ABCB1 overexpression through locus amplification represents an actionable target to combat paclitaxel resistance in pancreatic cancer cells.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest types of cancer and the chemotherapies such as gemcitabine/nab-paclitaxel are confronted with intrinsic or acquired resistance. The aim of this study was to investigate mechanisms underlying paclitaxel resistance in PDAC and explore strategies to overcome it. METHODS: Three paclitaxel (PR) and gemcitabine resistant (GR) PDAC models were established. Transcriptomics and proteomics were used to identify conserved mechanisms of drug resistance. Genetic and pharmacological approaches were used to overcome paclitaxel resistance. RESULTS: Upregulation of ABCB1 through locus amplification was identified as a conserved feature unique to PR cells. ABCB1 was not affected in any of the GR models and no cross resistance was observed. The ABCB1 inhibitor verapamil or siRNA-mediated ABCB1 depletion sensitized PR cells to paclitaxel and prevented efflux of ABCB1 substrates in all models. ABCB1 expression was associated with a trend towards shorter survival in patients who had received gemcitabine/nab-paclitaxel treatment. A pharmacological screen identified known and novel kinase inhibitors that attenuate efflux of ABCB1 substrates and sensitize PR PDAC cells to paclitaxel. CONCLUSION: Upregulation of ABCB1 through locus amplification represents a novel, conserved mechanism of PDAC paclitaxel resistance. Kinase inhibitors identified in this study can be further (pre) clinically explored as therapeutic strategies to overcome paclitaxel resistance in PDAC.

Differential Sensitivity to Ionizing Radiation in Gemcitabine-Resistant and Paclitaxel-Resistant Pancreatic Cancer Cells.

PURPOSE: Chemoresistance remains a major challenge in treating pancreatic ductal adenocarcinoma (PDAC). Although chemoradiation has proven effective in other tumor types, such as head and neck squamous cell carcinoma, its role in PDAC and effect on acquired chemoresistance have yet to be fully explored. In this study, we investigated the sensitivity of gemcitabine-resistant (GR) and paclitaxel-resistant (PR) PDAC cells to ionizing radiation (IR) and their underlying mechanisms. METHODS AND MATERIALS: GR and PR clones were generated from PANC-1, PATU-T, and SUIT2-007 pancreatic cancer cell lines. Cell survival after radiation was assessed using clonogenic assay, sulforhodamine B assay, apoptosis, and spheroid growth by bioluminescence. Radiation-induced DNA damage was assessed using Western blot, extra-long polymerase chain reaction, reactive oxygen species production, and immunofluorescence. Autophagy and modulation of the Hippo signaling pathway were investigated using proteomics, Western blot, immunofluorescence, and reverse-transcription quantitative polymerase chain reaction. RESULTS: In both 2- and 3-dimensional settings, PR cells were more sensitive to IR and showed decreased ß-globin amplification, indicating more DNA damage accumulation compared with GR or wild-type cells after 24 hours. Proteomic analysis of PR PATU-T cells revealed that the protein MST4, a kinase involved in autophagy and the Hippo signaling pathway, was highly downregulated. A differential association was found between autophagy and radiation treatment depending on the cell model. Interestingly, increased yes-associated protein nuclear localization and downstream Hippo signaling pathway target gene expression were observed in response to IR. CONCLUSIONS: This was the first study investigating the potential of IR in targeting PDAC cells with acquired chemoresistance. Our results demonstrate that PR cells exhibit enhanced sensitivity to IR due to greater accumulation of DNA damage. Additionally, depending on the specific cellular context, radiation-induced modulation of autophagy and the Hippo signaling pathway emerged as potential underlying mechanisms, findings with potential to inform personalized treatment strategies for patients with acquired chemoresistance.

Seeding the aggregation of TDP-43 requires post-fibrillization proteolytic cleavage.

