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Abstract Background Transcript profiling of closely related species provides a means for identifying genes potentially important in species diversification. However, the predictive value of transcript profiling for inferring downstream-physiological processes has been unclear. In the present study we use shotgun proteomics to validate inferences from microarray studies regarding physiological differences in three Pachycladon species. We compare transcript and protein profiling and evaluate their predictive value for inferring glucosinolate chemotypes characteristic of these species. Results Evidence from heterologous microarrays and shotgun proteomics revealed differential expression of genes involved in glucosinolate hydrolysis (myrosinase-associated proteins) and biosynthesis (methylthioalkylmalate isomerase and dehydrogenase), the interconversion of carbon dioxide and bicarbonate (carbonic anhydrases), water use efficiency (ascorbate peroxidase, 2 cys peroxiredoxin, 20 kDa chloroplastic chaperonin, mitochondrial succinyl CoA ligase) and others (glutathione-S-transferase, serine racemase, vegetative storage proteins, genes related to translation and photosynthesis). Differences in glucosinolate hydrolysis products were directly confirmed. Overall, prediction of protein abundances from transcript profiles was stronger than prediction of transcript abundance from protein profiles. Protein profiles also proved to be more accurate predictors of glucosinolate profiles than transcript profiles. The similarity of species profiles for both transcripts and proteins reflected previously inferred phylogenetic relationships while glucosinolate chemotypes did not. Conclusions We have used transcript and protein profiling to predict physiological processes that evolved differently during diversification of three Pachycladon species. This approach has also identified candidate genes potentially important in adaptation, which are now the focus of ongoing study. Our results indicate that protein profiling provides a valuable tool for validating transcript profiles in studies of adaptive divergence.

Western flower thrips, Frankliniella occidentalis (Thysanoptera: Thripidae) is an invasive agricultural pest with developed resistance to abamectin in some strains due to frequent treatment with the pesticide. In this study, we examined differentially expressed proteins (DEPs) between abamectin-resistant (Aba R ; under abamectin selective pressure) and susceptible strains (Aba S ; without abamectin selective pressure) of F. occidentalis. Proteins were isolated from second instar larvae of both strains and separated via two-dimensional polyacrylamide gel electrophoresis. Nano-flow liquid chromatography-tandem mass spectrometry identified selected protein spot features. From 70 DEPs, 43 spot features were identified: A total of 23 showed an increase in abundance, and 20 were down-regulated in response to abamectin pressure. The enzymatic and structural proteins were classified into the functional groups of macromolecular metabolisms, signaling and cellular processes, immune system, genetic information processing, and exoskeleton-related proteins. The up-regulation of exoskeleton-related proteins may contribute to forming a thicker cuticle, potentially hindering abamectin penetration, which is an interesting finding that needs further investigation. Two novel proteins, triacylglycerol lipase and cuticle protein CPF 2, were only expressed in Aba R . This work provides insights into abamectin resistance mechanisms in F. occidentalis, which will provide important information for developing insecticide resistance management approaches for this pest., (© 2025 The Author(s). ELECTROPHORESIS published by Wiley‐VCH GmbH.)

Identifying proteins from living organisms helps us understand the biological functions of cells, discover new molecular mechanisms, and interrogate known mechanisms for improving our understanding. For a comprehensive understanding of cellular functions, identifying the whole protein content, or proteome, of a cell is desirable but challenging. Here, we describe in detail two methods of proteome fractionation at either the protein (SDS-PAGE) or peptide (high-pH reversed-phase fractionation) level, which can be used to maximize the identification of proteins from complex biological samples. We apply these methods to two different sample types commonly processed in our laboratory to demonstrate the versatility of these protocols. These methods produce many more peptide identifications when compared to conventional single-shot analysis and can also be used in combination to generate larger complementary datasets with greater depth of proteome coverage., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The remobilization of stored assimilates from stems to seeds plays a pivotal role in augmenting barley yield, particularly under water stress conditions. This study examines the molecular mechanisms underlying stem reserve utilization by conducting a comparative analysis of the proteome and metabolome across three barley contrasting genotypes: Yousef, Morocco, and PBYT17. Evaluations were performed at 21 and 28 days after anthesis (DAA) under both water stress and control conditions. The results indicate that the Yousef genotype exhibits superior remobilization of stem reserves, thereby demonstrating its potential to thrive even in adverse environmental conditions. Utilizing advanced quantitative proteomics and targeted metabolomics, this investigation identified a significant number of metabolites and proteins exhibiting differential accumulation across the genotypes. Specifically, 17 metabolites and 1580 proteins were catalogued, highlighting the intricate biochemical responses to water stress. Noteworthy enzymes such as sucrose synthase, inositol monophosphatase 3, and galactokinase were found to be closely associated with remobilization efficiency. In the drought-tolerant genotype, these enzymes maintained stable levels, in stark contrast to the decline observed in the susceptible genotype. This stability is crucial for promoting seed development through ascorbic acid synthesis and for mitigating oxidative stress, which is exacerbated by drought conditions. The elevated levels of certain metabolites, including glucose 6-phosphate, and UDP-glucose, in the drought-tolerant genotype suggest a robust mechanism for maintaining signalling molecules for carbon availability, which is then instrumental in regulating plant growth and seed size development. The findings from this study strongly imply that the drought-tolerant genotype, through enhanced antioxidant capacity, can effectively produce energy-rich storage compounds, thereby optimizing carbon allocation under water stress. Such insights are invaluable for future breeding strategies aimed at improving barley resilience in the face of climate variability., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

