Proteomic approach for purification of seminal plasma proteins involved in tumor proliferation

Human seminal plasma contains a large array of proteins required for the normal physiology and metabolism of spermatozoa. These are mainly secreted from prostate epithelium, testes, and seminal vesicles. Fortunately, many of these are found to be present at elevated concentration in seminal plasma and act as a biomarker of different carcinomas as their levels are also enhanced in serum and are found to be involved in tumor progression and metastasis apart from fertility. The proteins which were overexpressed in the seminal plasma of prostate carcinoma patients were identified by 2-DE and MALDI-TOF/MS. We have designed a strategy to purify these four proteins prostate specific antigen (PSA), prostatic acid phosphatase (PAP), Zinc alpha2-glycoprotein (ZAG), and progastricsin (PG), together in homogeneity by using simple chromatographic techniques. Acidic and basic fractions of human seminal plasma were separated by ion exchange chromatography and further purified by gel permeation chromatography. Our results form a new and valuable resource for those attempting structure-based drug designing for prostate and other cancers where the amount of proteins is required in plenty and in native form.     

Top–down mass spectrometry reveals new sequence variants of the major bovine seminal plasma protein PDC‐109

The major protein of bovine seminal plasma, PDC‐109, is a 109‐residue polypeptide that exists as a polydisperse aggregate under native conditions. The oligomeric state of this aggregate varies with ionic strength and the presence of lipids. Binding of PDC‐109 to choline phospholipids on the sperm plasma membrane results in an efflux of cholesterol and choline phospholipids, which is an important step in sperm capacitation. In this study, Fourier transform ion cyclotron resonance mass spectrometry was used to analyze PDC‐109 purified from bovine seminal plasma. In addition to the previously known PDC‐109 variants, four new sequence variants were identified by top–down mass spectrometry. For example, a protein variant containing point mutations P10L and G14R was identified along with another form having a 14‐residue truncation in the N‐terminal region. Two other minor variants could also be identified from the affinity‐purified PDC‐109. These results demonstrate that PDC‐109 is naturally produced as a mixture of several protein forms, most of which have not been detected in previous studies. Native mass spectrometry revealed that PDC‐109 is exclusively monomeric at low protein concentrations, suggesting that the protein oligomers are weakly bound and can easily be disrupted. Ligand binding to PDC‐109 was also investigated, and it was observed that two molecules of O‐phosphorylcholine bind to each PDC‐109 monomer, consistent with previous reports. Copyright © 2012 John Wiley & Sons, Ltd.     

Alpha‐1 antitrypsin variants in plasma from HIV‐infected patients revealed by proteomic and glycoproteomic analysis

Novel tools are necessary to explore proteins related to human immunodeficiency virus (HIV) infection. In this work, proteomic and glycoproteomic technology were employed to examine plasma samples from HIV‐positive patients. Through comparative proteome analysis of normal and HIV‐positive plasma samples, 19 differentially expressed protein spots related to 12 non‐redundant proteins were identified by ESI‐ion trap MS. Among these, the 130‐kDa isoform of α‐1‐antitrypsin was found to be decreased in HIV‐positive patients while another variant with a molecular weight of 40 kDa was increased. SWISS‐2‐D‐PAGE reference gel and protein sequence comparisons of the 40‐kDa protein showed homology with α‐1‐antitrypsin minus the N‐terminus, and its identity was further confirmed by 1‐D Western blotting and glycoproteomic analysis. In all, our results showed that proteomics and glycoproteomics are powerful tools for discovering proteins related to HIV infection. Furthermore, this 40‐kDa variant of α‐1‐antitrypsin found in the plasma of HIV‐positive individuals may prove to be a potentially useful biomarker for anti‐HIV research according to bioinformatics analysis.     

BOP: A basic phenylalanine‐rich oligo‐peptide located on the surface of glycolate oxidase influences its pI values

Glycolate oxidase (GO) consists of identical subunits and therefore should show one definite pI value, but the isolated GO exhibited a range of pIs. This study investigated the underlying cause of this phenomenon. GO was purified and showed a molecular weight of 40 kDa by SDS‐PAGE. Elution behavior on DEAE‐cellulose chromatography and cellulose acetate membrane electrophoresis indicated that the purified GO was highly basic (pI>10.0). Repeated IEF and cIEF analysis showed that the pI of the purified GO was in the range of 10.0–3.25, either in a smear form or as distinct bands. 2‐DE analysis showed that the 40 kDa subunit of GO displayed variable pIs from 9.6 to 3.65. It was likely that the purified GO was actually a complex consisted of GO and an unknown protein. CE‐SDS, SDS‐cellulose acetate membrane electrophoresis and amino acid compositions indicated that the unknown protein was a highly basic polymer (BP) consisting of basic and phenylalanine‐rich oligo‐peptide (BOP). Many BOPs are located on the surface of the acidic GO via ionic and hydrophobic interactions and formed GO‐BOP complex (GC), resulting in a highly basic GC although GO itself was acidic. This hypothesis was further supported by the facts that anti‐GC serum failed to recognize GO, and GC showed a peak at 257 nm although GO has few phenylalanine residues. Irregular and incomplete disassociation between GO and BOP was observed in IEF and cIEF, resulting in various intermediates with different ratios of GO/BOP, which could be the reason for the range of pIs observed for GO.     

