Inflammatory and antioxidant pattern unbalance in "clopidogrel-resistant" patients during acute coronary syndrome.

BACKGROUND: In acute coronary syndrome (ACS), inflammation and redox response are associated with increased residual platelet reactivity (RPR) on clopidogrel therapy. We investigated whether clopidogrel interaction affects platelet function and modulates factors related to inflammation and oxidation in ACS patients differently responding to clopidogrel. MATERIAL AND METHODS: Platelet aggregation was measured in 29 ACS patients on dual (aspirin/clopidogrel) antiplatelet therapy. Nonresponders (NR) were defined as RPR ≥70% by ADP. Several inflammatory and redox parameters were assayed and platelet proteome was determined. RESULTS: Eight (28%) out of 29 ACS patients resulted NR to clopidogrel. At 24 hours, the levels of Th2-type cytokines IL-4, IFNγ, and MCP-1 were higher in NR, while blood GSH (r-GSHbl) levels were lower in NR than responders (R). Proteomic analysis evidenced an upregulated level of platelet adhesion molecule, CD226, and a downregulation of the antioxidant peroxiredoxin-4. In R patients the proinflammatory cytokine IL-6 decreased, while the anti-inflammatory cytokine IL-1Ra increased. CONCLUSIONS: In patients with high RPR on clopidogrel therapy, an unbalance of inflammatory factors, platelet adhesion molecules, and circulatory and platelet antioxidant molecules was observed during the acute phase. Proinflammatory milieu persists in nonresponders for a long time after the acute event while antioxidant blood factors tend to conform to normal responsiveness.

Low T3 State Is Correlated with Cardiac Mitochondrial Impairments after Ischemia Reperfusion Injury: Evidence from a Proteomic Approach.

Mitochondria are major determinants of cell fate in ischemia/reperfusion injury (IR) and common effectors of cardio-protective strategies in cardiac ischemic disease. Thyroid hormone homeostasis critically affects mitochondrial function and energy production. Since a low T3 state (LT3S) is frequently observed in the post infarction setting, the study was aimed to investigate the relationship between 72 h post IR T3 levels and both the cardiac function and the mitochondrial proteome in a rat model of IR. The low T3 group exhibits the most compromised cardiac performance along with the worst mitochondrial activity. Accordingly, our results show a different remodeling of the mitochondrial proteome in the presence or absence of a LT3S, with alterations in groups of proteins that play a key role in energy metabolism, quality control and regulation of cell death pathways. Overall, our findings highlight a relationship between LT3S in the early post IR and poor cardiac and mitochondrial outcomes, and suggest a potential implication of thyroid hormone in the cardio-protection and tissue remodeling in ischemic disease.

Secreted proteins from carotid endarterectomy: an untargeted approach to disclose molecular clues of plaque progression.

BACKGROUND: Atherosclerosis is the main cause of morbidity and mortality in Western countries and carotid plaque rupture is associated to acute events and responsible of 15-20% of all ischemic strokes. Several proteomics approaches have been up to now used to elucidate the molecular mechanisms involved in plaque formation as well as to identify markers of pathology severity for early diagnosis or target of therapy. The aim of this study was to characterize the plaque secretome. The advantage of this approach is that secretome mimics the in vivo condition and implies a reduced complexity compared to the whole tissue proteomics allowing the detection of under-represented potential biomarkers. METHODS: Secretomes from carotid endarterectomy specimens of 14 patients were analyzed by a liquid chromatography approach coupled with label free mass spectrometry. Differential expression of proteins released from plaques and from their downstream distal side segments were evaluated in each specimen. Results were validated by Western blot analysis and ELISA assays. Histology and immunohistochemistry were performed to characterize plaques and to localise the molecular factors highlighted by proteomics. RESULTS: A total of 463 proteins were identified and 31 proteins resulted differentially secreted from plaques and corresponding downstream segments. A clear-cut distinction in the distribution of cellular- and extracellular-derived proteins, evidently related to the higher cellularity of distal side segments, was observed along the longitudinal axis of carotid endarterectomy samples. The expressions of thrombospondin-1, vitamin D binding protein, and vinculin, as examples of extracellular and intracellular proteins, were immunohistologically compared between adjacent segments and validated by antibody assays. ELISA assays of plasma samples from 34 patients and 10 healthy volunteers confirmed a significantly higher concentration of thrombospondin-1 and vitamin D binding protein in atherosclerotic subjects. CONCLUSIONS: Taking advantage of the optimized workflow, a detailed protein profile related to carotid plaque secretome has been produced which may assist and improve biomarker discovery of molecular factors in blood. Distinctive signatures of proteins secreted by adjacent segments of carotid plaques were evidenced and they may help discriminating markers of plaque complication from those of plaque growth.

