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Importance: Biomarkers that are predictive of outcomes in individuals at risk of psychosis would facilitate individualized prognosis and stratification strategies., Objective: To investigate whether proteomic biomarkers may aid prediction of transition to psychotic disorder in the clinical high-risk (CHR) state and adolescent psychotic experiences (PEs) in the general population., Design, Setting, and Participants: This diagnostic study comprised 2 case-control studies nested within the European Network of National Schizophrenia Networks Studying Gene-Environment Interactions (EU-GEI) and the Avon Longitudinal Study of Parents and Children (ALSPAC). EU-GEI is an international multisite prospective study of participants at CHR referred from local mental health services. ALSPAC is a United Kingdom-based general population birth cohort. Included were EU-GEI participants who met CHR criteria at baseline and ALSPAC participants who did not report PEs at age 12 years. Data were analyzed from September 2018 to April 2020., Main Outcomes and Measures: In EU-GEI, transition status was assessed by the Comprehensive Assessment of At-Risk Mental States or contact with clinical services. In ALSPAC, PEs at age 18 years were assessed using the Psychosis-Like Symptoms Interview. Proteomic data were obtained from mass spectrometry of baseline plasma samples in EU-GEI and plasma samples at age 12 years in ALSPAC. Support vector machine learning algorithms were used to develop predictive models., Results: The EU-GEI subsample (133 participants at CHR (mean [SD] age, 22.6 [4.5] years; 68 [51.1%] male) comprised 49 (36.8%) who developed psychosis and 84 (63.2%) who did not. A model based on baseline clinical and proteomic data demonstrated excellent performance for prediction of transition outcome (area under the receiver operating characteristic curve [AUC], 0.95; positive predictive value [PPV], 75.0%; and negative predictive value [NPV], 98.6%). Functional analysis of differentially expressed proteins implicated the complement and coagulation cascade. A model based on the 10 most predictive proteins accurately predicted transition status in training (AUC, 0.99; PPV, 76.9%; and NPV, 100%) and test (AUC, 0.92; PPV, 81.8%; and NPV, 96.8%) data. The ALSPAC subsample (121 participants from the general population with plasma samples available at age 12 years (61 [50.4%] male) comprised 55 participants (45.5%) with PEs at age 18 years and 61 (50.4%) without PEs at age 18 years. A model using proteomic data at age 12 years predicted PEs at age 18 years, with an AUC of 0.74 (PPV, 67.8%; and NPV, 75.8%)., Conclusions and Relevance: In individuals at risk of psychosis, proteomic biomarkers may contribute to individualized prognosis and stratification strategies. These findings implicate early dysregulation of the complement and coagulation cascade in the development of psychosis outcomes.

Stenotrophomonas maltophilia is a Gram-negative opportunistic pathogen that can chronically colonize the lungs of people with cystic fibrosis (CF) and is associated with lethal pulmonary hemorrhage in immunocompromised patients. Its secreted virulence factors include the extracellular serine proteases StmPR1, StmPR2, and StmPR3. To explore the impact of secreted virulence determinants on pulmonary mucosal defenses in CF, we examined the secretome of human CFBE41o- bronchial epithelial cells in response to treatment with S. maltophilia K279a cell culture supernatant (CS) using a liquid-chromatography-tandem mass spectrometry (LC-MS/MS) based label-free quantitative (LFQ) shotgun proteomics approach for global profiling of the cell secretome. Secretome analysis identified upregulated pathways mainly relating to biological adhesion and epithelial cell signaling in infection, whereas no specific pathways relating to the immune response were enriched. Further exploration of the potentially harmful effects of K279a CS on CF bronchial epithelial cells, demonstrated that K279a CS caused CFBE41o- cell condensation and detachment, reversible by the serine protease inhibitor PMSF. K279a CS also decreased trans-epithelial electrical resistance in CFBE41o- cell monolayers suggestive of disruption of tight junction complexes (TJC). This finding was corroborated by an observed increase in fluorescein isothiocyanate (FITC) dextran permeability and by demonstrating PMSF-sensitive degradation of the tight junction proteins ZO-1 and occludin, but not JAM-A or claudin-1. These observations demonstrating destruction of the CFBE41o- TJC provide a novel insight regarding the virulence of S. maltophilia and may explain the possible injurious effects of this bacterium on the CF bronchial epithelium and the pathogenic mechanism leading to lethal pulmonary hemorrhage., (Copyright © 2020 Molloy, Cagney, Dillon, Wynne, Greene and McElvaney.)

