Quantitative Proteomics Reveal Region-Specific Alterations in Neuroserpin-Deficient Mouse Brain and Retina: Insights into Serpini1 Function.

Neural regeneration and neuroprotection represent strategies for future management of neurodegenerative disorders such as Alzheimer's disease (AD) or glaucoma. However, the complex molecular mechanisms that are involved in neuroprotection are not clearly understood. A promising candidate that maintains neuroprotective signaling networks is neuroserpin (Serpini1), a serine protease inhibitor expressed in neurons which selectively inhibits extracellular tissue-type plasminogen activator (tPA)/plasmin and plays a neuroprotective role during ischemic brain injury. Abnormal function of this protein has been implicated in several conditions including stroke, glaucoma, AD, and familial encephalopathy with neuroserpin inclusion bodies (FENIB). Here, we explore the potential biochemical roles of Serpini1 by comparing proteome changes between neuroserpin-deficient (NS-/-) and control mice, in the retina (RE), optic nerve (ON), frontal cortex (FC), visual cortex (VC), and cerebellum (CB). To achieve this, a multiple-plex quantitative proteomics approach using isobaric tandem mass tag (TMT) technology was employed followed by functional enrichment and protein-protein interaction analysis. We detected around 5000 proteins in each tissue and a pool of 6432 quantified proteins across all regions, resulting in a pool of 1235 differentially expressed proteins (DEPs). Principal component analysis and hierarchical clustering highlighted similarities and differences in the retina compared to various brain regions, as well as differentiating NS-/- proteome signatures from control samples. The visual cortex revealed the highest number of DEPs, followed by cerebellar regions. Pathway analysis unveiled region-specific changes, including visual perception, focal adhesion, apoptosis, glutamate receptor activation, and supramolecular fiber organization in RE, ON, FC, VC, and CB, respectively. These novel findings provide comprehensive insights into the region-specific networking of Serpini1 in the central nervous system, further characterizing its potential role as a neuroprotective agent. Data are available via ProteomeXchange with identifier PXD046873.

Proteome profiling of enriched membrane-associated proteins unraveled a novel sophorose and cello-oligosaccharide transporter in Trichoderma reesei.

BACKGROUND: Trichoderma reesei is an organism extensively used in the bioethanol industry, owing to its capability to produce enzymes capable of breaking down holocellulose into simple sugars. The uptake of carbohydrates generated from cellulose breakdown is crucial to induce the signaling cascade that triggers cellulase production. However, the sugar transporters involved in this process in T. reesei remain poorly identified and characterized. RESULTS: To address this gap, this study used temporal membrane proteomics analysis to identify five known and nine putative sugar transporters that may be involved in cellulose degradation by T. reesei. Docking analysis pointed out potential ligands for the putative sugar transporter Tr44175. Further functional validation of this transporter was carried out in Saccharomyces cerevisiae. The results showed that Tr44175 transports a variety of sugar molecules, including cellobiose, cellotriose, cellotetraose, and sophorose. CONCLUSION: This study has unveiled a transporter Tr44175 capable of transporting cellobiose, cellotriose, cellotetraose, and sophorose. Our study represents the first inventory of T. reesei sugar transportome once exposed to cellulose, offering promising potential targets for strain engineering in the context of bioethanol production.

Pre-Treatment of Rice Plants with ABA Makes Them More Tolerant to Multiple Abiotic Stress.

Multiple abiotic stress is known as a type of environmental unfavourable condition maximizing the yield and growth gap of crops compared with the optimal condition in both natural and cultivated environments. Rice is the world's most important staple food, and its production is limited the most by environmental unfavourable conditions. In this study, we investigated the pre-treatment of abscisic acid (ABA) on the tolerance of the IAC1131 rice genotype to multiple abiotic stress after a 4-day exposure to combined drought, salt and extreme temperature treatments. A total of 3285 proteins were identified and quantified across the four treatment groups, consisting of control and stressed plants with and without pre-treatment with ABA, with 1633 of those proteins found to be differentially abundant between groups. Compared with the control condition, pre-treatment with the ABA hormone significantly mitigated the leaf damage against combined abiotic stress at the proteome level. Furthermore, the application of exogenous ABA did not affect the proteome profile of the control plants remarkably, while the results were different in stress-exposed plants by a greater number of proteins changed in abundance, especially those which were increased. Taken together, these results suggest that exogenous ABA has a potential priming effect for enhancing the rice seedlings' tolerance against combined abiotic stress, mainly by affecting stress-responsive mechanisms dependent on ABA signalling pathways in plants.