Despite the strong evidence linking the transactive response DNA-binding protein 43 (TDP-43) aggregation to the pathogenesis of frontotemporal lobar degeneration with TDP-43, amyotrophic lateral sclerosis and several neurodegenerative diseases, our knowledge of the sequence and structural determinants of its aggregation and neurotoxicity remains incomplete. Herein, we present a new method for producing recombinant full-length TDP-43 filaments that exhibit sequence and morphological features similar to those of brain-derived TDP-43 filaments. We show that TDP-43 filaments contain a ß-sheet-rich helical amyloid core that is fully buried by the flanking structured domains of the protein. We demonstrate that the proteolytic cleavage of TDP-43 filaments and exposure of this amyloid core are necessary for propagating TDP-43 pathology and enhancing the seeding of brain-derived TDP-43 aggregates. Only TDP-43 filaments with exposed amyloid core efficiently seeded the aggregation of endogenous TDP-43 in cells. These findings suggest that inhibiting the enzymes mediating cleavage of TDP-43 aggregates represents a viable disease-modifying strategy to slow the progression of amyotrophic lateral sclerosis and other TDP-43 proteinopathies.

Brain histone beta-hydroxybutyrylation couples metabolism with gene expression.

Little is known about the impact of metabolic stimuli on brain tissue at a molecular level. The ketone body beta-hydroxybutyrate (BHB) can be a signaling molecule regulating gene transcription. Thus, we assessed lysine beta-hydroxybutyrylation (K-bhb) levels in proteins extracted from the cerebral cortex of mice undergoing a ketogenic metabolic challenge (48 h fasting). We found that fasting enhanced K-bhb in a variety of proteins including histone H3. ChIP-seq experiments showed that K9 beta-hydroxybutyrylation of H3 (H3K9-bhb) was significantly enriched by fasting on more than 8000 DNA loci. Transcriptomic analysis showed that H3K9-bhb on enhancers and promoters correlated with active gene expression. One of the most enriched functional annotations both at the epigenetic and transcriptional level was "circadian rhythms''. Indeed, we found that the diurnal oscillation of specific transcripts was modulated by fasting at distinct zeitgeber times both in the cortex and suprachiasmatic nucleus. Moreover, specific changes in locomotor activity daily features were observed during re-feeding after 48-h fasting. Thus, our results suggest that fasting remarkably impinges on the cerebral cortex transcriptional and epigenetic landscape, and BHB acts as a powerful epigenetic molecule in the brain through direct and specific histone marks remodeling in neural tissue cells.

Transcriptional level evaluation of osteopontin/miRNA-181a axis in hepatocellular carcinoma cell line-secreted extracellular vesicles.

Recent evidence suggested the role of secreted extracellular vesicles (EVs) in the intracellular signalling within the liver becoming a promising candidate as biomarker in hepatocellular carcinoma (HCC). Osteopontin (OPN) seems to play a relevant role both for early diagnosis of HCC than on the mechanisms that drive oncogenesis but, to date, information on the expression levels of OPN in EVs secreted by HCC tumor cell line are missing. The study aimed to verify, by transcriptional and proteomic study, the presence of OPN in EVs secreted by tumorigenic (HepG2) and non-tumorigenic hepatocyte cell line (WRL68), and to analyse the expression variations of OPN, its isoforms and miRNA-181a in both these EVs. "In silico analysis" was also performed via the Gene expression Profiling Interactive analysis (GEPIA) and Hepatocellular Carcinoma Database (HCCDB). An up-regulation of OPN in EVs secreted by HepG2 with respect to WRL68 was found in line with the results obtained by the "in silico analysis". The study demonstrates, for the first time, the OPN isoforms and its modulator miRNA-181a expression in EVs secreted by both cell lines, highlighting high levels of OPN isoforms in EVs secreted by HepG2 and identifying OPN as a promising biomarker for HCC diagnosis.

Apolipoprotein Signature of HDL and LDL from Atherosclerotic Patients in Relation with Carotid Plaque Typology: A Preliminary Report.

