Phosphoproteomic Analysis of Platelets in Severe Obesity Uncovers Platelet Reactivity and Signaling Pathways Alterations.

OBJECTIVE: Obesity is associated with a proinflammatory and prothrombotic state that supports atherosclerosis progression. The goal of this study was to gain insights into the phosphorylation events related to platelet reactivity in obesity and identify platelet biomarkers and altered activation pathways in this clinical condition. Approach and Results: We performed a comparative phosphoproteomic analysis of resting platelets from obese patients and their age- and gender-matched lean controls. The phosphoproteomic data were validated by mechanistic, functional, and biochemical assays. We identified 220 differentially regulated phosphopeptides, from at least 175 proteins; interestingly, all were up-regulated in obesity. Most of the altered phosphoproteins are involved in SFKs (Src-family kinases)-related signaling pathways, cytoskeleton reorganization, and vesicle transport, some of them validated by targeted mass spectrometry. To confirm platelet dysfunction, flow cytometry assays were performed in whole blood indicating higher surface levels of GP (glycoprotein) VI and CLEC (C-type lectin-like receptor) 2 in platelets from obese patients correlating positively with body mass index. Receiver operator characteristics curves analysis suggested a much higher sensitivity for GPVI to discriminate between obese and lean individuals. Indeed, we also found that obese platelets displayed more adhesion to collagen-coated plates. In line with the above data, soluble GPVI levels-indicative of higher GPVI signaling activation-were almost double in plasma from obese patients. CONCLUSIONS: Our results provide novel information on platelet phosphorylation changes related to obesity, revealing the impact of this chronic pathology on platelet reactivity and pointing towards the main signaling pathways dysregulated.

A Combination of Proteomic Approaches Identifies A Panel of Circulating Extracellular Vesicle Proteins Related to the Risk of Suffering Cardiovascular Disease in Obese Patients.

Plasma-derived extracellular vesicles (EVs) have been extensively described as putative biomarkers in different diseases. Interestingly, increased levels of EVs subpopulations are well known to associate with obesity. The goal of this study is to identify EVs-derived biomarkers in plasma from obese patients in order to predict the development of pathological events associated with obesity. Samples are obtained from 22 obese patients and their lean-matched controls are divided into two cohorts: one for a 2D fluorescence difference gel electrophoresis (2D-DIGE)-based study, and the other one for a label free LC-MS/MS-based approach. EVs are isolated following a serial ultracentrifugation protocol. Twenty-two and 23 differentially regulated features are detected from 2D-DIGE and label free LC-MS/MS, respectively; most of them involve in the coagulation and complement cascades. Remarkably, there is an upregulation of complement C4, complement C3, and fibrinogen in obese patients following both approaches, the latter two also validated by 2D-western-blotting in an independent cohort. These results correlate with a proinflammatory and prothrombotic state of those individuals. On the other hand, a downregulation of adiponectin leading to an increased risk of suffering cardiovascular diseases has been shown. The results suggest the relevance of plasma-derived-EVs proteins as a source of potential biomarkers for the development of atherothrombotic events in obesity.

Involvement of exosomes in lung inflammation associated with experimental acute pancreatitis.

A frequent complication of acute pancreatitis is the lung damage associated with the systemic inflammatory response. Although various pro-inflammatory mediators generated at both local and systemic levels have been identified, the pathogenic mechanisms of the disease are still poorly understood. In recent years, exosomes have emerged as a new intercellular communication system able to transfer encapsulated proteins and small RNAs and protect them from degradation. Using an experimental model of taurocholate-induced acute pancreatitis in rats, we aimed to evaluate the role of exosomes in the extent of the systemic inflammatory response. Induction of pancreatitis increased the concentration of circulating exosomes, which showed a different proteomic profile to those obtained from control animals. A series of tracking experiments using PKH26-stained exosomes revealed that circulating exosomes effectively reached the alveolar compartment and were internalized by macrophages. In vitro experiments revealed that exosomes obtained under inflammatory conditions activate and polarize these alveolar macrophages towards a pro-inflammatory phenotype. Interestingly, the proteomic analysis of circulating exosomes during acute pancreatitis suggested a multi-organ origin with a relevant role for the liver as a source of these vesicles. Tracking experiments also revealed that the liver retains the majority of exosomes from the peritoneal cavity. We conclude that exosomes are involved in the lung damage associated with experimental acute pancreatitis and could be relevant mediators in the systemic effects of pancreatitis. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Secretome analysis of human mesenchymal stem cells undergoing chondrogenic differentiation.

