Proteomic analysis of ovarian cancer proximal fluids: validation of elevated peroxiredoxin 1 in patient peripheral circulation

Background

Epithelial ovarian cancer (EOC) is the deadliest gynecologic malignancy in the United States. Unfortunately, a validated protein biomarker-screening test to detect early stage disease from peripheral blood has not yet been developed. The present investigation assesses the ability to identify tumor relevant proteins from ovarian cancer proximal fluids, including tissue interstitial fluid (TIF) and corresponding ascites, from patients with papillary serous EOC and translates these findings to targeted blood-based immunoassays.

Methodology/Principal Findings

Paired TIF and ascites collected from four papillary serous EOC patients at the time of surgery underwent immunodepletion, resolution by 1D gel electrophoresis and in-gel digestion for analysis by liquid chromatography-tandem mass spectrometry, which resulted in an aggregate identification of 569 and 171 proteins from TIF and ascites, respectively. Of these, peroxiredoxin I (PRDX1) was selected for validation in serum by ELISA and demonstrated to be present and significantly elevated (p = 0.0188) in 20 EOC patients with a mean level of 26.0 ng/mL (±9.27 SEM) as compared to 4.19 ng/mL (±2.58 SEM) from 16 patients with normal/benign ovarian pathology.

Conclusions/Significance

We have utilized a workflow for harvesting EOC-relevant proximal biofluids, including TIF and ascites, for proteomic analysis. Among the differentially abundant proteins identified from these proximal fluids, PRDX1 was demonstrated to be present in serum and shown by ELISA to be elevated by nearly 6-fold in papillary serous EOC patients relative to normal/benign patients. Our findings demonstrate the facile ability to discover potential EOC-relevant proteins in proximal fluids and confirm their presence in peripheral blood serum. In addition, our finding of elevated levels of PRDX1 in the serum of EOC patients versus normal/benign patients warrants further evaluation as a tumor specific biomarker for EOC.     

The proline-rich peptide Bac7(1-35) reduces mortality from <Emphasis Type="Italic">Salmonella typhimurium</Emphasis> in a mouse model of infection

Background  

Bac7 is a proline-rich peptide with a potent in vitro antimicrobial activity against Gram-negative bacteria. Here we investigated its activity in biological fluids and in vivo using a mouse model of S. typhimurium infection.     

Qualitative and quantitative characteristics of the extracellular DNA delivered to the nucleus of a living cell

Background  

The blood plasma and other intertissue fluids usually contain a certain amount of DNA, getting there due to a natural cell death in the organism. Cells of this organism can capture the extracellular DNA, whereupon it is delivered to various cell compartments. It is hypothesized that the extracellular DNA is involved in the transfer of genetic information and its fixation in the genome of recipient cell.     

Harshlight: a "corrective make-up" program for microarray chips

Background  

Microscopists are familiar with many blemishes that fluorescence images can have due to dust and debris, glass flaws, uneven distribution of fluids or surface coatings, etc. Microarray scans do show similar artifacts, which might affect subsequent analysis. Although all but the starkest blemishes are hard to find by the unaided eye, particularly in high-density oligonucleotide arrays (HDONAs), few tools are available to help with the detection of those defects.     

Peptides OFFGEL electrophoresis: a suitable pre-analytical step for complex eukaryotic samples fractionation compatible with quantitative iTRAQ labeling

Background  

The proteomes of mammalian biological fluids, cells and tissues are complex and composed of proteins with a wide dynamic range. The effective way to overcome the complexity of these proteomes is to combine several fractionation steps. OFFGEL fractionation, recently developed by Agilent Technologies, provides the ability to pre-fractionate peptides into discrete liquid fractions and demonstrated high efficiency and repeatability necessary for the analysis of such complex proteomes.     

<Emphasis Type="Italic">In vitro</Emphasis>effects of relaxin on gene expression in porcine cumulus-oocyte complexes and developing embryos

Background  

Relaxin hormone peptide is found in porcine follicular and utero-tubal fluids, but its possible actions during early embryo development are still undetermined. Here, we investigated the effects of porcine relaxin during oocyte maturation and embryo development, and gene expression in the pig.     

Life cycle assessment of mineral oil-based and vegetable oil-based hydraulic fluids including comparison of biocatalytic and conventional production methods

Background, aim and scope  

Lubricants are used in numerous applications in our society, for instance, as hydraulic fluids. When used in forestry, 60–80% of these hydraulic fluids are released into the environment. This is one of the reasons for the growing interest for developing and utilising hydraulic fluids with good environmental performance. Another driving force in the development of hydraulic fluids is to replace fossil products with renewable ones. The aim of this paper is to investigate the environmental impact of two types of hydraulic fluids, one based on mineral oil and one on vegetable oil. The difference in environmental impact of using chemical or biocatalytic production methods is also assessed.     

A Targeted Proteomics Approach for Biomarker Discovery Using Bilateral Matched Nipple Aspiration Fluids

Introduction  

The objective of this study was to identify cancer-associated protein expression patterns in bilateral matched nipple aspiration fluids using nanoscale reciprocal Cy3/Cy5 labeling and high-content antibody microarrays. This novel platform allows the pair-wise comparisons of the relative abundance of 512 different antigens using minimal NAF sample containing 1 μg of total protein.     