This experiment was carried out to provide a comprehensive insight into the protein activities involved in dormancy establishment in seeds of common cocklebur (Xanthium strumarium), an annual plant with two dimorphic seeds contained in one casing known as a burr. These consist of a smaller dormant seed and a larger non-dormant seed. The proteome profile was compared between developing dormant and non-dormant seeds of Xanthium strumarium at five consecutive stages including three, 10, 20, 30, and 45 days after burr emergence (stages 1 to 5). We identified 6524 proteins in total, and approximately 3.6% of these were differentially abundant proteins (DAPs) between the two seed types. Both seed types showed fundamental changes in developmental programs during the examined stages. More than 38% of all DAPs were observed at the first stage, supporting the importance of the early developmental stage in seed fate determination. The detected DAPs at stage 1 were mainly associated with the cell division phase, which showed a delay in the dormant seeds. Over-representation of proteins responsible for cell wall biosynthesis, cytokinesis, and seed development were detected for non-dormant seeds at the first stage, while dormancy-associated proteins showed less abundance. Stage 3 was the critical stage for switching processes toward seed maturation and abscisic acid (ABA) signaling. Interestingly, higher abundance proteins in the mature non-dormant seed were mainly involved in the facilitation of seed germination. Taken together, the temporal pattern of the accumulated proteins in developing dormant seeds demonstrated a delay in the initiation of active cell division, enriched response to ABA, and defects in seed maturation. Moreover, stored proteins in the mature dormant seed delay germination but not dormancy induction. Finally, our results suggest that dormancy may be established at a stage of seed development earlier than previously thought., (© 2024 Scandinavian Plant Physiology Society.)

Characterising inner ear disorders represents a significant challenge due to a lack of reliable experimental procedures and identified biomarkers. It is also difficult to access the complex microenvironments of the inner ear and investigate specific pathological indicators through conventional techniques. Omics technologies have the potential to play a vital role in revolutionising the diagnosis of ear disorders by providing a comprehensive understanding of biological systems at various molecular levels. These approaches reveal valuable information about biomolecular signatures within the cochlear tissue or fluids such as the perilymphatic and endolymphatic fluid. Proteomics identifies changes in protein abundance, while metabolomics explores metabolic products and pathways, aiding the characterisation and early diagnosis of diseases. Although there are different methods for identifying and quantifying biomolecules, mass spectrometry, as part of proteomics and metabolomics analysis, could be utilised as an effective instrument for understanding different inner ear disorders. This study aims to review the literature on the application of proteomic and metabolomic approaches by specifically focusing on Meniere's disease, ototoxicity, noise-induced hearing loss, and vestibular schwannoma. Determining potential protein and metabolite biomarkers may be helpful for the diagnosis and treatment of inner ear problems.

Abstract: The aggregation of amyloid-beta peptide and tau protein dysregulation are implicated to play key roles in Alzheimer's disease pathogenesis and are considered the main pathological hallmarks of this devastating disease. Physiologically, these two proteins are produced and expressed within the normal human body. However, under pathological conditions, abnormal expression, post-translational modifications, conformational changes, and truncation can make these proteins prone to aggregation, triggering specific disease-related cascades. Recent studies have indicated associations between aberrant behavior of amyloid-beta and tau proteins and various neurological diseases, such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis, as well as retinal neurodegenerative diseases like Glaucoma and age-related macular degeneration. Additionally, these proteins have been linked to cardiovascular disease, cancer, traumatic brain injury, and diabetes, which are all leading causes of morbidity and mortality. In this comprehensive review, we provide an overview of the connections between amyloid-beta and tau proteins and a spectrum of disorders., (Copyright © 2024 Copyright: © 2024 Neural Regeneration Research.)