[Proteomics and personalized medicine]. / Proteomica e medicina personalizzata.

With the disclosure of the human genome a new era for bio-medicine has arisen, characterized by the challenge to investigate pathogenic mechanisms, studying simultaneously metabolites, DNA, RNA, and proteins. As a result, the "omics" revolution boomed, giving birth to a new medicine named "omics-based medicine". Among the other "omics", proteomics has been widely used in medicine, since it can produce a more "holistic" overview of a disease and provide a "constellation" of possible specific markers, a molecular fingerprinting that defines the clinical condition of an individual. Endpoint of this comprehensive and detailed analysis is the "diagnostic-omics", i.e. the achievement of personalized diagnoses with obvious benefits for prevention and therapy and this goal can be reached only with a perfect integration between clinicians and proteomists. To impact on the possible key factors involved in the pathological processes, oligonucleotide-based knock-down strategies can be helpful. They exploit omics-derived molecular tools (antisense, siRNA, ribozymes, decoys, and aptamers) that can be used to inhibit, at transcriptional or post-transcriptional levels, the events leading to protein synthesis, thus decreasing its expression. The identification of the pivotal mechanisms involved in diseases using global, "scenic" approaches such as the "omics" ones, and the subsequent validation and detailed description of the processes by specific molecular tools, can result in a more preventive, predictive and personalized medicine.

Endocellular polyamine availability modulates epithelial-to-mesenchymal transition and unfolded protein response in MDCK cells.

Epithelial-to-mesenchymal transition (EMT) is involved in embryonic development as well as in several pathological conditions. Literature indicates that polyamine availability may affect transcription of c-myc, matrix metalloproteinase (MMP)1, MMP2, TGFbeta(1), and collagen type I mRNA. The aim of this study was to elucidate polyamines role in EMT in vitro. Madin-Darby canine kidney (MDCK) cells were subjected to experimental manipulation of intracellular levels of polyamines. Acquisition of mesenchymal phenotype was evaluated by means of immunofluorescence, western blots, and zymograms. MDCK cells were then subjected to 2D gel proteomic study and incorporation of a biotinilated polyamine (BPA). Polyamine endocellular availability modulated EMT process. Polyamine-depleted cells treated with TGFbeta(1) showed enhanced EMT with a marked decrease of E-cadherin expression at plasma membrane level and an increased expression of mesenchymal markers such as fibronectin and alpha-smooth muscle actin. Polyamine-depleted cells showed a twofold increased expression of the rough endoplasmic reticulum (ER)-stress proteins GRP78, GRP94, and HSP90 alpha/beta in 2D gels. The latter data were confirmed by western blot analysis. Administration of BPA showed that polyamines are covalently linked, within the cell, to ER-stress proteins. Intracellular polyamine availability affects EMT in MDCK cells possibly through the modulation of ER-stress protein homeostasis.

A gel-free approach in vascular smooth muscle cell proteome: perspectives for a better insight into activation.

BACKGROUND: The use of chromatography coupled with mass spectrometry (MS) analysis is a powerful approach to identify proteins, owing to its capacity to fractionate molecules according to different chemical features. The first protein expression map of vascular smooth muscle cells (VSMC) was published in 2001 and since then other papers have been produced. The most detailed two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) map was presented by Mayr et al who identified 235 proteins, corresponding to the 154 most abundant unique proteins in mouse aortic VSMC. A chromatographic approach aimed at fractionating the VSMC proteome has never been used before. RESULTS: This paper describes a strategy for the study of the VSMC proteome. Our approach was based on pre-fractionation with ion exchange chromatography coupled with matrix assisted laser desorption-time of flight mass spectrometry analysis assisted by a liquid chromatography (LC-MALDI-TOF/TOF). Ion exchange chromatography resulted in a good strategy designed to simplify the complexity of the cellular extract and to identify a large number of proteins. Selectivity based on the ion-exchange chemical features was adequate if evaluated on the basis of protein pI. The LC-MALDI approach proved to be highly reproducible and sensitive since we were able to identify up to 815 proteins with a concentration dynamic range of 7 orders of magnitude. CONCLUSIONS: In our opinion, the large number of identified proteins and the promising quantitative reproducibility made this approach a powerful method to analyze complex protein mixtures in a high throughput way and to obtain statistical data for the discovery of key factors involved in VSMC activation and to analyze a label-free differential protein expression.