Human mesenchymal stem cells (hMSCs) can be triggered to differentiate toward chondrocytes and thus harbor great therapeutic potential for the repair of cartilage defects in osteoarthritis (OA) and other articular diseases. However, the molecular mechanisms underlying the chondrogenesis process are still in part unknown. In this work, we followed a double-stable isotope labeling by amino acids in cell culture (SILAC) strategy to evaluate the quantitative modulation of the secretome of stem cells isolated from bone marrow (hBMSCs) during the first steps of their chondrogenic differentiation. Analysis by LC-ESI-MS/MS led to the identification of 221 proteins with a reported extracellular localization. Most of them were characteristic of cartilage extracellular matrix, and 34 showed statistically significant quantitative alterations during chondrogenesis. These include, among others, cartilage markers such as Proteoglycan 4 or COMP, anticatabolic markers (TIMP1), reported markers of cartilage development (Versican), and a suggested marker of chondrogenesis, CRAC1. Altogether, our work demonstrates the usefulness of secretome analysis for understanding the mechanisms responsible for cartilage matrix formation, and it reports a panel of extracellular markers potentially useful for the evaluation of tissue development in cell therapy- or tissue engineering-based approaches for cartilage repair.

Characterization of the human plasma phosphoproteome using linear ion trap mass spectrometry and multiple search engines.

Major plasma protein families play different roles in blood physiology and hemostasis and in immunodefense. Other proteins in plasma can be involved in signaling as chemical messengers or constitute biological markers of the status of distant tissues. In this respect, the plasma phosphoproteome holds potentially relevant information on the mechanisms modulating these processes through the regulation of protein activity. In this work we describe for the first time a collection of phosphopeptides identified in human plasma using immunoaffinity separation of the seven major serum protein families from other plasma proteins, SCX fractionation, and TiO(2) purification prior to LC-MS/MS analysis. One-hundred and twenty-seven phosphosites in 138 phosphopeptides mapping 70 phosphoproteins were identified with FDR < 1%. A high-confidence collection of phosphosites was obtained using a combined search with the OMSSA, SEQUEST, and Phenyx search engines.

A Comprehensive Tyrosine Phosphoproteomic Analysis Reveals Novel Components of the Platelet CLEC-2 Signaling Cascade.

C-type lectin-like receptor 2 (CLEC-2) plays a crucial role in different platelet-related physiological and pathological processes. It signals through a tyrosine kinase-mediated pathway that is highly dependent on the positive feedback exerted by the platelet-derived secondary mediators, adenosine diphosphate (ADP) and thromboxane A2 (TXA2). Here, we aimed to analyze the tyrosine phosphoproteome of platelets activated with the CLEC-2 agonist rhodocytin to identify relevant phosphorylated tyrosine residues (p-Tyr) and proteins involved in platelet activation downstream of this receptor. We identified 363 differentially p-Tyr residues, corresponding to the majority of proteins previously known to participate in CLEC-2 signaling and also novel ones, including adaptors (e.g., DAPP1, Dok1/3, CASS4, Nck1/2), kinases/phosphatases (e.g., FAK1, FES, FGR, JAK2, SHIP2), and membrane proteins (e.g., G6F, JAM-A, PECAM-1, TLT-1). To elucidate the contribution of ADP and TXA2 at different points of the CLEC-2 signaling cascade, we evaluated p-Tyr levels of residues identified in the analysis and known to be essential for the catalytic activity of kinases Syk(p-Tyr525+526) and Src(p-Tyr419), and for PLCγ2 activity (p-Tyr759). We demonstrated that Syk phosphorylation at Tyr525+526 also happens in the presence of ADP and TXA2 inhibitors, which is not the case for Src-pTyr419 and PLCγ2-pTyr759. Kinetics studies for the three phosphoproteins show some differences in the phosphorylation profile. Ca2+ mobilization assays confirmed the relevance of ADP and TXA2 for full CLEC-2-mediated platelet activation. The present study provides significant insights into the intracellular events that take place following CLEC-2 activation in platelets, contributing to elucidate in detail the CLEC-2 signalosome.

LymPHOS: design of a phosphosite database of primary human T cells.