Next-generation sequencing demands high-quality nucleic acid, yet isolating DNA and RNA is often challenging, particularly from plant tissues. Despite advances in developing various kits and reagents, these products are tailored to isolation of nucleic acid from model plant tissues. Here we introduce a universal lysis buffer to separate nucleic acid from various plant species, including recalcitrant plants, to facilitate molecular analyses, such as quantitative PCR (qPCR), transcriptomics, and whole-genome sequencing (WGS). The protocol is a modification of the original CTAB methods, which leads to nucleic acid isolation from many plant species, including monocots and eudicots. The lysis buffer consists of hexadecyltrimethylammonium bromide (CTAB), sodium chloride (NaCl), Tris base, ethylenediaminetetraacetic acid (EDTA) and β-mercaptoethanol (βME). The modified CTAB method enables the isolation of nucleic acid from small amounts of plant tissues (e.g., 15-100 mg) in a timely manner, which is well-suited for a large number of samples and also when adequate sample collection is a limiting factor. The protocol isolates not only DNA from various plant species but also RNA. This makes it highly effective for molecular analyses compared to previously described CTAB methods optimised for DNA isolation. The appropriate concentration of the components enables high-quality DNA and RNA isolation from plant tissues simultaneously. Additionally, this protocol is compatible with commercially available columns. For DNA and RNA to be qualified for next-generation sequencing platforms, the protocol is supplemented with columns to purify either DNA or RNA from the same tissue to meet high standards for sequencing analyses. This protocol provides an ideal approach to overcome potential obstacles in isolating high-quality DNA or RNA from a wide range of plant species for downstream molecular analysis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Masoomi-Aladizgeh et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Cannabis has been used historically for both medicinal and recreational purposes, with the most notable cannabinoids being cannabidiol (CBD) and tetrahydrocannabinol (THC). Although their therapeutic effects have been well studied and their recreational use is highly debated, the underlying mechanisms of their biological effects remain poorly defined. In this study, we use isobaric tag-based sample multiplexed proteome profiling to investigate protein abundance differences in the human neuroblastoma SH-SY5Y cell line treated with CBD and THC. We identified significantly regulated proteins by each treatment and performed a pathway classification and associated protein-protein interaction analysis. Our findings suggest that these treatments may lead to mitochondrial dysfunction and induce endoplasmic reticulum stress. These data can potentially be interrogated further to investigate the potential role of CBD and THC in various biological and disease contexts, providing a foundation for future studies.

Half of the world's population depends on rice plant cultivation, yet environmental stresses continue to substantially impact the production of one of our most valuable staple foods. The aim of this study was to investigate the changes in the transcriptome of the IAC1131 rice genotype when exposed to a suite of multiple abiotic stresses, either with or without pre-treatment with the plant hormone ABA (Abscisic acid). Four groups of IAC1131 rice plants were grown including control plants incubated with ABA, non-ABA-incubated control plants, stressed plants incubated with ABA, and non-ABA-incubated stressed plants, with leaf samples harvested after 0 days (control) and 4 days (stressed). We found that high concentrations of ABA applied exogenously to the control plants under normal conditions did not alter the IAC1131 transcriptome profile significantly. The observed changes in the transcriptome of the IAC1131 plants in response to multiple abiotic stress were made even more pronounced by ABA pre-treatment, which induced the upregulation of a significant number of additional genes. Although ABA application impacted the plant transcriptome, multiple abiotic stress was the dominant factor in modifying gene expression in the IAC1131 plants. Exogenous ABA application may mitigate the effects of stress through ABA-dependent signalling pathways related to biological photosynthesis functions. Pre-treatment with ABA alters the photosynthesis function negatively by reducing stomatal conductance, therefore helping plants to conserve the energy required for survival under unfavourable environmental conditions.

As the first in-person Asia Oceania Human Proteomics Organization (AOHUPO) congress since 2018, the 11th AOHUPO congress was an opportune time for the research community to reconnect and to renew friendships after the long period of restricted travel due to the global pandemic. Moreover, this congress was a great opportunity for the many AO regional proteomics and mass spectrometry scientists to meet in Singapore to exchange ideas and to present their latest findings. Cohosted by the Singapore Society for Mass Spectrometry and the Malaysian Proteomics Society and held in conjunction with the seventh Asia Oceania Agricultural Proteomics Organization Congress and Singapore Society for Mass Spectrometry 2023, the meeting featured both human and agricultural proteomics. Over five hundred scientists from the AO region converged on the MAX Atria @ Singapore EXPO, Changi, Singapore from May 8 to 10 for the main congress. The diverse program was made up of 64 invited speakers and panellists for seven plenary lectures, 27 concurrent symposia, precongress and postcongress workshops, and 174 poster presentations. The AOHUPO society were able to celebrate not only their 20th anniversary but also the outstanding academic research from biological and agricultural proteomics and related 'omics fields being conducted across the Asia-Oceania region., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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 (Aβ) and tau pathology, inflammation, vascular integrity, glucose metabolism and vascular endothelial growth factor (VEGF) signaling is also discussed.