Immunoaffinity chromatographic isolation of prostate-specific antigen from seminal plasma for capillary electrophoresis analysis of its isoforms

Prostate-specific antigen (PSA) concentration in serum has been the biomarker employed for prostate cancer diagnosis in the last two decades. However, new more specific biomarkers allowing a better differentiation of cancer from non-malignant prostate diseases are necessary. Glycosylation of PSA gives rise to different forms of the protein which can be separated into several isoforms by analytical techniques, such as CE. Because PSA glycosylation is influenced by pathological conditions, the CE pattern of PSA isoforms could be different in prostate cancer than in non-malignant prostate diseases. To study this CE pattern of PSA, prior purification of the protein from the biological fluid is mandatory. In this study an immunoaffinity chromatography method which allows PSA purification without altering the CE pattern is developed. An in-house prepared column produced with commercial anti-PSA antibodies is employed. The use of 1 M propionic acid as elution agent provides higher than 40% recovery of high purity PSA. CE analysis of PSA immunopurified from seminal plasma of a healthy individual shows the same 8 peaks as the commercially available PSA standard. Sample preparation only requires dilution with phosphate buffered saline prior to immunoaffinity purification. High repeatability for the sample preparation step was achieved (RSD% for percentage of corrected peak area in the range 0.6–5.3 for CE analysis of three independently purified seminal plasma aliquots compared to range 0.8–4.9 for a given aliquot analyzed three times by CE). IAC of five microliters seminal plasma provided enough PSA to achieve signal/noise ratio larger than 5 for the smallest CE isoforms.     

Combined electrostatics and hydrogen bonding determine intermolecular interactions between polyphosphoinositides

Membrane lipids are active contributors to cell function as key mediators in signaling pathways controlling cell functions including inflammation, apoptosis, migration, and proliferation. Recent work on multimolecular lipid structures suggests a critical role for lipid organization in regulating the function of both lipids and proteins. Of particular interest in this context are the polyphosphoinositides (PPI's), especially phosphatidylinositol (4,5) bisphosphate (PIP 2). The cellular functions of PIP 2 are numerous but the organization of PIP 2 in the inner leaflet of the plasma membrane, as well as the factors controlling targeting of PIP 2 to specific proteins, remains poorly understood. To analyze the organization of PIP 2 in a simplified planar system, we used Langmuir monolayers to study the effects of subphase conditions on monolayers of purified naturally derived PIP 2 and other anionic or zwitterionic phospholipids. We report a significant molecular area expanding effect of subphase monovalent salts on PIP 2 at biologically relevant surface densities. This effect is shown to be specific to PIP 2 and independent of subphase pH. Chaotropic agents (e.g., salts, trehalose, urea, temperature) that disrupt water structure and the ability of water to mediate intermolecular hydrogen bonding also specifically expanded PIP 2 monolayers. These results suggest a combination of water-mediated hydrogen bonding and headgroup repulsion in determining the organization of PIP 2, and may contribute to an explanation for the unique functionality of PIP 2 compared to other anionic phospholipids.     

Integrated selective enrichment target--a microtechnology platform for matrix-assisted laser desorption/ionization-mass spectrometry applied on protein biomarkers in prostate diseases

The performance of a miniaturized sample processing platform for matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS), manufactured by silicon microfabrication, called integrated selective enrichment target (ISET) technology was evaluated in a biological context. The ISET serves as both sample treatment device and MALDI-MS target, and contains an array of 96 perforated nanovials, which each can be filled with 40 nL of reversed-phase beads. This methodology minimizes the number of sample transfers and the total surface area available for undesired adsorption of the analytes in order to provide high-sensitivity analysis. ISET technology was successfully applied for characterization of proteins coisolated by affinity chromatography of prostate-specific antigen (PSA) from human seminal fluid. The application of ISET sample preparation enabled multiple analyses to be performed on a limited sample volume, which resulted in the discovery that prolactin inducible protein (PIP) was coisolated from the samples.     