Apolipoprotein ε in Brain and Retinal Neurodegenerative Diseases.

Alzheimer's disease (AD) is the most common form of dementia that remains incurable and has become a major medical, social, and economic challenge worldwide. AD is characterized by pathological hallmarks of senile plaques (SP) and neurofibrillary tangles (NFTs) that damage the brain up to twenty years before a clinical diagnosis is made. Interestingly these pathological features have also been observed in retinal neurodegenerative diseases including age related macular degeneration (ARMD), glaucoma and diabetic retinopathy (DR). An association of AD with these diseases has been suggested in epidemiological studies and several common pathological events and risk factors have been identified between these diseases. The E4 allele of Apolipoprotein E (APOE) is a well-established genetic risk factor for late onset AD. The ApoE ε4 allele is also associated with retinal neurodegenerative diseases however in contrast to AD, it is considered protective in AMD, likewise ApoE E2 allele, which is a protective factor for AD, has been implicated as a risk factor for AMD and glaucoma. This review summarizes the evidence on the effects of ApoE in retinal neurodegenerative diseases and discusses the overlapping molecular pathways in AD. The involvement of ApoE in regulating amyloid beta (Aß) and tau pathology, inflammation, vascular integrity, glucose metabolism and vascular endothelial growth factor (VEGF) signaling is also discussed.

One Step Forward with Dry Surface Biofilm (DSB) of Staphylococcus aureus: TMT-Based Quantitative Proteomic Analysis Reveals Proteomic Shifts between DSB and Hydrated Biofilm.

The Gram-positive bacterium Staphylococcus aureus is responsible for serious acute and chronic infections worldwide and is well-known for its biofilm formation ability. Recent findings of biofilms on dry hospital surfaces emphasise the failures in current cleaning practices and disinfection and the difficulty in removing these dry surface biofilms (DSBs). Many aspects of the formation of complex DSB biology on environmental surfaces in healthcare settings remains limited. In the present study, we aimed to determine how the protein component varied between DSBs and traditional hydrated biofilm. To do this, biofilms were grown in tryptic soy broth (TSB) on removable polycarbonate coupons in the CDC biofilm reactor over 12 days. Hydrated biofilm (50% TSB for 48 h, the media was then changed every 48 h with 20% TSB, at 37 °C with 130 rpm). DSB biofilm was produced in 5% TSB for 48 h at 35 °C followed by extended periods of dehydration (48, 66, 42 and 66 h at room temperature) interspersed with 6 h of 5% TSB at 35 °C. Then, we constructed a comprehensive reference map of 12-day DSB and 12-day hydrated biofilm associated proteins of S. aureus using a high-throughput tandem mass tag (TMT)-based mass spectrometry. Further pathway analysis of significantly differentially expressed identified proteins revealed that proteins significantly upregulated in 12-day DSB include PTS glucose transporter subunit IIBC (PtaA), UDP-N-acetylmuramate-L-alanine ligase (MurC) and UDP-N-acetylenolpyruvoylglucosamine (MurB) compared to 12-day hydrated biofilm. These three proteins are all linked with peptidoglycan biosynthesis pathway and are responsible for cell-wall formation and thicker EPS matrix deposition. Increased cell-wall formation may contribute to the persistence of DSB on dry surfaces. In contrast, proteins associated with energy metabolisms such as phosphoribosyl transferase (PyrR), glucosamine--fructose-6-phosphate aminotransferase (GlmS), galactose-6-phosphate isomerase (LacA), and argininosuccinate synthase (ArgG) were significantly upregulated whereas ribosomal and ABC transporters were significantly downregulated in the 12-day hydrated biofilm compared to DSB. However, validation by qPCR analysis showed that the levels of gene expression identified were only partially in line with our TMT-MS quantitation analysis. For the first time, a TMT-based proteomics study with DSB has shed novel insights and provided a basis for the identification and study of significant pathways vital for biofilm biology in this reference microorganism.