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.

Photoreceptor cells are highly susceptible to oxidative-stress-induced damage due to their high metabolic rate. Oxidative stress plays a key role in driving pathological events in several different ocular diseases, which lead to retinal degeneration and ultimately blindness. A growing number of studies have been performed to understand downstream events caused by ROS induced oxidative stress in photoreceptor cells; however, the underlying mechanisms of ROS toxicity are not fully understood. To shed light on ROS induced downstream pathological events, we employed a tandem mass tag (TMT) labelling-based quantitative mass-spectrometric approach to determine proteome changes in 661W photoreceptor cells following oxidative stress induction via the application of different concentrations of H 2 O 2 at different time points. Overall, 5920 proteins were identified and quantified, and 450 differentially expressed proteins (DEPs) were identified, which were altered in a dose and time dependent manner in all treatment groups compared to the control group. These proteins were involved in several biological pathways, including spliceosome and ribosome response, activated glutathione metabolism, decreased ECM-receptor interaction, oxidative phosphorylation, abnormally regulated lysosome, apoptosis, and ribosome biogenesis. Our results highlighted ECM receptor interaction, oxidative phosphorylation and spliceosome pathways as the major targets of oxidative stress that might mediate vascular dysfunction and cellular senescence.

Food safety of staple crops such as rice is of global concern and is at the top of the policy agenda worldwide. Abiotic stresses are one of the main limitations to optimizing yields for sustainability, food security and food safety. We analyzed proteome changes in Oryza sativa cv. Nipponbare in response to five adverse abiotic treatments, including three levels of drought (mild, moderate, and severe), soil salinization, and non-optimal temperatures. All treatments had modest, negative effects on plant growth, enabling us to identify proteins that were common to all stresses, or unique to one. More than 75% of the total of differentially abundant proteins in response to abiotic stresses were specific to individual stresses, while fewer than 5% of stress-induced proteins were shared across all abiotic constraints. Stress-specific and non-specific stress-responsive proteins identified were categorized in terms of core biological processes, molecular functions, and cellular localization.

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., (© 2022 The Authors. Proteomics published by Wiley-VCH GmbH.)

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., (© 2022 The Authors. Proteomics published by Wiley-VCH GmbH.)

Paleoproteomics typically involves the destructive sampling of precious bioarchaeological materials. This analysis aims to investigate the proteins identifiable via nanoLC-MS/MS from highly degraded 26th Dynasty Egyptian mummified human remains (NMR.29.1-8) after non-destructive sampling with commercially available dermatology-grade skin sampling tape strips. A collection of cranial and other bone fragments were sampled with the tape strips then subsequently analysed using a shotgun proteomics approach. The number of proteins identified using this method ranged from 18 to 437 at a peptide FDR of <1%. Deamidation ratios were assessed using an in-house R script, with asparagine deamidation averaging ∼20-30% and glutamine deamidation averaging ∼15-25%., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s). Published by Elsevier Inc.)

Heat stress specifically affects fertility by impairing pollen viability but cotton wild relatives successfully reproduce in hot savannas where they evolved. An Australian arid-zone cotton (Gossypium robinsonii) was exposed to heat events during pollen development then mature pollen was subjected to deep proteomic analysis using 57 023 predicted genes from a genomic database we assembled for the same species. Three stages of pollen development, including tetrads (TEs), uninucleate microspores (UNs) and binucleate microspores (BNs) were exposed to 36°C or 40°C for 5 days and the resulting mature pollen was collected at anthesis (p-TE, p-UN and p-BN, respectively). Using the sequential windowed acquisition of all theoretical mass spectra proteomic analysis, 2704 proteins were identified and quantified across all pollen samples analysed. Proteins predominantly decreased in abundance at all stages in response to heat, particularly after exposure of TEs to 40°C. Functional enrichment analyses demonstrated that extreme heat increased the abundance of proteins that contributed to increased messenger RNA splicing via spliceosome, initiation of cytoplasmic translation and protein refolding in p-TE40. However, other functional categories that contributed to intercellular transport were inhibited in p-TE40, linked potentially to Rab proteins. We ascribe the resilience of reproductive processes in G. robinsonii at temperatures up to 40°C, relative to commercial cotton, to a targeted reduction in protein transport., (© 2022 John Wiley & Sons Ltd.)

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.

