Mustafa Ayhan

Proteomic analysis and characterisation of human cervico-vaginal fluid proteins

Aim:  Cervico‐vaginal fluid (CVF) may provide insight into the biochemical pathways of human reproduction and parturition. The aim of this study was to establish a 2‐D electrophoretic map of human CVF in healthy, pregnant women at term.

A proteomic analysis of C-reactive protein stimulated THP-1 monocytes

BackgroundC-reactive protein (CRP) is a predictor of cardiovascular risk. It circulates as a pentameric protein in plasma. Recently, a potential dissociation mechanism from the disc-shaped pentameric CRP (pCRP) into single monomers (monomeric or mCRP) has been described. It has been shown that mCRP has strong pro-inflammatory effects on monocytes. To further define the role of mCRP in determining monocyte phenotype, the effects of CRP isoforms on THP-1 protein expression profiles were determined. The hypothesis to be tested was that mCRP induces specific changes in the protein expression profile of THP-1 cells that differ from that of pCRP.MethodsProtein cell lysates from control and mCRP, pCRP or LPS-treated THP-1 cells were displayed using 2-dimensional SDS PAGE and compared. Differentially expressed proteins were identified by MALDI-TOF MS and confirmed by Western blotting.ResultsmCRP significantly up-regulates ubiquitin-activating enzyme E1, a member of the ubiquitin-proteasome system in THP-1 monocytes. Furthermore, HSP 70, alpha-actinin-4 (ACTN4) and alpha-enolase/enolase 1 were upregulated. The proteomic profile of LPS and pCRP treated monocytes differ significantly from that of mCRP.ConclusionThe data obtained in this study support the hypothesis that isoform-specific effects of CRP may differentially regulate the phenotype of monocytes.

Proteomic Analysis of Human Cervico-Vaginal Fluid Displays Differential Protein Expression in Association with Labor Onset at Term

Human labor is characterized by dramatic physiological and structural alterations of the cervix and overlying fetal membranes, leading to myometrial activation and delivery. To investigate the potential mechanism of these changes, we performed 2D PAGE proteomic analysis on serial cervico-vaginal fluid samples obtained from women during late pregnancy and spontaneous labor. We identified 9 protein spots that were significantly altered ( p < 0.05) in association with spontaneous term labor. Eight protein spots were definitively characterized by electrospray ion-trap mass spectrometry yielding 7 different proteins: cystatin-A, interleukin-1 receptor antagonist, glutathione S-transferase P, peroxiredoxin-2, thioredoxin, copper-zinc superoxide dismutase, and epidermal fatty-acid binding protein. These proteins are involved in protease inhibition, anti-inflammatory cytokine activity, and oxidative stress defense. These findings may provide an insight into the biochemical processes and timing associated with extracellular matrix remodelling of the cervix, supracervical fetal membranes, and myometrial activation in association with spontaneous term labor. Application of these findings may lead to development of predictive biomarkers of labor onset.

Identification of ovarian cancer-associated proteins in symptomatic women: A novel method for semi-quantitative plasma proteomics

To evaluate the utility of an enhanced biomarker discovery approach in order to identify potential biomarkers relevant to ovarian cancer detection.

Temporal proteomic analysis of human cervicovaginal fluid with impending term labor.

This study utilized 2D-PAGE and MALDI-ToF to investigate labor-associated temporal protein changes in the human cervicovaginal fluid (>25 kDa). Monocyte/neutrophil elastase inhibitor, squamous cell carcinoma antigen 1, annexin A3, collagen and albumin demonstrated differential expression prior to and/or during term labor. These findings provide further insight into the extracellular matrix remodeling of the cervix and overlying fetal membranes and may be useful to develop predictive tools for labor.

Stage-specific analysis of plasma protein profiles in ovarian cancer: difference in-gel electrophoresis analysis of pooled clinical samples

Introduction: Ovarian cancer is the leading cause of death from gynecological cancer. Non-specific symptoms early in disease and the lack of specific biomarkers hinder early diagnosis. Multi-marker blood screening tests have shown promise for improving identification of early stage disease; however, available tests lack sensitivity, and specificity. Materials and Methods: In this study, pooled deeply-depleted plasma from women with Stage 1, 2 or 3 ovarian cancer and healthy controls were used to compare the 2-dimensional gel electrophoresis (2-DE) protein profiles and identify potential novel markers of ovarian cancer progression. Results/Discussion: Stage-specific variation in biomarker expression was observed. For example, apolipoprotein A1 expression is relatively low in control and Stage 1, but shows a substantial increase in Stage 2 and 3, thus, potential of utility for disease confirmation rather than early detection. A better marker for early stage disease was tropomyosin 4 (TPM4). The expression of TPM4 increased by 2-fold in Stage 2 before returning to “normal” levels in Stage 3 disease. Multiple isoforms were also identified for some proteins and in some cases, displayed stage-specific expression. An interesting example was fibrinogen alpha, for which 8 isoforms were identified. Four displayed a moderate increase at Stage 1 and a substantial increase for Stages 2 and 3 while the other 4 showed only moderate increases. Conclusion: Herein is provided an improved summary of blood protein profiles for women with ovarian cancer stratified by stage.