Hypothesis-free secretome analysis of thoracic aortic aneurysm reinforces the central role of TGF-ß cascade in patients with bicuspid aortic valve.

BACKGROUND: Ascending thoracic aortic aneurysm (ATAA) is a major cause of morbidity and mortality worldwide. The pathogenesis of medial degeneration of the aorta remains undefined. High-throughput secretome analysis by mass spectrometry may be useful to elucidate the molecular mechanisms involved in aneurysm formation as well as to identify biomarkers for early diagnosis or targets of therapy. The purpose of the present study was to analyze the secreted/released proteins from ATAA specimens of both tricuspid aortic valve (TAV) and bicuspid aortic valve (BAV) patients. METHODS: Aortic specimens were collected from patients undergoing elective surgery and requiring graft replacement of the ascending aorta. Each sample of the ascending aortic aneurysm, 4 BAV (3 males; aged 53.5±11.4 years) and 4 TAV (1 male; 78±7.5 years), was incubated for 24h in serum-free medium. Released proteins were digested with trypsin. Peptide mixtures were fractioned by nano-high performance liquid chromatography and analyzed by mass spectrometry. Following identification of differentially expressed proteins, quantitative real time polymerase chain reaction (qRT-PCR) analysis was performed. RESULTS: The comparison between the proteins released from BAV and TAV aneurysmatic tissues showed significantly diverging expression fingerprints in the two groups of patients. Bioinformatics analysis revealed 38 differentially released proteins; in particular 7 proteins were down-regulated while 31 were up-regulated in BAV with respect to TAV. Most of the proteins that were up-released in BAV were related to the activation of transforming growth factor (TGF)-ß signaling. Latent TGF-ß binding protein 4 (LTBP4) exhibited one of the highest significant under-expressions (10-fold change) in BAV secretomes with respect to TAV. qRT-PCR analysis validated this significant difference at LTBP4 gene level (BAV: 1.03±0.9 vs TAV: 3.6±3.2; p<0.05). CONCLUSION: Hypothesis-free secretome profiling clearly showed diverging expression fingerprints in the ATAA of TAV and BAV patients, confirming the crucial role of TGF-ß signaling in modulating ATAA development in bicuspid patients.

Resting smooth muscle cells as a model for studying vascular cell activation.

Vascular smooth muscle (VSM) cells constitute the main structural components of tunica media. Under physiological conditions, these cells display a contractile phenotype and a low proliferative activity. However, they may also acquire a synthetic phenotype and become predominantly proliferative if stimulated under certain stress conditions. This capacity plays a major role in the inception and progression of such cardiovascular diseases as atherosclerosis, hypertension and restenosis. Porcine coronary smooth muscle (PCSM) cells exhibit a synthetic phenotype (ON cells) under standard culturing conditions, but they can be reverted to a contractile phenotype (OFF cells) in a serum-free medium. However, OFF cells can also re-acquire a synthetic phenotype (OFF/ON cells) upon serum administration. In the present study, proliferative and contractile behaviors were characterized by expression of specific differentiation markers. Taken together, these results demonstrate that porcine vascular smooth muscle cells can retain their phenotypic plasticity in culture, and thus mimic in vitro their in vivo differentiation states. OFF cells may thus provide a suitable model system in studying the mechanism(s) by which either known or unknown serum factors may trigger vascular smooth muscle activation. In the present study, this possibility was actually tested by exposing OFF cells to fetal bovine serum (FBS), PDGF-BB and IGF-I. Data show that only FBS could induce a synthetic phenotype in OFF cells, while both PDGF-BB and IGF-I failed to induce any VSM activation.

Inhibition of BUB1 results in genomic instability and anchorage-independent growth of normal human fibroblasts.

The relative contribution of aneuploidy and gene mutations to human tumorigenesis is not yet known. Studies in mice have demonstrated that even single point mutations in oncogenes and tumor suppressor genes can dramatically increase tumor frequency. However, models to evaluate the definitive role of aneuploidy and genomic instability are not yet available. Human fibroblast cells have long been used as a tool for investigating proliferation, senescence, immortalization, and tumorigenesis, all processes that are strongly interrelated. We have now used antisense and ribozyme-mediated temporary inhibition of BUB1 to study the consequences of mitotic checkpoint failure on the development of aneuploidy. The analysis of cell colonies selected by soft agar growth showed evidence of chromosome instability and delayed senescence, without being tumorigenic in nude mice. Our data suggest that chromosomal instability and aneuploidy are early changes that precede tumorigenicity in the multistep process leading to neoplastic transformation.