In the past years, it has become increasingly clear that the protein cargo of the different lipoprotein classes is largely responsible for carrying out their various functions, also in relation to pathological conditions, including atherosclerosis. Accordingly, detailed information about their apolipoprotein composition and structure may contribute to the revelation of their role in atherogenesis and the understanding of the mechanisms that lead to atherosclerotic degeneration and toward vulnerable plaque formation. With this aim, shotgun proteomics was applied to identify the apolipoprotein signatures of both high-density and low-density lipoproteins (HDL and LDL) plasma fractions purified from healthy volunteers and atherosclerotic patients with different plaque typologies who underwent carotid endarterectomy. By this approach, two proteins with potential implications in inflammatory, immune, and hemostatic pathways, namely, integrin beta-2 (P05107) and secretoglobin family 3A member 2 (Q96PL1), have been confirmed to belong to the HDL proteome. Similarly, the list of LDL-associated proteins has been enriched with 21 proteins involved in complement and coagulation cascades and the acute-phase response, which potentially double the protein species of LDL cargo. Moreover, differential expression analysis has shown protein signatures specific for patients with "hard" or "soft" plaques.

Lipid biomarkers in statin users with coronary artery disease annotated by coronary computed tomography angiography.

Molecular markers are suggested to improve the diagnostic and prognostic accuracy in patients with coronary artery disease (CAD) beyond current clinical scores based on age, gender, symptoms and traditional risk factors. In this context, plasma lipids are emerging as predictors of both plaque composition and risk of future events. We aim to identify plasma lipid biomarkers associated to CAD indexes of stenosis severity, plaque lipid content and a comprensive score of CAD extent and its risk. We used a simple high performance liquid chromatography-tandem mass spectrometry method to identify 69 plasma lipids in 132 subjects referred to Coronary Computed Tomography Angiography (CCTA) for suspected CAD, all under statin treatment. Patients were stratified in groups using three different CCTA-based annotations: CTA-risk score, lipid plaque prevalence (LPP) ratio and the coronary artery disease-reporting and data system (CAD-RADS). We identified a common set of lipid biomarkers composed of 7 sphingomyelins and 3 phosphatidylethanolamines, which discriminates between high risk CAD patients and controls regardless of the CAD annotations used (CTA score, LPP ratio, or CAD-RADS). These results highlight the potential of circulating lipids as biomarkers of stenosis severity, non calcified plaque composition and overall plaque risk of events.

Characterization of Extracellular Vesicle Cargo in Sjögren's Syndrome through a SWATH-MS Proteomics Approach.

Primary Sjögren's syndrome (pSS) is a complex heterogeneous disease characterized by a wide spectrum of glandular and extra-glandular manifestations. In this pilot study, a SWATH-MS approach was used to monitor extracellular vesicles-enriched saliva (EVs) sub-proteome in pSS patients, to compare it with whole saliva (WS) proteome, and assess differential expressed proteins between pSS and healthy control EVs samples. Comparison between EVs and WS led to the characterization of compartment-specific proteins with a moderate degree of overlap. A total of 290 proteins were identified and quantified in EVs from healthy and pSS patients. Among those, 121 proteins were found to be differentially expressed in pSS, 82% were found to be upregulated, and 18% downregulated in pSS samples. The most representative functional pathways associated to the protein networks were related to immune-innate response, including several members of S100 protein family, annexin A2, resistin, serpin peptidase inhibitors, azurocidin, and CD14 monocyte differentiation antigen. Our results highlight the usefulness of EVs for the discovery of novel salivary-omic biomarkers and open novel perspectives in pSS for the identification of proteins of clinical relevance that could be used not only for the disease diagnosis but also to improve patients' stratification and treatment-monitoring. Data are available via ProteomeXchange with identifier PXD025649.

Protein Delivery by Peptide-Based Stealth Liposomes: A Biomolecular Insight into Enzyme Replacement Therapy.