Root system architecture (RSA) is an important agronomic trait with vital roles in plant productivity under water stress conditions. A deep and branched root system may help plants to avoid water stress by enabling them to acquire more water and nutrient resources. Nevertheless, our knowledge of the genetics and molecular control mechanisms of RSA is still relatively limited. In this study, we analyzed the transcriptome response of root tips to water stress in two well-known genotypes of rice: IR64, a high-yielding lowland genotype, which represents a drought-susceptible and shallow-rooting genotype; and Azucena, a traditional, upland, drought-tolerant and deep-rooting genotype. We collected samples from three zones (Z) of root tip: two consecutive 5 mm sections (Z1 and Z2) and the following next 10 mm section (Z3), which mainly includes meristematic and maturation regions. Our results showed that Z1 of Azucena was enriched for genes involved in cell cycle and division and root growth and development whereas in IR64 root, responses to oxidative stress were strongly enriched. While the expansion of the lateral root system was used as a strategy by both genotypes when facing water shortage, it was more pronounced in Azucena. Our results also suggested that by enhancing meristematic cell wall thickening for insulation purposes as a means of confronting stress, the sensitive IR64 genotype may have reduced its capacity for root elongation to extract water from deeper layers of the soil. Furthermore, several members of gene families such as NAC , AP2/ERF , AUX/IAA , EXPANSIN , WRKY , and MYB emerged as main players in RSA and drought adaptation. We also found that HSP and HSF gene families participated in oxidative stress inhibition in IR64 root tip. Meta-quantitative trait loci (QTL) analysis revealed that 288 differentially expressed genes were colocalized with RSA QTLs previously reported under drought and normal conditions. This finding warrants further research into their possible roles in drought adaptation. Overall, our analyses presented several major molecular differences between Azucena and IR64, which may partly explain their differential root growth responses to water stress. It appears that Azucena avoided water stress through enhancing growth and root exploration to access water, whereas IR64 might mainly rely on cell insulation to maintain water and antioxidant system to withstand stress. We identified a large number of novel RSA and drought associated candidate genes, which should encourage further exploration of their potential to enhance drought adaptation in rice., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Abdirad, Ghaffari, Majd, Irian, Soleymaniniya, Daryani, Koobaz, Shobbar, Farsad, Yazdanpanah, Sadri, Mirzaei, Ghorbanzadeh, Kazemi, Hadidi, Haynes and Salekdeh.)

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.

Reproductive performance in plants is impaired as maximum temperatures consistently approach 40°C. However, the timing of heatwaves critically affects their impact. We studied the molecular responses during pollen maturation in cotton to investigate the vulnerability to high temperature. Tetrads (TEs), uninucleate and binucleate microspores, and mature pollen were subjected to SWATH-MS and RNA-seq analyses after exposure to 38/28°C (day/night) for 5 days. The results indicated that molecular signatures were downregulated progressively in response to heat during pollen development. This was even more evident in leaves, where three-quarters of differentially changed proteins decreased in abundance during heat. Functional analysis showed that translation of genes increased in TEs after exposure to heat; however, the reverse pattern was observed in mature pollen and leaves. For example, proteins involved in transport were highly abundant in TEs whereas in later stages of pollen formation and leaves, heat suppressed synthesis of proteins involved in cell-to-cell communication. Moreover, a large number of heat shock proteins were identified in heat-affected TEs, but these proteins were less abundant in mature pollen and leaves. We speculate that the sensitivity of TE cells to heat is related to high rates of translation targeted to pathways that might not be essential for thermotolerance. Molecular signatures during stages of pollen development after heatwaves could provide markers for future genetic improvement., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