Hypogelsolinemia,a disorder of the extracellular actin scavenger system,in patients with multiple sclerosis

Background  

Extracellular gelsolin (GSN) and GC-globulin/Vitamin D-binding protein (DBP) appear to play an important role in clearing the actin from extracellular fluids and in modulating cellular responses to anionic bioactive lipids. In this study we hypothesized that cellular actin release and/or increase in bioactive lipids associated with multiple sclerosis (MS) development will translate into alteration of the actin scavenger system protein concentrations in blood and cerebrospinal fluid (CSF) of patients with MS.     

Microparticle-induced release of B-lymphocyte regulators by rheumatoid synoviocytes

Introduction  

In the present study, we investigated the ability of microparticles isolated from synovial fluids from patients with rheumatoid arthritis or osteoarthritis to induce the synthesis and release of key cytokines of B-lymphocyte modulation such as B cell-activating factor, thymic stroma lymphopoietin, and secretory leukocyte protease inhibitor by rheumatoid fibroblast-like synoviocytes.     

Estradiol,progesterone, testosterone profiles in human follicular fluid and cultured granulosa cells from luteinized pre-ovulatory follicles

Background  

The production of sex steroids by follicular cells is proposed to be influenced by the maturity of the incumbent oocyte. Thus steroid levels may reflect suitability of an oocyte for IVF. We examined follicular fluids and granulosa cell production of steroid from IVF patients in order to test the relationship between steroid levels and fertilization.     

A novel R-package graphic user interface for the analysis of metabonomic profiles

Background  

Analysis of the plethora of metabolites found in the NMR spectra of biological fluids or tissues requires data complexity to be simplified. We present a graphical user interface (GUI) for NMR-based metabonomic analysis. The "Metabonomic Package" has been developed for metabonomics research as open-source software and uses the R statistical libraries.     

A role for p38 MAPK in the regulation of ciliary motion in a eukaryote

Background  

Motile cilia are essential to the survival and reproduction of many eukaryotes; they are responsible for powering swimming of protists and small multicellular organisms and drive fluids across respiratory and reproductive surfaces in mammals. Although tremendous progress has been made to comprehend the biochemical basis of these complex evolutionarily-conserved organelles, few protein kinases have been reported to co-ordinate ciliary beat. Here we present evidence for p38 mitogen-activated protein kinase (p38 MAPK) playing a role in the ciliary beat of a multicellular eukaryote, the free-living miracidium stage of the platyhelminth parasite Schistosoma mansoni.     

Plasma and synovial fluid microRNAs as potential biomarkers of rheumatoid arthritis and osteoarthritis

Introduction  

MicroRNAs (miRNAs), endogenous small noncoding RNAs regulating the activities of target mRNAs and cellular processes, are present in human plasma in a stable form. In this study, we investigated whether miRNAs are also stably present in synovial fluids and whether plasma and synovial fluid miRNAs could be biomarkers of rheumatoid arthritis (RA) and osteoarthritis (OA).     

Ovine serum biomarkers of early and late phase scrapie

Background  

Transmissible spongiform encephalopathies are fatal neurodegenerative disease occurring in animals and humans for which no ante-mortem diagnostic test in biological fluids is available. In such pathologies, detection of the pathological form of the prion protein (i.e., the causative factor) in blood is difficult and therefore identification of new biomarkers implicated in the pathway of prion infection is relevant.     

Prostasome-like vesicles stimulate acrosome reaction of pig spermatozoa

Background  

The presence of small membranous particles characterizes the male genital fluids of different mammalian species. The influence of semen vesicles, denominated prostasomes, on sperm functional properties has been well documented in humans, but their biological activity is scarcely known in other species. The present work investigated prostasome-like vesicles in pig semen for their ability to interact with spermatozoa and to affect acrosome reaction.     

"Harshlighting" small blemishes on microarrays

Background  

Microscopists are familiar with many blemishes that fluorescence images can have due to dust and debris, glass flaws, uneven distribution of fluids or surface coatings, etc. Microarray scans show similar artefacts, which affect the analysis, particularly when one tries to detect subtle changes. However, most blemishes are hard to find by the unaided eye, particularly in high-density oligonucleotide arrays (HDONAs).     

Disulfide-bonded multimers of proteoglycan 4 (PRG4) are present in normal synovial fluids

Background

The proteoglycan 4 (PRG4) gene encodes for a mucin-like O-linked glycosylated protein with several names, including lubricin and superficial zone protein. The objective of this study was to analyze PRG4 in normal bovine calf and steer synovial fluids for evidence of native multimers formed by intermolecular disulfide bonds.

Methods

A combination of mucin biochemical techniques, with antibodies to both terminal domains and the mucin-like domain of PRG4, were used for analyses.

Results

Multimers were present in both calf and steer fluids, and reduction and alkylation converts the multimeric complex (likely dimeric) into monomeric subunits. Tandem mass spectrometry analyses supported the Western blot data and identified PRG4 in the reduced ∼ 345 kDa monomeric form. Interestingly, ∼ 70 kDa fragments released upon reduction contained peptides from both the N and C terminal regions, which most likely represent fragments of a sparsely glycosylated PRG4 population that are disulfide-linked to extensively glycosylated, intact monomers.

Conclusions

The analyses described here have demonstrated the presence of native disulfide-bonded multimers of PRG4 in normal bovine synovial fluids.

General significance

These structures are similar to those described for multimerization of mucins in general. Such multimerization and proteolytic cleavage of PRG4 may have functional significance in joint health and disease.