Current proteomic technology is capable of producing huge amounts of analytical information, which is often difficult to manage in a comprehensive form. Curation, further annotation and public communication of proteomic data require the development of standard data formats and efficient, multimedia database structures. We have implemented a workflow for the annotation of a phosphopeptide database (LymPHOS) that includes tools for MS data filtering and phosphosite assignation, mass spectrum visualization, experimental description and accurate phosphorylation site assignation. Experimental annotations were fitted to current minimum information about a proteomics experiment guidelines. A new guideline for phosphoprotein sample preparation is also proposed. Currently, the database describes 342 phosphorylation sites mapping to more than 200 gene sequences, and it can be accessed through the net (http://www.lymphos.org).

Platelet membrane lipid rafts protein composition varies following GPVI and CLEC-2 receptors activation.

Lipid rafts are membrane microdomains that have been proposed to play an important role in several platelet-signalling cascades, including those mediated by the receptors Glycoprotein VI (GPVI), and C-type lectin domain family 1 member B (CLEC-2), both involved in thrombus formation. We have performed a LC-MS/MS proteomic analysis of lipid rafts isolated from platelets activated through GPVI and CLEC-2 as well as from resting platelets. Our aim was to determine the magnitude of changes in lipid rafts protein composition and to elucidate the relevance of these alterations in platelet function. A number of relevant signalling proteins were found enriched in lipid rafts following platelet activation (such as the tyrosine protein kinases Fyn, Lyn and Yes; the G proteins G(i) and G(z); and cAMP protein kinase). Interestingly, our results indicate that the relative enrichment of lipid rafts in these signalling proteins may not be a consequence of protein translocation to these domains upon platelet stimulation, but the result of a massive loss in cytoskeletal proteins after platelet activation. Thus, this study may help to better understand the effects of platelet activation in the reorganization of lipid rafts and set the basis for further proteomic studies of these membrane microdomains in platelets. SIGNIFICANCE: We performed the first proteomic comparative analysis of lipid rafts- protein composition in platelets activated through GPVI and CLEC-2 receptors and in resting state. We identified a number of signalling proteins essential for platelet activation relatively enriched in platelets activated through both receptors, and we show that lipid rafts reorganization upon platelet activation leads to a loss in cytoskeletal proteins, highly associated to these domains in resting platelets.

Brachyspira hyodysenteriae and B. pilosicoli Proteins Recognized by Sera of Challenged Pigs.

The spirochetes Brachyspira hyodysenteriae and B. pilosicoli are pig intestinal pathogens that are the causative agents of swine dysentery (SD) and porcine intestinal spirochaetosis (PIS), respectively. Although some inactivated bacterin and recombinant vaccines have been explored as prophylactic treatments against these species, no effective vaccine is yet available. Immunoproteomics approaches hold the potential for the identification of new, suitable candidates for subunit vaccines against SD and PIS. These strategies take into account the gene products actually expressed and present in the cells, and thus susceptible of being targets of immune recognition. In this context, we have analyzed the immunogenic pattern of two B. pilosicoli porcine isolates (the Spanish farm isolate OLA9 and the commercial P43/6/78 strain) and one B. hyodysenteriae isolate (the Spanish farm V1). The proteins from the Brachyspira lysates were fractionated by preparative isoelectric focusing, and the fractions were analyzed by Western blot with hyperimmune sera from challenged pigs. Of the 28 challenge-specific immunoreactive bands detected, 21 were identified as single proteins by MS, while the other 7 were shown to contain several major proteins. None of these proteins were detected in the control immunoreactive bands. The proteins identified included 11 from B. hyodysenteriae and 28 from the two B. pilosicoli strains. Eight proteins were common to the B. pilosicoli strains (i.e., elongation factor G, aspartyl-tRNA synthase, biotin lipoyl, TmpB outer membrane protein, flagellar protein FlaA, enolase, PEPCK, and VspD), and enolase and PEPCK were common to both species. Many of the identified proteins were flagellar proteins or predicted to be located on the cell surface and some of them had been previously described as antigenic or as bacterial virulence factors. Here we report on the identification and semiquantitative data of these immunoreactive proteins which constitute a unique antigen collection from these bacteria.

A multicentric study to evaluate the use of relative retention times in targeted proteomics.