Upon activation, platelets release a powerful cocktail of soluble and vesicular signals, collectively termed the "platelet releasate" (PR). Although several studies have used qualitative/quantitative proteomic approaches to characterize PR; with debated content and significant inter-individual variability reported, confident, and reliable insights have been hindered. Using label-free quantitative (LFQ)-proteomics analysis, a reproducible, quantifiable investigation of the 1U mL -1 thrombin-induced PR from 32 healthy adults was conducted. MS proteomics data are available via ProteomeXchange, identifier PXD009310. Of the 894 proteins identified, 277 proteins were quantified across all donors and form a "core" PR. Bioinformatics and further LFQ-proteomic analysis revealed that the majority (84%) of "core" PR proteins overlapped with the protein composition of human platelet-derived exosomes. Vesicles in the exosomal-size range were confirmed in healthy-human PR and reduced numbers of similar-sized vesicles were observed in the PR of a mouse model of gray platelet syndrome, known to be deficient in platelet alpha-granules. Lastly, the variability of proteins in the PR was assessed, and reproducible secretion levels were found across all 32 healthy donors. Taken together, the PR contains valuable soluble and vesicular cargo and has low-population variance among healthy adults, rendering it a potentially useful platform for diagnostic fingerprinting of platelet-related disease., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

It was previously demonstrated that the WNT/β-catenin pathway is present and active in platelets and established that the canonical WNT ligand, WNT-3a, suppresses platelet adhesion and activation. In nucleated cells, β-catenin, the key downstream effector of this pathway, is a dual function protein, regulating the coordination of gene transcription and cell-cell adhesion. The specific role of β-catenin in the anucleate platelet however remains elusive. Here, a label-free quantitative proteomic analysis of β-catenin immunoprecipitates from human platelets is performed and nine co-immunoprecipitating proteins are identified. Three of the co-immunoprecipitating proteins (α-catenin-1, cadherin-6, and β-catenin-interacting protein 1) are common to both resting and activated conditions. Bioinformatics analysis of proteomics data reveal a strong association of the dataset with both cadherin adherens junctions and regulators of WNT signaling. It is then verified that platelet β-catenin and cadherin-6 interact and that this interaction is regulated by the activation state of the platelet. Taken together, this proteomics study suggests a novel role for β-catenin in human platelets where it interacts with platelet cadherins and associated junctional proteins., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The identification of early biological changes associated with the psychotic disorder (PD) is important as it may provide clues to the underlying pathophysiological mechanisms. We undertook the first proteomic profiling of blood plasma samples of children who later develop a PD. Participants were recruited from the UK Avon Longitudinal Study of Parents and Children (ALSPAC) cohort who also participated in psychiatric assessment interviews at age 18. Protein expression levels at age 11 were compared between individuals who developed PD at age 18 (n = 37) with population-based age-matched controls (n = 38). Sixty out of 181 plasma proteins profiled were found to be differentially expressed (P < .05) in children with an outcome of the PD. Thirty-four of these proteins were found to be differentially expressed following correction for multiple comparisons. Pathway analysis implicated the complement and coagulation cascade. A second, targeted proteomic approach was used to verify these findings in age 11 plasma from subjects who reported psychotic experiences at age 18 (n = 40) in comparison to age-matched controls (n = 66). Our findings indicate that the complement and coagulation system is dysregulated in the blood during childhood before the development of the PD.