Infantile neural ceroid lipofuscinosis (INCL) is a lysosomal storage disorder characterized by mutations in the CLN1 gene that leads to lack of the lysosomal enzyme palmitoyl-protein thioesterase-1 (PPT1), which causes the progressive death of cortical neurons. Enzyme replacement therapy (ERT) is one of the most promising treatments, but its translation toward a clinical use is hampered by the need to deliver the enzyme to the central nervous system and a more detailed understanding of its capability to restore physiologic conditions at the biochemical and protein level, beyond the simple regulation of enzymatic activity. Targeted nanoparticles can promote protein delivery to the central nervous system and affect biological pathways inside cells. Here, we describe an innovative peptide-based stealth nanoparticle that inhibits serum protein adsorption exploiting transferrin-driven internalization to convey the PPT1 enzyme to transferrin receptor-mediated pathways (endocytosis in this work, or transcytosis, in perspective, in vivo). These enzyme-loaded nanoparticles were able to restore stable levels of enzymatic activity in CLN1 patient's fibroblasts, comparable with the free enzyme, demonstrating that delivery after encapsulation in the nanocarrier does not alter uptake or intracellular trafficking. We also investigate, for the first time, dysregulated pathways of proteome and palmitoylome and their alteration upon enzyme delivery. Our nanoparticles were able of halving palmitoylated protein levels restoring conditions similar to the normal cells. From proteomic analysis, we also highlighted the reduction of the different groups of proteins after treatments with the free or encapsulated enzyme. In conclusion, our system is able to deliver the enzyme to a model of CLN1 disease restoring normal conditions in cells. Investigation of molecular details of pathologic state and enzyme-based correction reveals dysregulated pathways with unprecedented details for CLN1. Finally, we unveil for the first time the dysregulation landscape of palmitoylome and proteome in primary patient-derived fibroblasts and their modifications in response to enzyme administration. These findings will provide a guideline for the validation of future therapeutic strategies based on enzyme replacement therapy or acting at different metabolic levels.

Proteomics pipeline for phosphoenrichment and its application on a human melanoma cell model.

Cell signalling is tightly regulated by post-translational modification of proteins. Among them, phosphorylation is one of the most interesting and important. Identifying phosphorylation sites on proteins is challenging and requires strategies for pre-separation and enrichment of the phosphorylated species. We applied four different methods for phospho-enrichment involving TiO2 and IMAC matrix to human melanoma cell lysates of starved A375 induced for 1 h with 1% FBS. Comparison of protocol efficiency was evaluated through peptide concentration, sulphur and phosphorus content and peptide analysis by LC-MS in the collected fractions. Our results underlined that each single method is not sufficient for a comprehensive phosphoproteome analysis. In fact, each methodology permits to identify only a fraction of the phosphoproteome contained in a whole cell lysate. The selection of the most efficient protocols and a combination of two phospho-enrichment methods allowed the assessment of this workflow able to pinpoint the main actors in the phospho-proteome cascade of A375 human melanoma cells treated with Vemurafenib.

Homotypic targeting and drug delivery in glioblastoma cells through cell membrane-coated boron nitride nanotubes.

Glioblastoma multiforme (GBM) is one of the most aggressive types of brain cancer, characterized by rapid progression, resistance to treatments, and low survival rates; the development of a targeted treatment for this disease is still today an unattained objective. Among the different strategies developed in the latest few years for the targeted delivery of nanotherapeutics, homotypic membrane-membrane recognition is one of the most promising and efficient. In this work, we present an innovative drug-loaded nanocarrier with improved targeting properties based on the homotypic recognition of GBM cells. The developed nanoplatform consists of boron nitride nanotubes (BNNTs) loaded with doxorubicin (Dox) and coated with cell membranes (CM) extracted from GBM cells (Dox-CM-BNNTs). We demonstrated as Dox-CM-BNNTs are able to specifically target and kill GBM cells in vitro, leaving unaffected healthy brain cells, upon successful crossing an in vitro blood-brain barrier model. The excellent targeting performances of the nanoplatform can be ascribed to the protein component of the membrane coating, and proteomic analysis of differently expressed membrane proteins present on the CM of GBM cells and of healthy astrocytes allowed the identification of potential candidates involved in the process of homotypic cancer cell recognition.

