Geert Van Raemdonck

Comprehensive proteomic analysis of human cervical-vaginal fluid using colposcopy samples.

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.

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.

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.

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 carps 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.

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.

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.

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 cells 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.

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.

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.

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.