First syntheses of natural products with the 2,7-dihydroxy-2H-1,4-benzoxazin-3(4H)-one skeleton

2,7-Dihydroxy-2H-1,4-benzoxazin-3(4H)-one 11 (DHBOA) and 2,4,7-trihydroxy-2H-1,4-benzoxazin-3(4H)-one 14 (TRIBOA) representing aglucones of naturally occurring acetal glucoside type allelo chemicals found in Gramineae have been for the first time synthesized by two pathways both involving selective reductive cyclizations of appropriate 7-benzyloxy-2-nitrophenol derivatives as precursors. TRIBOA 14 and its bioactive naturally occurring 7-methyl ether DIMBOA have been found to undergo a hitherto unknown transformation to the corresponding 2,6-dibromo substituted lactam forms 20 and 21 in the presence of hydrogen bromide in acetic acid, which is of value in a better understanding of the possible mode of bioactivity.     

Cysteine carboxyl O-methylation of human placental 23 kDa protein

C-Terminal carboxyl methylation of a human placental 23 kDa protein catalyzed by membrane-associated methyltransferase has been investigated. The 23 kDa protein substrate methylated was partially purified by DEAE-Sephacel, hydroxyapatite and Sephadex G-100 gel filtration chromatographies. The substrate protein was eluted on Sephadex G-100 gel filtration chromatography as a protein of about 29 kDa. In the absence of Mg2+, the methylation was stimulated by guanine nucleotides (GTP, GDP and GTPgammaS), but in the presence of Mg2+, only GTPgammaS stimulated the methylation which was similar to the effect on the G25K/rhoGDI complex. AFC, an inhibitor of C-terminal carboxyl methylation, inhibited the methylation of human placental 23 kDa protein. These results suggests that the substrate is a small G protein different from the G25K and is methylated on C-terminal isoprenylated cysteine residue. This was also confirmed by vapor phase analysis. The methylated substrate protein was redistributed to membrane after in vitro methylation, suggesting that the methylation of this protein is important for the redistribution of the 23 kDa small G protein for its putative role in intracellular signaling.     

Design and synthesis of novel thiazole-containing cross-linked polyamides related to the antiviral antibiotic distamycin

A family of naturally occurring oligopeptides includes netropsin, distamycin, anthelvencin, kikumycin B, amidinomycin, and norformycin. Netropsin (I) and distamycin (II) express their biological activities by targeting specific sequences of chemical functionalities in the minor groove of DNA. Both netropsin and distamycin can be regarded as polyamide chains in which each alpha-carbon has been replaced by a five-membered pyrrole ring. The repeat distance in such an augmented polyamide chain is almost the same as the distance from one base pair to the next along the floor of a minor groove within beta-DNA. In this paper we report the synthesis of 16-21 cross-linked polyamides containing a thiazole heterocyclic ring bearing the active functionalites NH(2), NHCHO, or H. 16 and 17 were synthesized by DCC and HOBt catalyzed reaction of 5 with 14 and 15, while the formylation products 18 and 19 were obtained by coupling the formylated 4-methyl-thiazolated acid 6 with 14 and 15. The deaminated compounds 20 and 21 were obtained by the coupling of 5-trichloroacetyl-4-methylthiazole 7 synthesized from 4-methylthiazole. All the six cross-linked polyamides 16-21 were tested for their DNA gyrase inhibition. The studies have shown these polyamides have better sequence recognition and a greater percentage of inhibition than the corresponding monomers. The compound 17 shows complete inhibition of gyrase at 0.5 microM concentration as compared to the naturally occurring distamycin at 1.0 microM.     

The N-terminal 1-16 peptide derived in vivo from protein seminal vesicle protein IV modulates alpha-thrombin activity: potential clinical implications

We have previously shown that seminal vesicle protein IV (SV-IV) and its 1-70 N-terminal fragment have anti-inflammatory activity and modulate anti-thrombin III (AT) activity. Moreover, mass spectrometry analysis of purified SV-IV has shown that the protein was found to be highly heterogeneous and 14% of the total SV-IV molecules are truncated forms, of particular interest the 1-16, 1-17, and 1-18 peptides. In this work we report experimental data which demonstrate that the 1-16 peptide (P1-16) possesses a marked effect on the AT activity by preventing the formation of the thrombin-AT complex. We found that the formation of thrombin-AT complex is markedly decreased in the presence of P1-16 used at equimolar concentration with thrombin as evaluated with SDS-PAGE. We also monitored the conformational changes of thrombin in the presence of different P1-16 concentrations, and calculated the K(d) of thrombin/P1-16 system by circular dichroism technique. The probable interaction sites of P1-16 with thrombin have been also evaluated by molecular graphics and computational analyses. These results have potential implications in the treatment of sterility and thrombotic diseases.     