One year in review 2019: Sjögren's syndrome.

Primary Sjögren's syndrome (pSS) is a complex and heterogeneous disorder characterised by a wide spectrum of glandular and extra-glandular features. Novel insights into disease pathogenesis and the discovery of novel biomarkers are allowing us to characterise the disease not only phenotypically on the basis of clinical presentation, but also on the basis of the endotype. Ultimately, a better stratification of patients may pave new avenues for novel targeted therapies, opening new possibilities for the application of personalised medicine in pSS.

Salivary extracellular vesicles versus whole saliva: new perspectives for the identification of proteomic biomarkers in Sjögren's syndrome.

In the era of personalised medicine new biomarkers are required to early diagnose Sjögren's syndrome (SS), to define different disease subsets and to direct patients' clinical management and therapeutic intervention. In the last few years, several efforts have evaluated saliva proteome to detect and monitor primary SS. Although clinically valuable, these studies presented some limitations that have partially prevented the use of salivary biomarkers in clinical practice. Nowadays, proteomic of extracellular vesicle (EV) represents an emerging and promising field in the discovery of -omic biomarkers for pSS. EV is a relatively new term that includes exosomes, microvesicles and apoptotic body. EVs are packed with proteins, growth factors, cytokines, bioactive lipids, but also nucleic acids and in particular: mRNA, microRNA, long non-coding RNA, tRNA and rRNA. Therefore, they may represent a useful source for diagnostic, prognostic and therapeutic biomarkers in several conditions. In this review we will specifically focus on EV proteomics as a tool for the identification of novel biomarkers for pSS. In the first part we focused on the state of the art of the studies on proteomics in SS existing in the literature. In the second part we provided a definition of EV with an update on biological sample collection and processing for EV proteomic studies. Finally, we summarised the state of the art of EV -omics in SS highlighting the potential advantages of this novel approach compared to the overall traditional concept of analysing the proteome of blood or saliva.

Phenotyping multiple subsets in Sjögren's syndrome: a salivary proteomic SWATH-MS approach towards precision medicine.

BACKGROUND: This proof of concept study was aimed at characterizing novel salivary biomarkers specific for different subsets in primary Sjögren's syndrome (pSS) in order to improve patients' profiling. METHODS: pSS patients were stratified in three subgroups according to both (a) focus score in the minor salivary gland biopsies (i.e. intensity of immune cell infiltration in the tissue) and (b) unstimulated salivary flow rate. Healthy volunteers were included as controls. A nano-HPLC-SWATH-MS approach was used for the analysis of saliva proteome of different subsets. RESULTS: We found 203 differentially expressed proteins in pSS patients with respect to controls with evident differences in the expression of normal constituents of the human salivary proteome (i.e. prolactin-inducible protein, proline-rich proteins, cystatins) and several mediators of inflammatory processes. The comparative analysis of the pSS phenotypes unrevealed 63 proteins that were shared and specifically modulated in the three subsets of pSS patients converging on several inflammatory pathways. Among them S100A protein appeared of particular interest merging on IL-12 signaling and being significantly influenced by either salivary flow impairment or intensity of immune cell infiltration in the tissue. CONCLUSIONS: Constellations of proteins, including S100A proteins, characterize different pSS subsets reflecting either salivary gland dysfunction or inflammation. Salivary proteomics may foster future research projects ultimately aimed at developing personalized treatments for pSS patients.

A high glucose level is associated with decreased aspirin-mediated acetylation of platelet cyclooxygenase (COX)-1 at serine 529: A pilot study.

