Chronic exposure to multiple stressors alters the salivary proteome of piglets.

Monitoring chronic stress in pigs is not only essential in view of animal welfare but is also important for the farmer, given that stress influences the zootechnical performance of the pigs and increases their susceptibility to infectious diseases. To investigate the use of saliva as a non-invasive, objective chronic stress monitoring tool, twenty-four 4-day-old piglets were transferred to artificial brooders. At the age of 7 days, they were assigned to either the control or the stressed group and reared for three weeks. Piglets in the stressed group were exposed to overcrowding, absence of cage enrichment, and frequent mixing of animals between pens. Shotgun analysis using an isobaric labelling method (iTRAQ) for tandem mass spectrometry performed on saliva samples taken after three weeks of chronic stress identified 392 proteins, of which 20 proteins displayed significantly altered concentrations. From these 20 proteins, eight were selected for further validation using parallel reaction monitoring (PRM). For this validation, saliva samples that were taken one week after the start of the experiment and samples that were taken at the end of the experiment were analysed to verify the profile over time. We wanted to investigate whether the candidate biomarkers responded fast or rather slowly to the onset of chronic exposure to multiple stressors. Furthermore, this validation could indicate whether age influenced the baseline concentrations of these salivary proteins, both in healthy and stressed animals. This targeted PRM analysis confirmed that alpha-2-HS-glycoprotein was upregulated in the stressed group after one and three weeks, while odorant-binding protein, chitinase, long palate lung and nasal epithelium protein 5, lipocalin-1, and vomeromodulin-like protein were present in lower concentrations in the saliva of the stressed pigs, albeit only after three weeks. These results indicate that the porcine salivary proteome is altered by chronic exposure to multiple stressors. The affected proteins could be used as salivary biomarkers to identify welfare problems at the farm and facilitate research to optimise rearing conditions.

Separation efficiency kinetics of capillary flow micro-pillar array columns for liquid chromatography.

We report on a comparative study of the basic separation kinetics of commercial packed bed columns and a micro-pillar array column (µPAC) working in the 1-10µL/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 µPAC and the packed bed columns have similar volumes (around 10µL) and hence also similar t0-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 µPAC produces significantly more theoretical plates (up to 3 times) in the 1-4µL/min range, while, the packed bed columns perform better at the higher flow rates because of the relatively large inter-pillar distance in the µPAC. Under gradient conditions, the µPAC 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 µPAC in order to maintain a similar elution window on all columns.

Starch biosynthesis contributes to the maintenance of photosynthesis and leaf growth under drought stress in maize.

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.

Redox homeostasis in the growth zone of the rice leaf plays a key role in cold tolerance.

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.

Improved Sensitivity in Low-Input Proteomics Using Micropillar Array-Based Chromatography.

Capitalizing on the massive increase in sample concentrations which are produced by extremely low elution volumes, nanoliquid chromatography-electrospray ionization-tandem mass spectrometry (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 nanoflow high-pressure liquid chromatography (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 micropillar array based chromatographic support materials, completely new chromatographic concepts for optimization toward the needs of ultrasensitive proteomics become available. Here we report on a series of benchmarking experiments comparing the performance of a commercially available 50 cm micropillar array column to a widely used nanoflow HPLC column for the proteomics analysis of 10 ng of tryptic HeLa cell digest. Comparative analysis of LC-MS/MS-data corroborated that micropillar array cartridges provide outstanding chromatographic performance, excellent retention time stability, and increased 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 nanoflow HPLC columns.

Connecting the Dots in the Neuroglobin-Protein Interaction Network of an Unstressed and Ferroptotic Cell Death Neuroblastoma Model.

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-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's involvement in cellular mechanisms that provide neuroprotection and the upkeep of homeostasis.

MALDI Mass Spectrometry Imaging Linked with Top-Down Proteomics as a Tool to Study the Non-Small-Cell Lung Cancer Tumor Microenvironment.

Advanced non-small-cell lung cancer (NSCLC) is generally linked with a poor prognosis and is one of the leading causes of cancer-related deaths worldwide. Since only a minority of the patients respond well to chemotherapy and/or targeted therapies, immunotherapy might be a valid alternative in the lung cancer treatment field, as immunotherapy attempts to strengthen the body's own immune response to recognize and eliminate malignant tumor cells. However, positive response patterns to immunotherapy remain unclear. In this study, we demonstrate how immune-related factors could be visualized from single NSCLC tissue sections (Biobank@UZA) while retaining their spatial information by using matrix assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI), in order to unravel the molecular profile of NSCLC patients. In this way, different regions in lung cancerous tissues could be discriminated based on the molecular composition. In addition, we linked visualization (MALDI MSI) and identification (based on liquid chromatography higher resolution mass spectrometry) of the molecules of interest for the correct biological interpretation of the observed molecular differences within the area in which these molecules are detected. This is of major importance to fully understand the underlying molecular profile of the NSCLC tumor microenvironment.