Ribozyme-mediated inhibition of survivin expression increases spontaneous and drug-induced apoptosis and decreases the tumorigenic potential of human prostate cancer cells.

Survivin is a member of the inhibitor of apoptosis protein (IAP) family, which has been implicated in inhibition of apoptosis and control of mitotic progression. The finding that survivin is overexpressed in most human tumors but absent in normal adult tissues has led to the proposal of survivin as a promising therapeutic target for anticancer therapies. We decided to evaluate the effects of a ribozyme-based strategy for survivin inhibition in androgen-independent human prostate cancer cells. We constructed a Moloney-based retroviral vector expressing a ribozyme targeting the 3' end of the CUA(110) triplet in survivin mRNA, encoded as a chimeric RNA within adenoviral VA1 RNA. Polyclonal cell populations obtained by infection with the retroviral vector of two androgen-independent human prostate cancer cell lines (DU145 and PC-3) were selected for the study. Ribozyme-expressing prostate cancer cells were characterized by a significant reduction of survivin expression compared to parental cells transduced with a control ribozyme; the cells became polyploid, underwent caspase-9-dependent apoptosis and showed an altered pattern of gene expression, as detected by oligonucleotide array analysis. Survivin inhibition also increased the susceptibility of prostate cancer cells to cisplatin-induced apoptosis and prevented tumor formation when cells were xenografted in athymic nude mice. These findings suggest that manipulation of the antiapoptotic survivin pathway may provide a novel approach for the treatment of androgen-independent prostate cancer.

An integrated approach for the design and synthesis of oligonucleotide probes and their interfacing to a QCM-based RNA biosensor.

The quantitative determination of specific cellular messenger-RNA is extremely important both in basic and applied research, especially in diagnostic and pharmacological fields. In order to perform a direct and easy quantification of transcripts on cell extracts, the feasibility of an analytical device able to selectively detect a defined target RNA in a complex mixture while avoiding labelling, retrotranscription and amplification steps, has been explored. In particular, several aspects necessary to obtain good selectivity in target recognition, stability, reusability and sensitivity of a gene specific biosensor were considered. For the development of suitable probe-receptors, analysis of the nucleotide sequence of the target mRNA was carried out to localise the preferred binding regions. As criteria for optimisation, we selected accessibility and uniqueness. Oligonucleotide probes, designed to specifically bind these sequences, were synthesised by using particular monomers producing nuclease-resistant RNA strands with high affinity towards the target. Quartz crystal microbalance (QCM) technology was selected to realise a microgravimetric sensor able to bind the RNA under investigation through a complementary oligonucleotide probe. Covalent immobilisation of bioreceptor molecules to the transducer sensitive surface ensured a stable integration between the two. The binding ability of immobilised probes was tested evaluating their annealing behaviour with both complementary oligonucleotides and full-length target mRNA. The conditions necessary for the regeneration of biosensor were also assessed. Measurements of shift in QCM resonant frequency, performed by hybridisation experiments in liquido, demonstrate that a label-free RNA-biosensor with high specificity, reusability and the ability to give quantitative information, can be realised.

Bcl2-low-expressing MCF7 cells undergo necrosis rather than apoptosis upon staurosporine treatment.

We present a ribozyme-based strategy for studying the effects of Bcl2 down-regulation. The anti-bcl2 hammerhead ribozyme Rz-bcl2 was stably transfected into MCF7 cancer cells and the cleavage of Bcl2 mRNA was demonstrated using a new assay for cleavage product detection, while Western blot analysis showed a concomitant depletion of Bcl2 protein. Rz-bcl2-expressing cells were more sensitive to staurosporine than control cells. Moreover, both molecular and cellular read-outs indicated that staurosporine-induced cell death was necrosis rather than apoptosis in these cells. The study of the effects of Bcl2 down-regulation was extended to the global MCF7 protein expression profile, exploiting a proteomic approach. Two reference electro-pherograms of Rz-bcl2-transfected cells, one with the ribozyme in a catalytically active form and the other with the ribozyme in a catalytically inactive form, were obtained. When comparing the two-dimensional maps, 53 differentially expressed spots were found, four of which were identified by MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS as calreticulin, nucleophosmin, phosphoglycerate kinase and pyruvate kinase. How the up-regulation of these proteins might help to explain the modification of Bcl2 activity is discussed.