Diabetes is a major risk factor for cardiovascular diseases. Although aspirin is considered a cornerstone of the prevention and treatment of atherothrombotic-related ischemic events, this antiplatelet drug appears to be less effective in patients with poorly controlled diabetes. It has been suggested that the glycation of platelet proteins plays a pivotal role in poor responsiveness to aspirin. However, a direct effect on the critical residue (serine 529, or Ser 529) of the catalytic pocket of cyclooxygenase 1 (COX-1) has never been demonstrated. This pilot study aimed to elucidate the impact of hyperglycaemia on aspirin acetylation of COX-1 using a targeted mass spectrometry approach. We observed that high glucose concentration had a direct impact on the level of acetylation of the COX-1 Ser 529 residue, whereas it's overall acetylation level remained unchanged. Moreover, the functional aspirin-induced inhibition of COX-1 was dose-dependently impaired as glucose concentrations increased. These in vitro findings were in line with data obtained using platelets from diabetic patients. These data provide new insights into the interplay between glucose and aspirin on platelet proteins and their effects on platelet COX-1. They also suggest a potential mechanistic explanation for the phenomenon of poor response to aspirin in diabetic patients. Data are available via ProteomeXchange with identifier PXD011204. SIGNIFICANCE: Deciphering the mutual interplay between glucose and aspirin-mediated acetylation on platelet COX-1, might be of great interest as there is still a lack of information of the mechanism underlying this process that may contribute to the less-than expected response of platelets to aspirin, often observed in diabetes.

Shotgun proteomics data on the impact of hyperglycaemia on platelet protein acetylation by aspirin.

This data article associated with the manuscript "A high glucose levels is associated with decreased aspirin-mediated acetylation of platelet cyclooxygenase (COX)-1 at serine 529: a pilot study" (Finamore et al., 2018) refers to the shotgun proteomics approach carried out on platelet protein extracts from diabetic patients and healthy controls. Platelet proteins were in vitro incubated with 500 µM aspirin for 30 min at 37 °C to enhance the acetylation process. After protein digestion with trypsin, DDA data were acquired on a Thermo QExactive plus using 3 technical replicate injections per sample. Here, we were able to elucidate the preferential sites of aspirin-induced acetylation on a significant fraction of the platelet proteome and to quantify the impact of diabetes on the effect of aspirin on several platelet proteins. Data are available via ProteomeXchange with identifier PXD011582.

Characterisation of the influences of aspirin-acetylation and glycation on human plasma proteins.

The competition effect between aspirin-mediated acetylation and protein glycation has been a matter of concern for decades. However, the exact interactions between these two post-translational modifications are still not well understood. Several efforts have been made to explain how aspirin prevents glycation, but the influence of prior protein glycation on the action of aspirin has never been investigated. This study involved qualitative and quantitative analyses to: 1) identify acetylated and glycated proteins; 2) quantify rates of acetylation and glycation; and 3) elucidate the common modification sites. Human plasma was incubated with 30mM glucose and then 500µM aspirin. A label-free mass spectrometry approach indicated an increase in the acetylation level after this sequential glucose-then-aspirin incubation; these results were also confirmed by Western blot. Interestingly, for several proteins, decreases in glycation levels were evidenced after aspirin incubation. The common modification sites, where both acetylation and glycation took place, were also identified. The influence that glycation and acetylation processes have on each other could reflect conformational changes induced by glucose and aspirin. In future studies, in order to better understand the interactions between these two PTMs, we intend to apply this strategy to other blood compartments and to diabetic patients. BIOLOGICAL SIGNIFICANCE: Non-enzymatic glycation represents an early stage in the development of the long-lasting complications that are associated with diabetes. Aspirin has been shown to prevent this process in a few reference proteins, but how the two post-translational modifications (PTMs) of aspirin-mediated acetylation and protein glycation interact with each other remains poorly investigated. This study used a label-free quantitative proteomic approach to characterise the extent of aspirin-induced acetylation and protein glycation in human plasma. The results clearly supported a mutual influence between these PTMs, which lead us to propose a potential model based on structural conformational changes.