The preconditioning of human sperm with sublethal nitrosative stress before cryopreservation can potentially improve the thawed sperm quality. However, the underlying mechanisms behind this protective strategy are not entirely understood. We compared the cryosurvival of human sperm exposed to 0.01 μM nitric oxide (NO) throughout the cryopreservation and used multiplexed quantitative proteomics approach to identify changes in the proteome profile of preconditioned sperm cells. Semen samples were obtained from 30 normospermia donors and then each sample was divided into three equal parts: fresh (F), frozen-control (C), and frozen exposed to nitric oxide (NO). The sperm undergoing mild sublethal stress showed higher values for motility and viability compared to the frozen control sperm. Moreover, out of 2912 identified proteins, 248 proteins were detected as differentially abundant proteins (DAPs) between cryopreserved groups and fresh group (F) (p < 0.05). Gene ontology (GO) analysis of differentially abundant proteins indicated that the abundance of proteins associated with glycolysis, gluconeogenesis, and fertilization processes was reduced while oxidative phosphorylation pathway was increased in abundance in cryopreserved sperm compared to the fresh sperm. Moreover, redox protein such as thioredoxin 17 was increased in abundance in the NO group compared to the control freezing group. Therefore, the pre-conditioning of sperm prior to cryopreservation may play an important role in maintaining the redox balance in mitochondria of sperm after freezing. Overall, our results indicate that arylsulfatase A (ARSA), serine protease 37 (PRSS37), and sperm surface protein (SP17) may potentially serve as protein biomarkers associated with screening the fertilization potential of the thawed sperm., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The scarcity of freshwater is an increasing concern in flood-irrigated rice, whilst excessive use of nitrogen fertilizers is costly and contributes to environmental pollution. To co-ordinate growth adaptation under prolonged exposure to limited water or excess nitrogen supply, plants employ complex systems for signalling and regulation of metabolic processes. There is limited information on the involvement of one of the most important post-translational modifications (PTMs), protein phosphorylation, in plant adaptation to long-term changes in resource supply. Oryza sativa cv. Nipponbare was grown under two regimes of nitrogen from the time of germination to final harvest. Twenty-five days after germination, water was withheld from half the pots in each nitrogen treatment and low water supply continued for an additional 26 days, while the remaining pots were well watered. Leaves from all four groups of plants were harvested after 51 days in order to test whether phosphorylation of leaf proteins responded to prior abiotic stress events. The dominant impact of these resources is exerted in leaves, where PTMs have been predicted to occur. Proteins were extracted and phosphopeptides were analysed by nanoLC-MS/MS analysis, coupled with label-free quantitation. Water and nitrogen regimes triggered extensive changes in phosphorylation of proteins involved in membrane transport, such as the aquaporin OsPIP2-6, a water channel protein. Our study reveals phosphorylation of several peptides belonging to proteins involved in RNA-processing and carbohydrate metabolism, suggesting that phosphorylation events regulate the signalling cascades that are required to optimize plant response to resource supply.

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.

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., (© 2020 John Wiley & Sons Ltd.)

Different parts of the brain are affected distinctively in various stages of the Alzheimer's disease (AD) pathogenesis. Identifying the biochemical changes in specific brain regions is key to comprehend the neuropathological mechanisms in early pre-symptomatic phases of AD. Quantitative proteomics profiling of four distinct areas of the brain of young APP/PS1 mouse model of AD was performed followed by biochemical pathway enrichment analysis. Findings revealed fundamental compositional and functional shifts even in the early stages of the disease. This novel study highlights unique proteome and biochemical pathway alterations in specific regions of the brain that underlie the early stages of AD pathology and will provide a framework for future longitudinal studies. The proteomics data were deposited into the ProteomeXchange Consortium via PRIDE with the identifier PXD019192., (© 2021 Wiley-VCH GmbH.)

Mitochondrial dysfunction is involved in Alzheimer's disease (AD) pathogenesis. Mitochondria have their own genetic material; however, most of their proteins (∼99%) are synthesized as precursors on cytosolic ribosomes, and then imported into the mitochondria. Therefore, exploring proteome changes in these organelles can yield valuable information and shed light on the molecular mechanisms underlying mitochondrial dysfunction in AD. Here, we review AD-associated mitochondrial changes including the effects of amyloid beta and tau protein accumulation on the mitochondrial proteome. We also discuss the relationship of ApoE genetic polymorphism with mitochondrial changes, and present a meta-analysis of various differentially expressed proteins in the mitochondria in AD. Area covered : Proteomics studies and their contribution to our understanding of mitochondrial dysfunction in AD pathogenesis. Expert opinion : Proteomics has proven to be an efficient tool to uncover various aspects of this complex organelle, which will broaden our understanding of mitochondrial dysfunction in AD. Evidently, mitochondrial dysfunction is an early biochemical event that might play a central role in driving AD pathogenesis.

Shotgun proteomics is a high-throughput technology which has been developed with the aim of investigating the maximum number of proteins in cells in a given experiment. However, protein discovery and data generation vary in depth and coverage when different technical strategies are selected. In this study, three different sample preparation approaches, and peptide or protein fractionation methods, were applied to identify and quantify proteins from log-phase yeast lysate: sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), filter-aided sample preparation coupled with gas phase fractionation (FASP-GPF), and FASP - high pH reversed phase fractionation (HpH). Fractions were initially analyzed and compared using nanoflow liquid chromatography - tandem mass spectrometry (nanoLC-MS/MS) employing data dependent acquisition on a linear ion trap instrument. The number of fractions and analytical replicates was adjusted so that each experiment used a similar amount of mass spectrometric instrument time. A second set of experiments was performed, comparing FASP-GPF, SDS-PAGE and FASP-HpH using a Q Exactive Orbitrap mass spectrometer. Compared with results from the linear ion trap mass spectrometer, the use of a Q Exactive Orbitrap mass spectrometer enabled a substantial increase in protein identifications, and an even greater increase in peptide identifications. This shows that the main advantage of using the higher resolution instrument is in increased proteome coverage. A total of 1035, 1357 and 2134 proteins were separately identified by FASP-GPF, SDS-PAGE and FASP-HpH. Combining results from the Orbitrap experiments, there were a total of 2269 proteins found, with 94% of them identified using the FASP-HpH method. Therefore, the FASP-HpH method is the optimal choice among these approaches, when applied to this type of sample., (Copyright © 2020 Elsevier B.V. All rights reserved.)