Proteomic analysis of the meristematic root zone in contrasting genotypes reveals new insights in drought tolerance in rice.

Drought is responsible for major losses in rice production. Root tips contain meristematic and elongation zones that play major roles in determination of root traits and adaptive strategies to drought. In this study we analysed two contrasting genotypes of rice: IR64, a lowland, drought-susceptible, and shallow-rooting genotype; and Azucena, an upland, drought-tolerant, and deep-rooting genotype. Samples were collected of root tips of plants grown under control and water deficit stress conditions. Quantitative proteomics analysis resulted in the identification of 7294 proteins from the root tips of IR64 and 6307 proteins from Azucena. Data are available via ProteomeXchange with identifier PXD033343. Using a Partial Least Square Discriminant Analysis on 4170 differentially abundant proteins, 1138 statistically significant proteins across genotypes and conditions were detected. Twenty two enriched biological processes showing contrasting patterns between two genotypes in response to stress were detected through gene ontology enrichment analysis. This included identification of novel proteins involved in root elongation with specific expression patterns in Azucena, including four Expansins and seven Class III Peroxidases. We also detected an antioxidant network and a metallo-sulfur cluster assembly machinery in Azucena, with roles in reactive oxygen species and iron homeostasis, and positive effects on root cell cycle, growth and elongation.

Fingolimod effects on the brain are mediated through biochemical modulation of bioenergetics, autophagy, and neuroinflammatory networks.

Fingolimod (FTY720) is an oral drug approved by the Food and Drug Administration (FDA) for management of multiple sclerosis (MS) symptoms, which has also shown beneficial effects against Alzheimer's (AD) and Parkinson's (PD) diseases pathologies. Although an extensive effort has been made to identify mechanisms underpinning its therapeutic effects, much remains unknown. Here, we investigated Fingolimod induced proteome changes in the cerebellum (CB) and frontal cortex (FC) regions of the brain which are known to be severely affected in MS, using a tandem mass tag (TMT) isobaric labeling-based quantitative mass-spectrometric approach to investigate the mechanism of action of Fingolimod. This study identified 6749 and 6319 proteins in CB and FC, respectively, and returned 2609 and 3086 differentially expressed proteins in mouse CB and FC, respectively, between Fingolimod treated and control groups. Subsequent bioinformatics analyses indicated a metabolic reprogramming in both brain regions of the Fingolimod treated group, where oxidative phosphorylation was upregulated while glycolysis and pentose phosphate pathway were downregulated. In addition, modulation of neuroinflammation in the Fingolimod treated group was indicated by upregulation of retrograde endocannabinoid signaling and autophagy pathways, and downregulation of neuroinflammation related pathways including neutrophil degranulation and the IL-12 mediated signaling pathway. Our findings suggest that Fingolimod may exert its protective effects on the brain by inducing metabolic reprogramming and neuroinflammation pathway modulation.

Proteome of Staphylococcus aureus Biofilm Changes Significantly with Aging.

Staphylococcus aureus is a notorious biofilm-producing pathogen that is frequently isolated from implantable medical device infections. As biofilm ages, it becomes more tolerant to antimicrobial treatment leading to treatment failure and necessitating the costly removal of infected devices. In this study, we performed in-solution digestion followed by TMT-based high-throughput mass spectrometry and investigated what changes occur in the proteome of S. aureus biofilm grown for 3-days and 12-days in comparison with 24 h planktonic. It showed that proteins associated with biosynthetic processes, ABC transporter pathway, virulence proteins, and shikimate kinase pathway were significantly upregulated in a 3-day biofilm, while proteins associated with sugar transporter, degradation, and stress response were downregulated. Interestingly, in a 3-day biofilm, we observed numerous proteins involved in the central metabolism pathways which could lead to biofilm growth under diverse environments by providing an alternative metabolic route to utilize energy. In 12-day biofilms, proteins associated with peptidoglycan biosynthesis, sugar transporters, and stress responses were upregulated, whereas proteins associated with ABC transporters, DNA replication, and adhesion proteins were downregulated. Gene Ontology analysis revealed that more proteins are involved in metabolic processes in 3dwb compared with 12dwb. Furthermore, we observed significant variations in the formation of biofilms resulting from changes in the level of metabolic activity in the different growth modes of biofilms that could be a significant factor in S. aureus biofilm maturation and persistence. Collectively, potential marker proteins were identified and further characterized to understand their exact role in S. aureus biofilm development, which may shed light on possible new therapeutic regimes in the treatment of biofilm-related implant-associated infections.