Screening of radical scavengers in Scutellaria baicalensis using HPLC with diode array and chemiluminescence detection

Scutellaria baicalensis Georgi is a well-known medical plant widely used as a famous traditional Chinese medicine. It has been reported that S. baicalensis can protect against oxidative stress and possess anti-inflammatory effect. In the present paper, an HPLC-diode array-chemiluminescence detection method for on-line detection was successfully developed to screen antioxidants in complex S. baicalensis extracts. Using the proposed approach, eight compounds in the S. baicalensis extracts were found to possess a potential antioxidant activity. Furthermore, the effects of purified compounds on protecting RAW 264.7 cells from hydrogen peroxide injury were also investigated in vitro to confirm the established method, which were consistent with the results of HPLC-diode array-chemiluminescence detection method. These results demonstrated that this method was a useful technique for rapidly screening and identifying bioactive components from complex herbal medicines.     

Synthesis of styrene‐g‐polyisoprene nanoparticles by emulsion polymerization and its effect on properties of polyisoprene composites

The graft copolymerization of styrene onto nanosized polyisoprene (PIP) was carried out by using cumene hydroperoxide and tetraethylene pentamine as redox initiators. The high conversion and high degree of grafting could be achieved when a small particle was used as the core polymer. The grafting efficiency and monomer conversion increased with increasing reaction temperature and monomer concentration. Transmission electron microscopy indicated that the small PIP nanoparticles were completely coated with polystyrene (PS) by grafting resulting in a core shell morphology of nanosized graft PIP. Nanosized PIP and nanosized PS‐g‐PIP could be used as compatibilizers for vulcanized rubber latex. The addition of nanosized PIP and PS‐g‐PIP strongly influenced the mechanical properties of the natural rubber (NR)‐based compound. Incorporation of nanosized PIP and PS‐g‐PIP resulted in an improvement of the resistance of the compounds to heat aging. Copyright © 2011 John Wiley & Sons, Ltd.     

Analysis of natural flavonoids by microchip-micellar electrokinetic chromatography with pulsed amperometric detection

Catechins (catechin and other derivatives) are naturally occurring flavonoids present in a number of plants and foods. They are also part of numerous nutraceutical formulations because they are believed to have antioxidant, cancer chemo-preventative, anti-inflammatory and antimicrobial properties. The determination of catechins has traditionally been performed by HPLC. However, this methodology is both time and sample intensive and generates large amounts of organic solvent waste. In the current report, an application of MEKC using a PDMS microchip is presented for the analysis of catechins. The system uses pulsed amperometric detection for direct analysis of important naturally occurring catechins. The effect of pH, surfactant concentration, detection potential and signal stability were analyzed. Linear relationships were found between the concentration and peak current, with good stability and limits of detection of 8 [micro sign]M for catechin, epigallocatechin gallate and epicatechin, and 14 [micro sign]M for epicatechin gallate. Optimum conditions were applied to the detection of selected catechins in a commercially available green tea extract nutraceutical and the results were compared to HPLC analysis. The analysis using microchip micellar electrokinetic chromatography and pulsed amperometric detection was completed in 4.5 min, 10 times faster than the HPLC analysis.     

Synthetic Methods for the Preparation of Conformationally Restricted Analogues of Nicotine

In the context of naturally occurring nitrogen heterocycles, nicotine is a chiral alkaloid present in tobacco plants, which can target and stimulate nicotinic acetylcholine receptors (nAChRs), a class of ligand-gated ion channels commonly located throughout the human brain. Due to its well-known toxicity for humans, there is considerable interest in the development of synthetic analogues; in particular, conformationally restricted analogues of nicotine have emerged as promising drug molecules for selective nAChR-targeting ligands. In the present mini-review, we will describe the synthesis of the conformationally restricted analogues of nicotine involving one or more catalytic processes. In particular, we will follow a systematic approach as a function of the heteroarene structure, considering: (a) 2,3-annulated tricyclic derivatives; (b) 3,4-annulated tricyclic derivatives; (c) tetracyclic derivatives; and (d) other polycyclic derivatives. For each of them we will also consider, when carried out, biological studies on their activity for specific nAChR subunits.     