Despite the maturity reached by targeted proteomic strategies, reliable and standardized protocols are urgently needed to enhance reproducibility among different laboratories and analytical platforms, facilitating a more widespread use in biomedical research. To achieve this goal, the use of dimensionless relative retention times (iRT), defined on the basis of peptide standard retention times (RT), has lately emerged as a powerful tool. The robustness, reproducibility and utility of this strategy were examined for the first time in a multicentric setting, involving 28 laboratories that included 24 of the Spanish network of proteomics laboratories (ProteoRed-ISCIII). According to the results obtained in this study, dimensionless retention time values (iRTs) demonstrated to be a useful tool for transferring and sharing peptide retention times across different chromatographic set-ups both intra- and inter-laboratories. iRT values also showed very low variability over long time periods. Furthermore, parallel quantitative analyses showed a high reproducibility despite the variety of experimental strategies used, either MRM (multiple reaction monitoring) or pseudoMRM, and the diversity of analytical platforms employed. BIOLOGICAL SIGNIFICANCE: From the very beginning of proteomics as an analytical science there has been a growing interest in developing standardized methods and experimental procedures in order to ensure the highest quality and reproducibility of the results. In this regard, the recent (2012) introduction of the dimensionless retention time concept has been a significant advance. In our multicentric (28 laboratories) study we explore the usefulness of this concept in the context of a targeted proteomics experiment, demonstrating that dimensionless retention time values is a useful tool for transferring and sharing peptide retention times across different chromatographic set-ups.

Multivariate optimization of the factors influencing the solid-phase microextraction of pyrethroid pesticides in water.

A method based on solid-phase microextraction (SPME) and gas chromatography with micro-electron capture detection (GC-microECD) has been optimized for the analysis of pyrethroids in water samples. The influence of parameters such as temperature, fibre coating, salting-out effect and sampling mode on the extraction efficiency has been studied by means of a mix-level factorial design, which allowed the study of main effects as well as two factor interactions. Finally, a method based on direct SPME at 50 degrees C, using polydimethylsiloxane fibre is proposed. The method showed good linearity (R2>0.995) and repeatability (RSD

New approach based on solid-phase microextraction to estimate polydimethylsiloxane fibre coating-water distribution coefficients for brominated flame retardants.

A depletion solid-phase microextraction (SPME) method based on multiple SPME extraction was applied to estimate fibre coating-water distribution constants (Kfs) of brominated flame retardants. Several polybrominated diphenyl ethers (PBDEs) including compounds present in the commercial mixture "Pentamix", and two polybrominated biphenyls (PBBs) were considered as target analytes. One hundred-micrometer poly(dimethylsiloxane) (PDMS) coating fibre was selected to estimate partition coefficients. SPME kinetics studies at 25 and 100 degrees C were performed. Kfs values obtained at both temperatures for brominated flame retardants were compared with the corresponding octanol-water partition coefficients (Kow) values found in literature. A linear log-log relationship between Kow with Kfs was found. To the best of our knowledge, this is the first study where brominated flame retardants Kfs values are estimated.

The Exposed Proteomes of Brachyspira hyodysenteriae and B. pilosicoli.

Brachyspira hyodysenteriae and Brachyspira pilosicoli are well-known intestinal pathogens in pigs. B. hyodysenteriae is the causative agent of swine dysentery, a disease with an important impact on pig production while B. pilosicoli is responsible of a milder diarrheal disease in these animals, porcine intestinal spirochetosis. Recent sequencing projects have provided information for the genome of these species facilitating the search of vaccine candidates using reverse vaccinology approaches. However, practically no experimental evidence exists of the actual gene products being expressed and of those proteins exposed on the cell surface or released to the cell media. Using a cell-shaving strategy and a shotgun proteomic approach we carried out a large-scale characterization of the exposed proteins on the bacterial surface in these species as well as of peptides and proteins in the extracellular medium. The study included three strains of B. hyodysenteriae and two strains of B. pilosicoli and involved 148 LC-MS/MS runs on a high resolution Orbitrap instrument. Overall, we provided evidence for more than 29,000 different peptides pointing to 1625 and 1338 different proteins in B. hyodysenteriae and B. pilosicoli, respectively. Many of the most abundant proteins detected corresponded to described virulence factors and vaccine candidates. The level of expression of these proteins, however, was different among species and strains, stressing the value of determining actual gene product levels as a complement of genomic-based approaches for vaccine design.

The phosphoproteome of human Jurkat T cell clones upon costimulation with anti-CD3/anti-CD28 antibodies.