Key Genes and Biochemical Networks in Various Brain Regions Affected in Alzheimer's Disease.

Alzheimer's disease (AD) is one of the most complicated progressive neurodegenerative brain disorders, affecting millions of people around the world. Ageing remains one of the strongest risk factors associated with the disease and the increasing trend of the ageing population globally has significantly increased the pressure on healthcare systems worldwide. The pathogenesis of AD is being extensively investigated, yet several unknown key components remain. Therefore, we aimed to extract new knowledge from existing data. Ten gene expression datasets from different brain regions including the hippocampus, cerebellum, entorhinal, frontal and temporal cortices of 820 AD cases and 626 healthy controls were analyzed using the robust rank aggregation (RRA) method. Our results returned 1713 robust differentially expressed genes (DEGs) between five brain regions of AD cases and healthy controls. Subsequent analysis revealed pathways that were altered in each brain region, of which the GABAergic synapse pathway and the retrograde endocannabinoid signaling pathway were shared between all AD affected brain regions except the cerebellum, which is relatively less sensitive to the effects of AD. Furthermore, we obtained common robust DEGs between these two pathways and predicted three miRNAs as potential candidates targeting these genes; hsa-mir-17-5p, hsa-mir-106a-5p and hsa-mir-373-3p. Three transcription factors (TFs) were also identified as the potential upstream regulators of the robust DEGs; ELK-1, GATA1 and GATA2. Our results provide the foundation for further research investigating the role of these pathways in AD pathogenesis, and potential application of these miRNAs and TFs as therapeutic and diagnostic targets.

Multiple Abiotic Stresses Applied Simultaneously Elicit Distinct Responses in Two Contrasting Rice Cultivars.

Rice crops are often subject to multiple abiotic stresses simultaneously in both natural and cultivated environments, resulting in yield reductions beyond those expected from single stress. We report physiological changes after a 4 day exposure to combined drought, salt and extreme temperature treatments, following a 2 day salinity pre-treatment in two rice genotypes-Nipponbare (a paddy rice) and IAC1131 (an upland landrace). Stomata closed after two days of combined stresses, causing intercellular CO2 concentrations and assimilation rates to diminish rapidly. Abscisic acid (ABA) levels increased at least five-fold but did not differ significantly between the genotypes. Tandem Mass Tag isotopic labelling quantitative proteomics revealed 6215 reproducibly identified proteins in mature leaves across the two genotypes and three time points (0, 2 and 4 days of stress). Of these, 987 were differentially expressed due to stress (cf. control plants), including 41 proteins that changed significantly in abundance in all stressed plants. Heat shock proteins, late embryogenesis abundant proteins and photosynthesis-related proteins were consistently responsive to stress in both Nipponbare and IAC1131. Remarkably, even after 2 days of stress there were almost six times fewer proteins differentially expressed in IAC1131 than Nipponbare. This contrast in the translational response to multiple stresses is consistent with the known tolerance of IAC1131 to dryland conditions.

Comparative Analysis of Aducanumab, Zagotenemab and Pioglitazone as Targeted Treatment Strategies for Alzheimer's Disease.