To facilitate selective breeding of polyphenol-rich peanuts, we looked for mass spectrometry-based proteomic evidence, investigating a subset of recombinant inbred lines (RILs) developed by the Australian peanut breeding program. To do this, we used label-free shotgun proteomics for protein and peptide quantitation, statistically analyzed normalized spectral abundance factors using R-package, as well as assayed important antioxidants. Results revealed statistically significant protein expression changes in 82 proteins classified between high or low polyphenols expressing RILs. Metabolic changes in polyphenol-rich RIL p27-362 point towards increased enzymatic breakdown of sugars and phenylalanine biosynthesis. The study revealed phenylpropanoid pathway overexpression resulting in increased polyphenols biosynthesis. Overexpression of antioxidant enzymes such as catalase, by 73.4 fold was also observed. A strong metabolic correlation exists with the observed phenotypic traits. Peanut RIL p27-362 presents a superior nutritional composition with antioxidant-rich peanut phenotype and could yield commercial profits. Data are available via ProteomeXchange with identifierPXD015493., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Current evidence suggests that exposure to chronically induced intraocular pressure (IOP) leads to neurodegenerative changes in the inner retina. This study aimed to determine retinal proteomic alterations in a rat model of glaucoma and compared findings with human retinal proteomics changes in glaucoma reported previously. We developed an experimental glaucoma rat model by subjecting the rats to increased IOP (9.3 ± 0.1 vs 20.8 ± 1.6 mm Hg) by weekly microbead injections into the eye (8 weeks). The retinal tissues were harvested from control and glaucomatous eyes and protein expression changes analysed using a multiplexed quantitative proteomics approach (TMT-MS3). Immunofluorescence was performed for selected protein markers for data validation. Our study identified 4304 proteins in the rat retinas. Out of these, 139 proteins were downregulated (≤0.83) while the expression of 109 proteins was upregulated (≥1.2-fold change) under glaucoma conditions (P ≤ .05). Computational analysis revealed reduced expression of proteins associated with glutathione metabolism, mitochondrial dysfunction/oxidative phosphorylation, cytoskeleton, and actin filament organisation, along with increased expression of proteins in coagulation cascade, apoptosis, oxidative stress, and RNA processing. Further functional network analysis highlighted the differential modulation of nuclear receptor signalling, cellular survival, protein synthesis, transport, and cellular assembly pathways. Alterations in crystallin family, glutathione metabolism, and mitochondrial dysfunction associated proteins shared similarities between the animal model of glaucoma and the human disease condition. In contrast, the activation of the classical complement pathway and upregulation of cholesterol transport proteins were exclusive to human glaucoma. These findings provide insights into the neurodegenerative mechanisms that are specifically affected in the retina in response to chronically elevated IOP., (© 2020 Wiley Periodicals LLC.)

Extracellular deposits of the amyloid-beta peptide (Aβ) 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 Aβ neurotoxicity is considered to play an important role. However, the molecular mechanisms underlying Aβ toxicity that lead to neurodegeneration are not clearly established. Here we have elucidated the molecular pathways and networks which are impacted by Aβ in neurons using SH-SY5Y human neuroblastoma cells as a model. These cells were treated with Aβ 1-42 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 Aβ 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. Aβ 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 Aβ on neurons, with implications for better understanding the impacts of Aβ on early neurodegeneration in Alzheimer's disease pathology., Competing Interests: None

PeptideWitch is a python-based web module that introduces several key graphical and technical improvements to the Scrappy software platform, which is designed for label-free quantitative shotgun proteomics analysis using normalised spectral abundance factors. The program inputs are low stringency protein identification lists output from peptide-to-spectrum matching search engines for 'control' and 'treated' samples. Through a combination of spectral count summation and inner joins, PeptideWitch processes low stringency data, and outputs high stringency data that are suitable for downstream quantitation. Data quality metrics are generated, and a series of statistical analyses and graphical representations are presented, aimed at defining and presenting the difference between the two sample proteomes.

