from rabbits using complementary mass spectrometry techniques, including multidimensional peptide separation and protein identification via matrix-assisted laser desorption ionization-time of flight (MALDI-TOF/TOF) and electrospray ionization (ESI-LTQ-Orbitrap) tandem mass spectrometry. A total of 6033 peptides were detected, corresponding to 557 unique T. pallidum proteins at a high level of confidence, representing 54% of the predicted proteome. A previous gel-based T. pallidum MS proteome study detected 58 of these proteins. One hundred fourteen of the detected proteins were previously annotated as hypothetical or uncharacterized proteins; this is the first account of 106 of these proteins at the protein level. Detected proteins were characterized according to their predicted biological function and localization; half were allocated into a wide range of functional categories. Proteins annotated as potential membrane proteins and proteins with unclear functional annotations were subjected to an additional bioinformatics pipeline analysis to facilitate further characterization. A total of 116 potential membrane proteins were identified, of which 16 have evidence supporting outer membrane localization. We found 8/12 proteins related to the paralogous tpr gene family: TprB, TprC/D, TprE, TprG, TprH, TprI and TprJ. Protein abundance was semi-quantified using label-free spectral counting methods. A low correlation (r = 0.26) was found between previous microarray signal data and protein abundance. Conclusions This is the most comprehensive description of the global T. pallidum proteome to date. These data provide valuable insights into in vivo T. pallidum protein expression, paving the way for improved understanding of the pathogenicity of this enigmatic organism., Author Summary Syphilis remains a major cause of morbidity and mortality worldwide. The bacterium causing syphilis, Treponema pallidum ssp. pallidum, has evolved into a highly distinctive organism that is only able survive (and be propagated) in mammals. In humans it can evade the immune system for decades with devastating consequences. Much remains to be learned about how it accomplishes this. Only a minority of its predicted proteins have been detected experimentally thus far. We aimed to more comprehensively characterize the proteins of this organism. Since it cannot be cultured in vitro, we cultured T. pallidum in rabbits and analyzed extracted proteins using different mass spectrometry methods, a manner of detecting proteins with high accuracy. In total, we detected more than half of the predicted number of proteins that could be expressed by this bacterium (N = 557). For approximately half of the proteins, we succeeded in characterizing their predicted cellular location using an array of bioinformatic tools and catalogued their function. This is the most comprehensive analysis of the T. pallidum proteome to date. This study lays the groundwork for other protein investigations of this unique organism.

Background:Current syphilis diagnostic strategies are lacking a sensitive manner of directly detectingTreponema pallidumantigens. A diagnostic test that could directly detectT. pallidumantigens in individuals with syphilis would be of considerable clinical utility, especially for the diagnosis of reinfections and for post-treatment serological follow-up.Methods:In this study, 11 candidateT. pallidumbiomarker proteins were chosen according to their physiochemical characteristics,T. pallidumspecificity and predicted abundance. Thirty isotopically labelled proteotypic surrogate peptides (hPTPs) were synthesized and incorporated into a scheduled multiple reaction monitoring assay. Protein extracts from undepleted/unenriched plasma (N = 18) and urine (N = 4) samples from 18 individuals with syphilis in various clinical stages were tryptically digested, spiked with the hPTP mixture and analysed with a triple quadruple mass spectrometer.Results:No endogenous PTPs corresponding to the eleven candidate biomarkers were detected in any samples analysed. To estimate the Limit of Detection (LOD) of a comparably sensitive mass spectrometer (LTQ-Orbitrap), two dilution series of rabbit cultured purifiedT. pallidumwere prepared in PBS. Polyclonal anti-T. pallidumantibodies coupled to magnetic Dynabeads were used to enrich one sample series; no LOD improvement was found compared to the unenriched series. The estimated LOD of MS instruments is 300T. pallidum/ml in PBS.Conclusions:Biomarker protein detection likely failed due to the low (femtomoles/liter) predicted concentration ofT. pallidumproteins. Alternative sample preparation strategies may improve the detectability ofT. pallidumproteins in biofluids.

HIV-exposed seronegative individuals (HESNs) are persons who remain seronegative despite repeated exposure to HIV, suggesting an in vivo resistance mechanism to HIV. Elucidation of endogenous factors responsible for this phenomenon may aid in the development of new classes of microbicides and therapeutics. We compared cervicovaginal protein abundance profiles between high-risk HESN and two control groups: low-risk HESN and HIV-positives. Four iTRAQ-based quantitative experiments were performed using samples classified based on presence/absence of particular gynaecological conditions. After statistical analysis, two proteins were shown to be differentially abundant between high-risk HESNs and control groups. Serpin A5, a serine proteinase inhibitor and Myeloblastin, a serine protease, were up- and downregulated, respectively. Commercially available ELISA assays were used to confirm differential Serpin A5 levels. These results suggest that HIV resistance in CVF of HESNs is the result of a delicate balance between two complementary mechanisms: downregulation of serine proteinases and upregulation of their inhibitors.