Quantitative analysis of glycated proteins.

The proposed protocol presents a comprehensive approach for large-scale qualitative and quantitative analysis of glycated proteins (GP) in complex biological samples including biological fluids and cell lysates such as plasma and red blood cells. The method, named glycation isotopic labeling (GIL), is based on the differential labeling of proteins with isotopic [(13)C6]-glucose, which supports quantitation of the resulting glycated peptides after enzymatic digestion with endoproteinase Glu-C. The key principle of the GIL approach is the detection of doublet signals for each glycated peptide in MS precursor scanning (glycated peptide with in vivo [(12)C6]- and in vitro [(13)C6]-glucose). The mass shift of the doublet signals is +6, +3 or +2 Da depending on the peptide charge state and the number of glycation sites. The intensity ratio between doublet signals generates quantitative information of glycated proteins that can be related to the glycemic state of the studied samples. Tandem mass spectrometry with high-energy collisional dissociation (HCD-MS2) and data-dependent methods with collision-induced dissociation (CID-MS3 neutral loss scan) are used for qualitative analysis.

Proteomics investigation of human platelets in healthy donors and cystic fibrosis patients by shotgun nUPLC-MSE and 2DE: a comparative study.

Platelets are of pathophysiological relevance in haemostasis, wound repair, inflammation and cardiovascular disease. We have shown that human platelets express a biologically active Cystic Fibrosis Transmembrane Conductance Regulator, which is dysfunctional in Cystic Fibrosis (CF) patients, and regulate platelet responses related to inflammation and its resolution. In order to further elucidate platelet involvement in CF inflammation, we pursued a comparative proteomic analysis of cells from healthy donors and CF patients, in association with a non-supervised comparative analysis of the Gene Ontology. Our results, showing changes in the integrin signalling in CF, support a pro-inflammatory profile of CF platelets.

Proteomics investigation of human platelets by shotgun nUPLC-MSE and 2DE experimental strategies: a comparative study.

BACKGROUND: Platelets, the smallest human blood cells component, have a key role in the control of haemostasis and thrombosis but they have also been shown to be implicated in a number of different pathological states because of their involvement also in the process of inflammation end its resolution. Their peculiar anucleated morphology render the proteomics an intriguing approach to understand their biology. Given the high impact of platelet in different diseases we have started a systematic investigation of protein repertoire in controlled platelet preparation. MATERIAL AND METHODS: Platelets have been extracted from blood of healthy donors (n=6) collected by venipuncture in Vacutainer. The quality of the preparation was assessed by observation and enumeration in a Bürker chamber with a conventional tissue culture microscope. To characterize human platelets proteome we analysed the pool of purified platelets combining two proteomic approaches: 2-DE separation combined with Mass Spectrometry and nanoscale ultra performances LC-MS(E) shotgun proteomics experiments. RESULTS: The 2D gel analysis leads an average of 1900 protein spots, after the filtering of "noise" and "false positive" spots, over 500 were selected to be eligible for further analysis given their optimal spot quality value. To perform the analysis by ion accounting shotgun proteomic approach, based on nano ultra performance liquid chromatography (nUPLC) coupled to MS(E) processing of continuum LC-MS data, the same pool of samples was subject to liquid phase tryptic digestion and the peptide obtained used for the experiments. All the data obtained were analysed using ProteinLynx GlobalServer v2.3 (PLGS, Waters). Three analytical replicates run were acquire in high/low energy modes and associated to a human protein database returning the identification of 100 distinct genes. Comparative analysis of the Gene Ontology has been performed to evaluate the differential functional representation of the molecular repertoire investigated with these two orthogonal approaches. DISCUSSION: The overall molecular function classification revealed differences between the two proteomic approaches. In particular, we found significant differences in cytoskeletal proteins (19.65% 2-DE versus 45.60 Shotgun) and receptors (0,92% 2-DE versus 6.90% Shotgun).