Phosphorylation is a reversible post-translational modification, playing a vital role in protein function. In T cells, protein phosphorylation is the key mechanism regulating T cell receptor-driven signaling pathways. In order to gain insights into the phosphoproteome evolution of T cell activation, we performed a large-scale quantitative phosphoproteomics study of Jurkat E6.1 (wild type) and Jurkat gamma1 (Phospholipase gamma1 null) cell clones upon costimulation with anti-CD3 and anti-CD28 antibodies at times ranging from 15min to as long as 120min. In total, we identified 5585 phosphopeptides belonging to 2008 phosphoproteins from both cell clones. We detected 130 and 114 novel phosphopeptides in Jurkat E6.1 and Jurkat gamma1 clones, respectively. A significantly lower number of proteins containing regulated phosphorylation sites were identified in Jurkat gamma1 in comparison to Jurkat E6.1, reflecting the vital role of Phospholipase gamma1 in T cell signaling. Several new phosphorylation sites from lymphocyte-specific protein tyrosine kinase (Lck) were identified. Of these, serine-121 showed significant changes in JE6.1 while only small changes in the Jgamma1 clone. Our data may contribute to the current human T cell phosphoproteome and provide a better understanding on T cell receptor signaling. Data are available via ProteomeXchange with identifier PXD002871.

A comprehensive Candida albicans PeptideAtlas build enables deep proteome coverage.

To provide new and expanded proteome documentation of the opportunistically pathogen Candida albicans, we have developed new protein extraction and analysis routines to provide a new, extended and enhanced version of the C. albicans PeptideAtlas. Two new datasets, resulting from experiments consisting of exhaustive subcellular fractionations and different growing conditions, plus two additional datasets from previous experiments on the surface and the secreted proteomes, have been incorporated to increase the coverage of the proteome. High resolution precursor mass spectrometry (MS) and ion trap tandem MS spectra were analyzed with three different search engines using a database containing allele-specific sequences. This approach, novel for a large-scale C. albicans proteomics project, was combined with the post-processing and filtering implemented in the Trans Proteomic Pipeline consistently used in the PeptideAtlas project and resulted in 49,372 additional peptides (3-fold increase) and 1630 more proteins (1.6-fold increase) identified in the new C. albicans PeptideAtlas with respect to the previous build. A total of 71,310 peptides and 4174 canonical (minimal non-redundant set) proteins (4115 if one protein per pair of alleles is considered) were identified representing 66% of the 6218 proteins in the predicted proteome. This makes the new PeptideAtlas build the most comprehensive C. albicans proteomics resource available and the only large-scale one with detections of individual alleles.

Phosphorylation analysis of primary human T lymphocytes using sequential IMAC and titanium oxide enrichment.

T lymphocytes mediate cellular and humoral defense against foreign bodies or autoantigens. An understanding of T-cell information processing furthers studies of the immunological response. We describe a large-scale phosphorylation analysis of primary T cells using a multidimensional separation strategy, involving preparative SDS-PAGE for prefractionation, in-gel digestion and sequential phosphopeptide enrichment using IMAC and TiO2. A total of 281 phosphorylation sites (197 of high confidence, Ascore > 15), mapping to 204 human gene sequences, were identified by LC-MS(n) analysis in an LTQ linear ion trap. Subsequently, we created the LymPHOS database (http://lymphos.org), which links mass spectrometric peptide information to phosphorylation sites and phosphoprotein sequences.

Effects of sample pretreatment and storage conditions in the determination of pyrethroids in water samples by solid-phase microextraction and gas chromatography-mass spectrometry.

The aqueous instability of pyrethroids and other compounds usually found in commercial pesticide formulations has been demonstrated in this work. Several types of sample treatment have been studied to avoid analyte losses during sample manipulation and storage. Analysis was performed by SPME-GC-MS. Addition of sodium thiosulfate to tap water prevented pyrethroid degradation as a result of oxidation by free chlorine. The amount added was optimized to minimize the effect of the salt on the analytical results. Analysis of samples that had been stored at 4 degrees C for several days revealed loss of some of the pyrethroids in the first period of storage. The effect of freezing the samples was studied and it was confirmed that samples could be stabilized for at least one week by freezing. Finally, addition of a miscible organic solvent, for example acetone, led to improvement of the analytical precision. The quality of the SPME-GC-MS method was studied. Linearity (R > 0.993), repeatability (RSD < 15%), and sensitivity (detection limits between 0.9 and 35 pg mL(-1)) were good. When the procedure was applied to real samples including run off and waste water some of the target compounds were identified and quantified.