Objectives Cervicovaginal fluid (CVF) can be considered as a potential source of biomarkers for diseases of the lower female reproductive tract. The fluid can easily be collected, thereby offering new opportunities such as the development of self tests. Our objective was to identify a CVF protein biomarker for cervical cancer or its precancerous state. Methods A differential proteomics study was set up using CVF samples from healthy and precancerous women. Label-free spectral counting was applied to quantify protein abundances. Results The proteome analysis revealed 16 candidate biomarkers of which alpha-actinin-4 (p = 0.001) and pyruvate kinase isozyme M1/M2 (p = 0.014) were most promising. Verification of alpha-actinin-4 by ELISA (n = 28) showed that this candidate biomarker discriminated between samples from healthy and both low-risk and high-risk HPV-infected women (p = 0.009). Additional analysis of longitudinal samples (n = 29) showed that alpha-actinin-4 levels correlated with virus persistence and clearing, with a discrimination of approximately 18 pg/ml. Conclusions Our results show that CVF is an excellent source of protein biomarkers for detection of lower female genital tract pathologies and that alpha-actinin-4 derived from CVF is a promising candidate biomarker for the precancerous state of cervical cancer. Further studies regarding sensitivity and specificity of this biomarker will demonstrate its utility for improving current screening programs and/or its use for a cervical cancer self-diagnosis test.

Species specific differences in transporters, chaperones, metal binding proteins and other targets are important in metal toxicity. Therefore, we have studied the effects of copper exposure on the proteome of gill tissue from Oncorhynchus mykiss, Cyprinus carpio and Carassius auratus gibelio, which have different sensitivities toward copper. Fish were exposed to the Flemish water quality standard for surface waters, being 50 μg/L, for 3 days. Sampled gill tissue was subjected to a 2D-Dige and an iTRAQ analysis. While gibel carp showed more positive responses such as increased apolipoprotein A-I, transferrin and heat shock protein 70, common carp's gill tissue on the other hand displayed a changed actin cytoskeleton, and indications of a changed metabolism. These last two traits were evident in rainbow trout as well, together with decreased expressions of transferrin and albumin. urthermore, the Weighted Gene Co-Expression Network Analysis of rainbow trout data revealed a network of 98 proteins related to Cu accumulation in gill, of which the occurrence of proteins related to oxidative stress, such as superoxide dismutase and cytochrome c were promising. Additionally, the outcome of the different proteomics techniques demonstrates the usefulness of iTRAQ analysis compared to 2D-Dige and the need for fully annotated genomes.

BackgroundCervical-vaginal fluid (CVF) plays an important role in the prevention of gynecological infections, although little is known about the contribution of CVF proteins to the immunity of the lower female genital tract. In order to analyze the protein composition of human CVF, we used CVF samples that are routinely collected during colposcopy, but are usually discarded. Since these samples are available in large quantities we aimed to analyze their usefulness for proteomics experiments. The samples were analyzed using different prefractionation techniques (ultrafiltration and C4(RP)-LC protein separation) followed by C18(RP)-LC peptide separation and identification by MALDI-TOF-TOF mass spectrometry. To determine the reproducibility of this proteomics platform we analyzed three technical replicates. Using spectral counting, protein abundances were estimated in a semiquantitative way. We also compared the results obtained in this study with those from previous studies derived from patients with different physiological conditions in order to determine an overlapping protein set.ResultsIn total, we were able to identify 339 proteins in human CVF of which 151 proteins were not identified in any other proteomics study on human CVF so far. Those included antimicrobial peptides, such as human beta-defensin 2 and cathelicidin, which were known to be present in CVF, and endometrial proteins such as glycodelin and ribonucleoprotein A. Comparison of our results with previously published data led to the identification of a common protein set of 136 proteins. This overlapping protein set shows increased fractions of immunological and extracellular proteins, confirming the extracellular immunological role of CVF.ConclusionWe demonstrated here that CVF colposcopy samples can be used in proteomics experiments and hence are applicable for biomarker discovery experiments. The delineation of an overlapping set of proteins that is identified in most proteomics studies on CVF may help in the description of a reference proteome when performing proteomics studies on human CVF.

Cervicovaginal fluid has an important function in the homeostasis and immunity of the lower female genital tract. Analysis of the cervicovaginal fluid proteome may therefore yield important information about the pathogenesis of numerous gynecological pathologies. Additionally, cervicovaginal fluid has great potential as a source of biomarkers for these conditions. This review provides a detailed discussion about the human cervicovaginal proteome and the proteomics studies performed to characterize this biological fluid. Furthermore, infection-correlated pathological conditions of the female genital tract are discussed for which cervicovaginal fluid has been used in order to identify potential biomarkers. Recent years, numerous studies have analyzed cervicovaginal fluid samples utilizing antibody-based technologies, such as ELISA or Western blotting, to identify biomarkers for preterm birth, premature preterm rupture of membranes, bacterial vaginosis and cervical cancer. The present article will discuss the importance of proteomic technologies as alternative techniques to gain additional meaningful information about these conditions. In addition, the review focuses on recent proteomic studies on cervicovaginal fluid samples for the identification of potential biomarkers. We conclude that the use of proteomic technology for analysis of human cervicovaginal fluid samples is promising and may lead to the discovery of new biomarkers which can improve disease prevention and therapy development.