Alzheimer's disease (AD) is the leading cause of dementia that has remained a major medical, sociocultural and economical challenge globally. Previously developed treatments like anticholinesterase inhibitors (AChEIs) and N-methyl-D-aspartate receptor (NMDAR) antagonists only provide short-term symptomatic improvement and do not prevent progression. Repeated setbacks and failures over the past 25 years in AD clinical trials have hindered efforts to develop effective AD treatments. Fortunately, Aducanumab, a specific anti-amyloid ß antibody, has shown promising clinical results and was recently approved by the Food and Drug Administration (FDA) through an accelerated approval pathway. This has raised hopes for AD patients; however post-approval trials are necessary to estimate the true scope of its clinical benefits. We have reviewed several AD clinical studies and summarized the experience to date with Aducanumab and two other potential AD drugs including Zagotenemab (an anti-tau antibody) and Pioglitazone (nuclear Peroxisome-Proliferator Activated Receptor γ (PPARγ) agonist). These have shown mixed results so far and the next few years will be critical to elucidate and interpret their broad long-term protective effects. A concerted effort is required to understand and strengthen the translation of pre-clinical findings from these drugs to routine clinical practice.

A Proteomic View of Cellular and Molecular Effects of Cannabis.

Cannabis (Cannabis sativa), popularly known as marijuana, is the most commonly used psychoactive substance and is considered illicit in most countries worldwide. However, a growing body of research has provided evidence of the therapeutic properties of chemical components of cannabis known as cannabinoids against several diseases including Alzheimer's disease (AD), multiple sclerosis (MS), Parkinson's disease, schizophrenia and glaucoma; these have prompted changes in medicinal cannabis legislation. The relaxation of legal restrictions and increased socio-cultural acceptance has led to its increase in both medicinal and recreational usage. Several biochemically active components of cannabis have a range of effects on the biological system. There is an urgent need for more research to better understand the molecular and biochemical effects of cannabis at a cellular level, to understand fully its implications as a pharmaceutical drug. Proteomics technology is an efficient tool to rigorously elucidate the mechanistic effects of cannabis on the human body in a cell and tissue-specific manner, drawing conclusions associated with its toxicity as well as therapeutic benefits, safety and efficacy profiles. This review provides a comprehensive overview of both in vitro and in vivo proteomic studies involving the cellular and molecular effects of cannabis and cannabis-derived compounds.

Pollen development in cotton (Gossypium hirsutum) is highly sensitive to heat exposure during the tetrad stage.

The development of gametes in plants is acutely susceptible to heatwaves as brief as a few days, adversely affecting pollen maturation and reproductive success. Pollen in cotton (Gossypium hirsutum) was differentially affected when tetrad and binucleate stages were exposed to heat, revealing new insights into the interaction between heat and pollen development. Squares were tagged and exposed to 36/25°C (day/night, moderate heat) or 40/30°C (day/night, extreme heat) for 5 days. Mature pollen grains and leaves were collected for physiological and proteomic responses. While photosynthetic competence was not compromised even at 40°C, leaf tissues became leakier. In contrast, pollen grains were markedly smaller after the tetrad stage was exposed to 40°C and boll production was reduced by 65%. Sugar levels in pollen grains were elevated after exposure to heat, eliminating carbohydrate deficits as a likely cause of poor reproductive capacity. Proteomic analysis of pure pollen samples revealed a particularly high abundance of 70-kDa heat shock (Hsp70s) and cytoskeletal proteins. While short-term bursts of heat had a minor impact on leaves, male gametophyte development was profoundly damaged. Cotton acclimates to maxima of 36°C at both the vegetative and reproductive stages but 5-days exposure to 40°C significantly impairs reproductive development.

Amyloid-beta peptide neurotoxicity in human neuronal cells is associated with modulation of insulin-like growth factor transport, lysosomal machinery and extracellular matrix receptor interactions.