Proteomic changes in oocytes after in vitro maturation in lipotoxic conditions are different from those in cumulus cells.

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.

On the characterisation of the porcine gland-specific salivary proteome.

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.

Needle lost in the haystack: multiple reaction monitoring fails to detect Treponema pallidum candidate protein biomarkers in plasma and urine samples from individuals with syphilis.

Background: Current syphilis diagnostic strategies are lacking a sensitive manner of directly detecting Treponema pallidum antigens. A diagnostic test that could directly detect T. pallidum antigens 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 candidate T. pallidum biomarker proteins were chosen according to their physiochemical characteristics, T. pallidum specificity 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 purified T. pallidum were prepared in PBS. Polyclonal anti- T. pallidum antibodies 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 300 T. pallidum/ml in PBS. Conclusions: Biomarker protein detection likely failed due to the low (femtomoles/liter) predicted concentration of T. pallidum proteins. Alternative sample preparation strategies may improve the detectability of T. pallidum proteins in biofluids.

Ethylene Receptors, CTRs and EIN2 Target Protein Identification and Quantification Through Parallel Reaction Monitoring During Tomato Fruit Ripening.

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.

Candidate Treponema pallidum biomarkers uncovered in urine from individuals with syphilis using mass spectrometry.

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.

The effect of reactive oxygen and nitrogen species on the structure of cytoglobin: A potential tumor suppressor.

Many current anti-cancer therapies rely on increasing the intracellular reactive oxygen and nitrogen species (RONS) contents with the aim to induce irreparable damage, which subsequently results in tumor cell death. A novel tool in cancer therapy is the use of cold atmospheric plasma (CAP), which has been found to be very effective in the treatment of many different cancer cell types in vitro as well as in vivo, mainly through the vast generation of RONS. One of the key determinants of the cell's fate will be the interaction of RONS, generated by CAP, with important proteins, i.e. redox-regulatory proteins. One such protein is cytoglobin (CYGB), a recently discovered globin proposed to be involved in the protection of the cell against oxidative stress. In this study, the effect of plasma-produced RONS on CYGB was investigated through the treatment of CYGB with CAP for different treatment times. Spectroscopic analysis of CYGB showed that although chemical modifications occur, its secondary structure remains intact. Mass spectrometry experiments identified these modifications as oxidations of mainly sulfur-containing and aromatic amino acids. With longer treatment time, the treatment was also found to induce nitration of the heme. Furthermore, the two surface-exposed cysteine residues of CYGB were oxidized upon treatment, leading to the formation of intermolecular disulfide bridges, and potentially also intramolecular disulfide bridges. In addition, molecular dynamics and docking simulations confirmed, and further show, that the formation of an intramolecular disulfide bond, due to oxidative conditions, affects the CYGB 3D structure, thereby opening the access to the heme group, through gate functioning of His117. Altogether, the results obtained in this study (1) show that plasma-produced RONS can extensively oxidize proteins and (2) that the oxidation status of two redox-active cysteines lead to different conformations of CYGB.

Candidate biomarkers in the cervical vaginal fluid for the (self-)diagnosis of cervical precancer.

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 30-40 years. A simple test that could be performed at home or at a doctor's 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.

Characterizing the Syphilis-Causing Treponema pallidum ssp. pallidum Proteome Using Complementary Mass Spectrometry.

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.

Identification of protein biomarkers for cervical cancer using human cervicovaginal fluid.

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.

Increased Serpin A5 levels in the cervicovaginal fluid of HIV-1 exposed seronegatives suggest that a subtle balance between serine proteases and their inhibitors may determine susceptibility to HIV-1 infection.

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.

IPA Analysis of Cervicovaginal Fluid from Precancerous Women Points to the Presence of Biomarkers for the Precancerous State of Cervical Carcinoma.

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 system-which is in many cases incomplete because of the counteraction by the HPV virus-we 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.

Comparative proteomics of copper exposure and toxicity in rainbow trout, common carp and gibel carp.

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 50µg/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.

Use of cervicovaginal fluid for the identification of biomarkers for pathologies of the female genital tract.

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.