Quantification of naturally occurring pyrrole acids in melanosomes

Three naturally occurring pyrrole acids were found in Sepia, human black hair, and bovine choroid and iris melanosomes using high-performance liquid chromatography and mass spectrometry--pyrrole-2,3-dicarboxylic acid (PDCA), pyrrole-2,3,5-tricarboxylic acid (PTCA) and pyrrole-2,3,4,5-tetracarboxylic acid (PTeCA). PDCA and PTCA are common markers quantified from oxidative degradation of eumelanins. Using standards, the amounts of naturally occurring PDCA and PTCA were determined and compared to those obtained following peroxide oxidation of the same samples. Because the naturally occurring acids are water soluble, these results indicate that care must be exercised when comparing PDCA and PTCA yields from the degradation analyses of melanins isolated and prepared by different methods. This work also establishes that PTeCA is a naturally occurring pyrrole acid in melanosomes.     

Structural studies on sugar chains of carbohydrate-deficient transferrin from patients with alcoholic liver disease using lectin affinity electrophoresis

It is well-known that microheterogeneity of human serum transferrin observed in alcoholics manifests as sialic acid-deficient transferrin isoforms, otherwise known as carbohydrate-deficient transferrin (CDT). A recent study demonstrated that serum CDT lacked one or both of the entire carbohydrate chains but the investigation required several troublesome procedures. The aim of the present study was to confirm the sugar chain structures of serum transferrin, and of serum CDT in particular, from patients with alcoholic liver disease (ALD) using conventional lectin affinity electrophoresis which might be useful in the clinical setting. The serum CDT obtained from ALD-patients was partially purified using an anion exchanger. Serum transferrin and the partially purified serum CDT were investigated by concanavalin A (Con A)- and Datura stramonium agglutinin (DSA)-affinity electrophoresis followed by antibody-affinity blotting and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with Western blotting. By Con A-affinity electrophoresis, serum CDT was separated into weakly reactive and nonreactive transferrins which showed slower electrophoretic mobilities than those from the healthy controls. Moreover, nearly all of the serum CDT was nonreactive with DSA. On SDS-PAGE, the molecular masses of serum CDT were estimated to be approximately 75 and 72 kDa, which corresponded to those of partially and completely deglycosylated transferrin obtained from the healthy controls (78 kDa), respectively. In conclusion, these results indicated that the sugar chain structures of serum CDT from patients with ALD show not merely a loss of terminal sialic acids, but also the absence of asparagine-N-linked oligosaccharides.     

A benchmark spike‐in data set for biomarker identification in metabolomics

The development and the validation of innovative approaches for biomarker selection are of paramount importance in many ‐omics technologies. Unfortunately, the actual testing of new methods on real data is difficult, because in real data sets, one can never be sure about the “true” biomarkers. In this paper, we present a publicly available metabolomic ultra performance liquid chromatography–mass spectrometry spike‐in data set for apples. The data set consists of 10 control samples and three spiked sets of the same size, where naturally occurring compounds are added in different concentrations. In this sense, the data set can serve as a test bed to assess the performance of new algorithms and compare them with previously published results. We illustrate some of the possibilities provided by this spike‐in data set by comparing the performance of two popular biomarker‐selection methods, the univariate t‐test and the multivariate variable importance in projection. To promote a widespread use of the data, raw data files as well as preprocessed peak lists are made available. Copyright © 2012 John Wiley & Sons, Ltd.     

Comparison of phosphatidylcholine vesicle properties related to geometrical isomerism

Glycerophosphatidylcholine containing trans-unsaturated fatty acid residues was prepared by reaction of the corresponding naturally occurring cis lipid with photochemically generated thiyl radicals. This modified lipid was chosen as the simplest model for gaining some insights of the complex scenario of membrane formation, in connection with the role of lipid geometry and the predominance of cis lipids in eukaryotic cells. The critical aggregation concentration for the spontaneous formation of vesicles was determined for cis and trans isomers with cis-parinaric acid used as a fluorescent probe and it was found to be similar for both lipids. Vesicle dimensions were investigated by light scattering and electron microscopy, and the type of fatty acid residues influenced the vesicle diameter, with a decrease along the series cis > trans > saturated. Fluorescence measurement of dye release from trans and cis vesicles showed also a different permeability. A picture emerged of the geometrical isomer preference in cells as a process driven by natural selection during the life evolution of different organisms, both in terms of compartment dimensions and membrane functionality.     

An Optimized Synthesis of 2-Methoxyestradiol,A Naturally Occurring Human Metabolite with Anticancer Activity

2-Methoxyestradiol, a naturally occurring human metabolite with demonstrated anticancer activity, has been synthesized in three steps and 76% yield from the bis(MOM) ether of 2-formylestradiol.