Extracellular deposits of the amyloid-beta peptide (Aß) are known as the main pathological hallmark of Alzheimer's disease. In Alzheimer's disease, neurons are injured and die throughout the brain, a process in which Aß neurotoxicity is considered to play an important role. However, the molecular mechanisms underlying Aß toxicity that lead to neurodegeneration are not clearly established. Here we have elucidated the molecular pathways and networks which are impacted by Aß in neurons using SH-SY5Y human neuroblastoma cells as a model. These cells were treated with Aß1-42 peptides to study changes in biochemical networks using tandem mass tag labeled quantitative proteomic technique followed by computational analysis of the data. The molecular impacts of Aß on cells were evident in a time- and dose-dependent manner, albeit the duration of treatment induced greater differential changes in cellular proteome compared to the effects of concentration. Aß induced early changes in proteins associated with lysosomes, collagen chain trimerization and extracellular matrix receptor interaction, complement and coagulation cascade, oxidative stress induced senescence, ribosome biogenesis, regulation of insulin-like growth factor transport and uptake by insulin-like growth factor-binding protein. These novel findings provide molecular insights on the effects of Aß on neurons, with implications for better understanding the impacts of Aß on early neurodegeneration in Alzheimer's disease pathology.

Proteomic Responses to Drought Vary Widely Among Eight Diverse Genotypes of Rice (Oryza sativa).

Rice is a critically important food source but yields worldwide are vulnerable to periods of drought. We exposed eight genotypes of upland and lowland rice (Oryza sativa L. ssp. japonica and indica) to drought stress at the late vegetative stage, and harvested leaves for label-free shotgun proteomics. Gene ontology analysis was used to identify common drought-responsive proteins in vegetative tissues, and leaf proteins that are unique to individual genotypes, suggesting diversity in the metabolic responses to drought. Eight proteins were found to be induced in response to drought stress in all eight genotypes. A total of 213 proteins were identified in a single genotype, 83 of which were increased in abundance in response to drought stress. In total, 10 of these 83 proteins were of a largely uncharacterized function, making them candidates for functional analysis and potential biomarkers for drought tolerance.

Proteomic and metabolomic analysis of desiccation tolerance in wheat young seedlings.

Young wheat seedlings are desiccation tolerant and have the capacity to withstand long dehydration period. In this study, we characterized the proteome and metabolome of wheat seedlings during desiccation and after recovery. Functional classification of differentially identified proteins revealed dynamic changes in the number and abundance of proteins observed during stress and recovery. Desiccation resulted in a decline in the abundance of proteins associated with photosynthesis and carbohydrate reserves, along with an increase in the presence of proteins associated with stress and defense response, such as peroxiredoxins and antioxidant enzymes. Following recovery, the abundance of stress-responsive proteins returned either partially or completely to their baseline level, confirming their importance to the seedling's desiccation response. Furthermore, proteins involved in carbohydrate metabolism, as well as fructose-bisphosphate aldolase and fructokinase-2 and phosphorylated metabolites as the substrate or the end-product, showed the inverse pattern during desiccation and after re-watering. This may reflect the fact that plants maintained energy supply during stress to protect seedlings from further damage, and for use in subsequent recovery after rewatering period. This study provides novel insights into the molecular mechanisms underlying the desiccation tolerance of wheat seedlings, and paves the way for more detailed molecular analysis of this remarkable phenomenon.

Amyloid ß Induces Early Changes in the Ribosomal Machinery, Cytoskeletal Organization and Oxidative Phosphorylation in Retinal Photoreceptor Cells.

Amyloid ß (Aß) accumulation and its aggregation is characteristic molecular feature of the development of Alzheimer's disease (AD). More recently, Aß has been suggested to be associated with retinal pathology associated with AD, glaucoma and drusen deposits in age related macular degeneration (AMD). In this study, we investigated the proteins and biochemical networks that are affected by Aß in the 661 W photoreceptor cells in culture. Time and dose dependent effects of Aß on the photoreceptor cells were determined utilizing tandem mass tag (TMT) labeling-based quantitative mass-spectrometric approach. Bioinformatic analysis of the data revealed concentration and time dependent effects of the Aß peptide stimulation on various key biochemical pathways that might be involved in mediating the toxicity effects of the peptide. We identified increased Tau phosphorylation, GSK3ß dysregulation and reduced cell viability in cells treated with Aß in a dose and time dependent manner. This study has delineated for the first-time molecular networks in photoreceptor cells that are impacted early upon Aß treatment and contrasted the findings with a longer-term treatment effect. Proteins associated with ribosomal machinery homeostasis, mitochondrial function and cytoskeletal organization were affected in the initial stages of Aß exposure, which may provide key insights into AD effects on the photoreceptors and specific molecular changes induced by Aß peptide.