Aptamer-Mediated Codelivery of Doxorubicin and NF-κB Decoy Enhances Chemosensitivity of Pancreatic Tumor Cells.

Aptamers able to bind efficiently cell-surface receptors differentially expressed in tumor and in healthy cells are emerging as powerful tools to perform targeted anticancer therapy. Here, we present a novel oligonucleotide chimera, composed by an RNA aptamer and a DNA decoy. Our assembly is able to (i) target tumor cells via an antitransferrin receptor RNA aptamer and (ii) perform selective codelivery of a chemotherapeutic drug (Doxorubicin) and of an inhibitor of a cell-survival factor, the nuclear factor κB decoy oligonucleotide. Both payloads are released under conditions found in endolysosomal compartments (low pH and reductive environment). Targeting and cytotoxicity of the oligonucleotidic chimera were assessed by confocal microscopy, cell viability, and Western blot analysis. These data indicated that the nuclear factor κB decoy does inhibit nuclear factor κB activity and ultimately leads to an increased therapeutic efficacy of Doxorubicin selectively in tumor cells.

Site-Specific Secretome Map Evidences VSMC-Related Markers of Coronary Atherosclerosis Grade and Extent in the Hypercholesterolemic Swine.

A major drawback in coronary atherosclerosis (ATS) research is the difficulty of investigating early phase of plaque growth and related features in the clinical context. In this study, secreted proteins from atherosclerotic coronary arteries in a hypercholesterolemic swine model were characterized by a proteomics approach and their expression was correlated to site-specific ATS stage and extent. A wide coronary artery map of secreted proteins has been obtained in high fat (HF) diet induced ATS swine model and a significantly different expression of many proteins related to vascular smooth muscle cell (VSMC) activation/migration has been identified. Significant associations with ATS stage of HF coronary lesions were found for several VSMC-derived proteins and validated for chitinase 3 like protein 1 (CHI3L1) by tissue immunoexpression. A direct correlation (R(2) = 0.85) was evidenced with intima to media thickness ratio values and ELISA confirmed the higher blood concentrations of CHI3L1 in HF cases. These findings confirmed the pivotal role of VSMCs in coronary plaque development and demonstrated a strong site-specific relation between VSMC-secreted CHI3L1 and lesion grade, suggesting that this protein could be proposed as a useful biomarker for diagnosing and staging of atherosclerotic lesions in coronary artery disease.

Radiosensitization of human melanoma cells by ribozyme-mediated inhibition of survivin expression.

Survivin is a structurally unique member of the inhibitors of apoptosis protein family and is involved in the control of cell division and inhibition of apoptosis. The notion that survivin is overexpressed in most human tumors but absent in normal adult tissues with only a few exceptions has led to the proposal of survivin as a promising therapeutic target for novel anticancer therapies. In this context, we generated a hammerhead ribozyme targeting the 3' end of the CUA110 triplet in the survivin mRNA. Two human melanoma cell lines (JR8 and M14) overexpressing survivin were stably transfected with the pRc/CMV vector carrying the ribozyme sequence. Two polyclonal cell populations proven to endogenously express ribozyme and characterized by a markedly lower survivin protein level (-60% and -50%, respectively) than JR8 and M14 parental cells were selected for the study. Ribozyme-expressing cells showed a significantly (p<0.01) increased sensitivity to gamma-irradiation (as detected by clonogenic cell survival) compared to JR8 and M14 cells. Moreover, in the JR8 cell line, the extent of radiation-induced apoptosis (in terms of percentage of apoptotic nuclei in cells stained with propidium iodide and level of caspase-3 catalytic activity) was markedly greater in ribozyme-expressing cells than in parental cells. These results demonstrate for the first time that attenuation of survivin expression renders human melanoma cells more susceptible to gamma-irradiation.

A method for prediction of accessible sites on an mRNA sequence for target selection of hammerhead ribozymes.