Prenatal iron deficiency (pID) has been described to increase the risk for neurodevelopmental disorders such as autism and schizophrenia; however, the precise molecular mechanisms are still unknown. Here, we utilized high-throughput MS to examine the proteomic effects of pID in adulthood on the rat frontal cortex area (FCA). In addition, the FCA proteome was examined in adulthood following risperidone treatment in adolescence to see if these effects could be prevented. We identified 1501 proteins of which 100 were significantly differentially expressed in the FCA at postnatal day 90. Pathway analysis of proteins affected by pID revealed changes in metabolic processes, including the tricyclic acid cycle, mitochondrial dysfunction, and P13K/Akt signaling. Interestingly, most of these protein changes were not present in the adult pID offspring who received risperidone in adolescence. Considering the link between pID and several neurodevelopmental disorders such as autism and schizophrenia these presented results bring new perspectives to understand the role of iron in metabolic pathways and provide novel biomarkers for future studies of pID., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Proteomic exploration of the effects of psychotropic drugs on specific brain areas in rodents has the potential to uncover novel molecular networks and pathways affected by psychotropic medications, and may inform etiologic hypotheses on mental disorders. Haloperidol, a widely used first-generation antipsychotic, has been shown to produce structural and functional changes of the hippocampus, a brain region also implicated in the neuropathology of disorders such as schizophrenia and bipolar disorder. Seven adult male C57BL/6 mice were injected daily intraperitoneally with 0.5 mg/kg of haloperidol, for 28 days. A control group of six animals was injected with vehicle only (saline). Protein levels of postmortem hippocampus homogenate were determined using label-free LC/MS/MS. In the treatment group, 216 differentially expressed hippocampal proteins were identified as compared to controls. Ingenuity pathway analysis implicated oxidative phosphorylation and mitochondrial function as top canonical pathways, and local networks involved in tubulin-mediated cytoskeleton dynamics, clathrin-mediated endocytosis, and extracellular signal-regulated kinase and c-Jun N-terminal kinase signaling. The findings of this study could stimulate further research into the cellular mechanisms associated with haloperidol treatment and the pathophysiology of psychotic disorders, assisting treatment biomarker discovery. All MS data have been deposited in the ProteomeXchange with identifier PXD002250 (http://proteomecentral.proteomexchange.org/dataset/PXD002250)., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The prefrontal cortex (PFC) is associated with mental health illnesses including schizophrenia, depression, bipolar disorder, and autism spectrum disorders. It richly expresses neuroreceptors which are the target for antipsychotics. However, as the precise mechanism of action of antipsychotic medications is not known, proteomic studies of the effects of antipsychotic drugs on the brain are warranted. In the current study, we aimed to characterize protein expression in the adult rodent PFC (n = 5 per group) following low-dose treatment with Risperidone or saline in adolescence (postnatal days 34-47). The PFC was examined by triplicate 1 h runs of label-free LC-MS/MS. The raw mass spectral data were analyzed with the MaxQuant(TM) software. Statistical analysis was carried out using SAS® Version 9.1. Pathway and functional analysis was performed with IngenuityPathway Analysis and in the Database for Annotation, Visualization and Integrated Discovery (DAVID), respectively, the most implicated pathways were found to be related to mitochondrial function, protein trafficking, and the cytoskeleton. This report adds to the current repertoire of data available concerning the effects of antipsychotic drugs on the brain and sheds light on their biological mechanisms. The MS data have been deposited with the ProteomeXchange Consortium with dataset identifier PXD000480., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We have analyzed the proteomes of two human melanoma cell lines (A375 and 526), and of the human melanocytes, (FOM 78), by two-dimensional electrophoresis (2D-PAGE) and liquid chromatography - tandem mass spectrometry (LC-MS/MS). Our comparative proteomic analysis revealed that six proteins were over-expressed in both melanoma cell lines as compared to melanocytes: galectin-1, inosine-5'-monophosphate dehydrogenase 2, serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A alpha isoform, protein DJ-1, cyclophilin A and cofilin-1. We show, for the first time, that only specific isoforms of these molecules are over-expressed in melanoma. Different protein profiles were also found between each individual melanoma cell line and the melanocytes. s-Methyl-5-thioadenosine phosphorylase, ubiquitin and ribosomal protein S27 a precursor, the basic form of protein DJ-1, annexin a1, proliferation associated protein 2g4, isoform alfa-enolase of alfa-enolase, protein disulfide-isomerase precursor, and elongation factor 2 were more strongly expressed in A375 cells compared to melanocytes. In 526 cells, 60s acidic ribosomal protein p1 and calreticulin precursor were more highly expressed than in melanocytes. These molecular differences may help in better understanding melanoma development and its different responsiveness to therapies. The identified proteins could be exploited as biomarkers or therapeutic targets for melanoma., (© 2011 Landes Bioscience)