Proteomic Profiling of Ovarian Cancer Plasma using Immunoaffinity Depleted Plasma and Two-Dimensional PAGE

ObjectiveThe aim of this study was to evaluate a multiple immunoaffinity protein depletion (multiple affinity removal system, MARS) pre-treatment strategy with subsequent two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and peptide mass finger printing analysis for the detection of ovarian cancer-associated plasma proteins.Materials and MethodsFollowing immunoaffinity depletion, total plasma protein content was reduced by 84.2 ± 1.8% (mean ± SE, n = 32). The number of proteins detected in the control and ovarian cancer groups was 349 and 357, respectively. This represented an increase in spot detection of almost twofold when compared to 2D PAGE displays of untreated plasma (174 spots). Of the proteins displayed, post-depletion, 300 (control) and 302 (ovarian cancer, OC) were common within each group. PDQuest analysis indicated that 109 protein spots were statistically different between the two groups and, of these, 59 exhibited greater than or equal to twofold difference in spot density (Student’s t test, p = 0.01). Thirty-nine of these proteins were successfully identified with reliable confidence.Results and DiscussionThe data obtained in this study demonstrates that immunodepletion of plasma before 2D PAGE profiling have generated identifiable plasma proteins that are differentially expressed in the high-grade ovarian cancer sample set compared to controls. This approach, therefore, may be useful in identifying candidate biomarkers for inclusion in multi-marker tests for ovarian cancer that may exhibit greater sensitivity and specificity than those currently available. It was evident, however, from the predominant identification of host response proteins that immunodepletion did not generate sufficient levels of enrichment of lower abundance tumor-specific proteins to facilitate detection.

Peptidomic profiles of post myocardial infarction rats affinity depleted plasma using matrix-assisted laser desorption/ionization time of flight (MALDI-ToF) mass spectrometry.

BackgroundDespite major advances in drug development, effective cardiovascular therapies and suitable cardiovascular biomarkers remain limited. The aim of this study was to leverage mass spectrometry (MS) based peptide profiling strategies to identify changes that occur in peptidomic profiles of rat plasma following coronary artery ligation generated myocardial infarction (MI).MethodsOne week after MI, rats were randomized to receive either an ACE inhibitor (ramipril, Ram-1 mg/kg/day), or vehicle (Veh) for 12 weeks. Echocardiography and hemodynamic measurements were made before sacrifice and plasma collection. High abundance proteins were depleted with affinity capture before MS profiling. Differentially expressed peptide ions were identified using proprietary software (ClinProtTools).ResultsMI increased heart/body weight (18%), lung/body weight (56%), and left ventricular (LV) end diastolic pressure (LVEDP, 247%); and significantly reduced percentage fractional shortening (FS, 75%) and rate of pressure rise in the LV (dP/dtmax, 20%). Ram treatment significantly attenuated the changes in LVEDP (61%) and FS (27%). Analysis of MALDI-ToF generated mass spectra demonstrated that peptide ions 1271, 1878, 1955, 2041 and 2254 m/z were consistently decreased by Ram treatment (p < 0.001) and thus may be associated with the agent’s therapeutic effects. Among peptides that were significantly changed, synapsin-2, adenomatous polyposis coli protein and transcription factor jun-D were identified as significantly reduced by Ram treatment.ConclusionsThis approach allows us to screen for potential biomarkers in a window of the blood proteome that previously has been difficult to access. The data obtained from such an approach may potentially useful in prognosis, diagnosis, and monitoring of treatment response.

Increased expression of alpha-enolase in cervico-vaginal fluid during labour

OBJECTIVES The aim of this study was (i) to characterise differentially expressed proteins in cervico-vaginal fluid (CVF) at the time of preterm labour onset and (ii) to confirm these studies in human CVF samples taken from women before and during spontaneous labour. STUDY DESIGN Preterm labour was induced in sheep (n = 5) via fetal dexamethasone infusion (1 mg/24 h). CVF samples were taken prior to dexamethasone infusion (0 h), 28 h after the start of dexamethasone infusion, and immediately prior to delivery. Two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) were used to identify differentially expressed proteins. For the human studies, paired CVF samples were taken 5-9 days before labour and during spontaneous labour onset (n = 7). RESULTS There was a 4.2-fold increase in α-enolase protein expression in sheep CVF during labour. Likewise, α-enolase protein expression was significantly increased during spontaneous human labour at term. CONCLUSIONS Alpha-enolase is known to be bound to neutrophils and interact in the immune response, and thus may play a role in inflammation associated with human labour.