Hammerhead ribozymes are short RNA molecules endowed with endoribonucleolytic activity. Their use as molecular tools for specific inhibition of gene translation is affected by many factors including the target accessibility. A method for the prediction of accessible target sites for hammerhead ribozymes within a given RNA sequence is described. This method maps all putative NUH cleavage sites (N = A, C, G, U and H = A, C, U) and picks out short flanking regions as the binding domain for the corresponding ribozyme. The probabilistic level of unfolding, accessibility score (AS), is then calculated for each target region on the basis of a comparison of all folding structures obtained for the target RNA and arranged according to the Boltzmann's distribution. At the end, a series of imposed limits gives the best target sequences endowed with highly probable accessibility and with a potentially active catalytic structure of the hammerhead sequence. A successive experimental approach to verify the effective accessibility of selected targets was used. For that, antisense oligonucleotides addressed to the coding region of bcl2 mRNA were synthesized and administered to the MCF7 human cell line. The inhibition of gene expression, as measured by western analysis of the BCL2 protein, demonstrated that all target sites selected on the basis of their putative accessibility were actually sensitive to antisense treatments while the inaccessible ones were not. The application of this target discovery method to ribozyme design is proposed in order to satisfy a crucial condition.

The energy profiling of short interfering RNAs is highly predictive of their activity.

The technology of knockdown of gene expression by synthetic short interfering RNAs (siRNAs) is currently diffused because of its relative simplicity and reliability. Despite the claims that active siRNAs can be easily identified, how to select active siRNAs with high efficiency it is not fully defined. Here, we report that energy profiling can be used in the guidelines for siRNA design.

Targeting of A701G nucleotide at the human ATP1A1 locus using a RNA/DNA chimera.

The single base substitution mediated by chimeric RNA/DNA oligonucleotide is a new promising approach of gene therapy for single base mutation diseases. We exploited this approach to render HeLa cells resistant to ouabain by introducing a single base substitution in the alpha 1 subunit of the NA+/K+ ATPase human gene. The chimeric oligonucleotide was administered to HeLa cells by electroporation and the frequency of ouabain resistant cells determined. The results showed that the chimeric RNA/DNA oligonucleotide failed to enhance the frequency of ouabain resistant cells supporting the controversy about the conflicting results of the technique.

Characterization of secreted vesicles from vascular smooth muscle cells.

The artery medial layer is mainly composed of vascular smooth muscle cells (VSMCs). These cells contribute to the formation of neointima and atherosclerotic plaques by switching from the quiescent-contractile to migratory-activated state. Apoptotic blebs, microvesicles and exosomes are secreted vesicles, with differences in composition and size, involved in cellular communication at multiple levels. In this article, an untargeted, proteomics approach was exploited to characterise VSMC released vesicles and a preliminary protein profile for microvesicles and exosomes of different cell phenotypes was obtained. Enriched samples of vesicles from serum-free and serum-activated VSMCs were analysed by a LC-MS/MS strategy leading to the identification of 349 proteins. In microvesicles, the most abundant classes of identified proteins were cytoplasmic or organelle associated, house keeping and metabolic factors. Otherwise, exosomes from different phenotypes revealed a sharper peculiarity thus, as suggested by the high percentage of ECM and ECM related proteins and cell adhesion molecules, they seem to play an important role in outward or cell-to-cell signalling. Comparison between exosomes or microvesicles from quiescent and activated VSMCs evidenced 29 differentially expressed proteins. Among these, in microvesicles there are several proteins that are involved in vesicle trafficking while in exosomes focal adhesion and ECM related factors are the most interesting. These data, although preliminary, are promising for a possible identification of potential circulating markers of a cell state.

A proteomic approach to identify seminal plasma proteins in roosters (Gallus gallus domesticus).

Considering the interest in avian semen processing and storage, the objective of this study was to identify the domestic fowl seminal plasma proteins using two-dimensional gel electrophoresis (2-DE) and mass spectrometry MS/MS. For three times in a 4-month period, seminal plasma was obtained from semen collected from four local male chickens (Gallus gallus domesticus) and prepared for two-dimensional polyacrylamide gel electrophoresis. A total of 83 spots were detected across all gels and analyzed by MALDI-TOF/TOF. Among these spots, 17 have been successfully identified. The most intensely stained spots were recognized as serum albumin, ovotransferrin, alpha-enolase, fatty acid binding protein, thioredoxin, trypsin inhibitor CITI-1 and gallinacin-9. From these proteins, two are characteristic of avian seminal plasma, the ovotransferrin and gallinacin-9, and one is specific of the Gallus species, the chicken trypsin inhibitor CITI-1.