Meiosis is the cell division that generates haploid gametes from diploid precursors. To provide insight into the functional proteome of budding yeast during meiosis, a 2-D DIGE kinetic approach was used to study proteins in the pH 6-11 range. Nearly 600 protein spots were visualised and 79 spots exhibited statistically significant changes in abundance as cells progressed through meiosis. Expression changes of up to 41-fold were detected and protein sequence information was obtained for 48 spots. Single protein identifications were obtained for 21 spots including different gel mobility forms of 5 proteins. A large number of post-translational events are suggested for these proteins, including processing, modification and import. The data are incorporated into an online 2-DE map of meiotic proteins in budding yeast, which extends our initial DIGE investigation of proteins in the pH 4-7 range. Together, the analyses provide peptide sequence data for 84 protein spots, including 50 single-protein identifications and gel mobility isoforms of 8 proteins. The largest classes of identified proteins include carbon metabolism, protein catabolism, protein folding, protein synthesis and the oxidative stress response. A number of the corresponding genes are required for yeast meiosis and recent studies have identified similar classes of proteins expressed during mammalian meiosis. This proteomic investigation and the resulting protein reference map make an important contribution towards a more detailed molecular view of yeast meiosis., (Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Skeletal muscle fibre transitions occur in many biological processes, in response to alterations in neuromuscular activity, in muscular disorders, during age-induced muscle wasting and in myogenesis. It was therefore of interest to perform a comprehensive proteomic profiling of muscle transformation. Chronic low-frequency stimulation of the rabbit tibialis anterior muscle represents an established model system for studying the response of fast fibres to enhanced neuromuscular activity under conditions of maximum activation. We have conducted a DIGE analysis of unstimulated control specimens versus 14- and 60-day conditioned muscles. A differential expression pattern was observed for 41 protein species with 29 increased and 12 decreased muscle proteins. Identified classes of proteins that are changed during the fast-to-slow transition process belong to the contractile machinery, ion homeostasis, excitation-contraction coupling, capillarization, metabolism and stress response. Results from immunoblotting agreed with the conversion of the metabolic, regulatory and contractile molecular apparatus to support muscle fibres with slower twitch characteristics. Besides confirming established muscle elements as reliable transition markers, this proteomics-based study has established the actin-binding protein cofilin-2 and the endothelial marker transgelin as novel biomarkers for evaluating muscle transformation.

Proteomics offers the opportunity to comprehensively investigate the anucleate platelet. Here, we present a detailed procedure for enrichment by immunoprecipitation, using the monoclonal antibody 4G10, of the dynamic phosphotyrosine proteome of human platelets. Such an approach offers the possibility of capturing the dynamic tyrosine phosphorylation events that occur upon platelet activation and aggregation, with an aim to identify novel signaling proteins.

Signalling cascades are regulated both positively and negatively by tyrosine phosphorylation. Integrin mediated platelet adhesion triggers signal transduction cascades involving translocation of proteins and tyrosine phosphorylation events, ultimately causing large signalling complexes to be assembled. In resting platelets, a small number of phosphorylated proteins are evident with molecular mass of 50-62 kDa and 120-130 kDa. In thrombin activated human platelets, however, there is a large increase in the number of tyrosine phosphorylated signalling proteins detected. These proteins include pCas (130 kDa), FAK (125 kDa), PI(3)k (85 kDa) and src (85 kDa). However, it is unlikely that this list of proteins represents all the dynamic changes that occur after platelet activation and it is understood that more proteins remain unidentified. In this study, we propose a method for the isolation of the phosphotyrosine proteome from both resting and thrombin activated human platelets. All the dynamic phosphotyrosine events that occur in the platelet after thrombin activation were isolated by immunoprecipitation, using the monoclonal antibody 4G10, allowing us to obtain higher concentrations of relatively low abundant proteins. The resulting phosphotyrosine proteomes were separated by two-dimensional gel electrophoresis. Sixty-seven proteins were reproducibly found to be unique in the thrombin activated platelet proteome when compared to resting platelets. We have positively identified ten of these proteins by Western blotting and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry and these include FAK, Syk, ALK-4, P2X6 and MAPK kinase kinase. This proteomics approach to understanding the signalling events following platelet activation may elucidate potential drug targets for the treatment of coronary thrombosis.

Psychosis risk prediction is one of the leading challenges in psychiatry. Previous investigations have suggested that plasma proteomic data may be useful in accurately predicting transition to psychosis in individuals at clinical high risk (CHR). We hypothesized that an a priori-specified proteomic prediction model would have strong predictive accuracy for psychosis risk and aimed to replicate longitudinal associations between plasma proteins and transition to psychosis. This study used plasma samples from participants in 3 CHR cohorts: the North American Prodrome Longitudinal Studies 2 and 3, and the NEURAPRO randomized control trial (total n = 754). Plasma proteomic data were quantified using mass spectrometry. The primary outcome was transition to psychosis over the study follow-up period. Logistic regression models were internally validated, and optimism-corrected performance metrics derived with a bootstrap procedure. In the overall sample of CHR participants (age: 18.5, SD: 3.9; 51.9% male), 20.4% (n = 154) developed psychosis within 4.4 years. The a priori-specified model showed poor risk-prediction accuracy for the development of psychosis (C-statistic: 0.51 [95% CI: 0.50, 0.59], calibration slope: 0.45). At a group level, Complement C8B, C4B, C5, and leucine-rich α-2 glycoprotein 1 (LRG1) were associated with transition to psychosis but did not surpass correction for multiple comparisons. This study did not confirm the findings from a previous proteomic prediction model of transition from CHR to psychosis. Certain complement proteins may be weakly associated with transition at a group level. Previous findings, derived from small samples, should be interpreted with caution.