Analytical performance of nano-LC-SRM using nondepleted human plasma over an 18-month period.

A standardized procedure for label-free nano-LC-SRM analysis of 32 high-medium abundance proteins from nondepleted human plasma was established and SRM data were acquired on 45 separate days for a control sample that was independently prepared on 39 distinct dates over an 18-month period (542 days). This case study enabled us to assess quantitative variance associated with nano-LC-SRM plasma analysis, mimicking experimental conditions that would be experienced with clinical trial biomarker studies. We assessed sample preparation variability attributed to different technicians and sample storage stability. Instrument performance varied over the 18-month period requiring ion path cleaning, so we assessed the impact of declining performance on specific peptide ion sensitivity and evaluated how various data normalization strategies could compensate for these changes. Our analysis demonstrated that while sample preparation was the main contributor for data variances when MS data were acquired within days, variability in SRM sensitivity was a far greater source of variance when data were acquired over a long period. The overall median multiplexed assay CV was 13% over the 18-month period. This case study is illustrative of large-scale plasma biomarker studies using nano-LC-SRM over extended periods and highlights aspects of bioanalysis that require careful attention to ensure reliable quantitation.

Phosphoproteomic Analysis of Cell-Based Resistance to BRAF Inhibitor Therapy in Melanoma.

The treatment of melanoma by targeted inhibition of the mutated kinase BRAF with small molecules only temporarily suppresses metastatic disease. In the face of chemical inhibition tumor plasticity, both innate and adaptive, promotes survival through the biochemical and genetic reconfiguration of cellular pathways that can engage proliferative and migratory systems. To investigate this process, high-resolution mass spectrometry was used to characterize the phosphoproteome of this transition in vitro. A simple and accurate, label-free quantitative method was used to localize and quantitate thousands of phosphorylation events. We also correlated changes in the phosphoproteome with the proteome to more accurately determine changes in the activity of regulatory kinases determined by kinase landscape profiling. The abundance of phosphopeptides with sites that function in cytoskeletal regulation, GTP/GDP exchange, protein kinase C, IGF signaling, and melanosome maturation were highly divergent after transition to a drug resistant phenotype.

Proteomics study reveals the molecular mechanisms underlying water stress tolerance induced by Piriformospora indica in barley.

Piriformospora indica is a mutualistic root endophytic fungus, which transfers several benefits to hosts including enhance plant growth and increase yield under both normal and stress conditions. It has been shown that P. indica root-colonization enhances water stress tolerance based on general and non-specific plant-species mechanism. To better understand the molecular mechanism of P. indica-mediated drought stress tolerance, we designed a set of comparative experiments to study the impact of P. indica on barely plants cultivar "Golden Promise" grown under different drought levels [Filed capacity (F.C.) and 25% F.C.]. P. indica enhanced root and shoot biomass of colonized plants under both well-watered and water-deficit conditions. Proteome analysis of P. indica-colonized barley leaves under well-treated and water-deficit conditions resulted in detection of 726 reproducibly protein spots. Mass spectrometry analysis resulted in the identification of 45 differentially accumulated proteins involved in photosynthesis, reactive oxygen scavenging, metabolisms, signal transduction, and plant defense responses. Interestingly, P. indica increased the level of proteins involved in photosynthesis, antioxidative defense system and energy transport. We propose that P. indica-mediated drought stress tolerance in barely is through photosynthesis stimulation, energy releasing and enhanced antioxidative capacity in colonized plants. BIOLOGICAL SIGNIFICANCE: Plant mutualistic symbionts offer long-term abiotic stress tolerance through the host adaptation to environmental stress. There have been a few published proteomic studies of plant symbionts to drought, and this is thought to be the first proteomic analysis, demonstrating the impact of endophyte on barley plant under drought stress. For some of identified proteins like TCTP and PCNA, a connection to physiological function in plants is novel, and can be the best candidates for sources of drought tolerance in future studies.