Drought often compromises yield in non-irrigated crops such as rainfed rice, imperiling the communities that depend upon it as a primary food source. In this study, two cultivated species ( Oryza sativa cv. Nipponbare and Oryza glaberrima cv. CG14) and an endemic, perennial Australian wild species ( Oryza australiensis ) were grown in soil at 40% field capacity for 7 d (drought). The hypothesis was that the natural tolerance of O. australiensis to erratic water supply would be reflected in a unique proteomic profile. Leaves from droughted plants and well-watered controls were harvested for label-free quantitative shotgun proteomics. Physiological and gene ontology analysis confirmed that O. australiensis responded uniquely to drought, with superior leaf water status and enhanced levels of photosynthetic proteins. Distinctive patterns of protein accumulation in drought were observed across the O. australiensis proteome. Photosynthetic and stress-response proteins were more abundant in drought-affected O. glaberrima than O. sativa , and were further enriched in O. australiensis . In contrast, the level of accumulation of photosynthetic proteins decreased when O. sativa underwent drought, while a narrower range of stress-responsive proteins showed increased levels of accumulation. Distinctive proteomic profiles and the accumulated levels of individual proteins with specific functions in response to drought in O. australiensis indicate the importance of this species as a source of stress tolerance genes.

We randomly selected 100 journal articles published in five proteomics journals in 2019 and manually examined each of them against a set of 13 criteria concerning the statistical analyses used, all of which were based on items mentioned in the journals' instructions to authors. This included questions such as whether a pilot study was conducted and whether false discovery rate calculation was employed at either the quantitation or identification stage. These data were then transformed to binary inputs, analyzed via machine learning algorithms, and classified accordingly, with the aim of determining if clusters of data existed for specific journals or if certain statistical measures correlated with each other. We applied a variety of classification methods including principal component analysis decomposition, agglomerative clustering, and multinomial and Bernoulli naïve Bayes classification and found that none of these could readily determine journal identity given extracted statistical features. Logistic regression was useful in determining high correlative potential between statistical features such as false discovery rate criteria and multiple testing corrections methods, but was similarly ineffective at determining correlations between statistical features and specific journals. This meta-analysis highlights that there is a very wide variety of approaches being used in statistical analysis of proteomics data, many of which do not conform to published journal guidelines, and that contrary to implicit assumptions in the field there are no clear correlations between statistical methods and specific journals.

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.

Increased amyloid β (Aβ) aggregation is a hallmark feature of Alzheimer's disease (AD) pathology. The APP/PS1 mouse model of AD exhibits accumulation of Aβ in the retina and demonstrates reduced retinal function and other degenerative changes. The overall molecular effects of AD pathology on the retina remain undetermined. Using a proteomics approach, this study assessed the molecular effects of Aβ accumulation and progression of AD pathology on the retina. Retinal tissues from younger (2.5 months) and older 8-month APP/PS1 mice were analysed for protein expression changes. A multiplexed proteomics approach using chemical isobaric tandem mass tags was applied followed by functional and protein-protein interaction analyses using Ingenuity pathway (IPA) and STRING computational tools. We identified approximately 2000 proteins each in the younger (upregulated 50; downregulated 36) and older set of APP/PS1 (upregulated 85; downregulated 79) mice retinas. Amyloid precursor protein (APP) was consistently upregulated two to threefold in both younger and older retinas (p < 0.0001). Mass spectrometry data further revealed that older APP/PS1 mice retinas had elevated levels of proteolytic enzymes cathepsin D, presenilin 2 and nicastrin that are associated with APP processing. Increased levels of proteasomal proteins Psma5, Psmd3 and Psmb2 were also observed in the older AD retinas. In contrast to the younger animals, significant downregulation of protein synthesis and elongation associated proteins such as Eef1a1, Rpl35a, Mrpl2 and Eef1e1 (p < 0.04) was identified in the older mice retinas. This study reports for the first time that not only old but also young APP/PS1 animals demonstrate increased amyloid protein levels in their retinas. Quantitative proteomics reveals new molecular insights which may represent a cellular response to clear amyloid build-up. Further, downregulation of ribosomal proteins involved in protein biosynthesis was observed which might be considered a toxicity effect.

Beneficial microbes have a positive impact on the productivity and fitness of the host plant. A better understanding of the biological impacts and underlying mechanisms by which the host derives these benefits will help to address concerns around global food production and security. The recent development of omics-based technologies has broadened our understanding of the molecular aspects of beneficial plant-microbe symbiosis. Specifically, proteomics has led to the identification and characterization of several novel symbiosis-specific and symbiosis-related proteins and post-translational modifications that play a critical role in mediating symbiotic plant-microbe interactions and have helped assess the underlying molecular aspects of the symbiotic relationship. Integration of proteomic data with other "omics" data can provide valuable information to assess hypotheses regarding the underlying mechanism of symbiosis and help define the factors affecting the outcome of symbiosis. Herein, an update is provided on the current and potential applications of symbiosis-based "omic" approaches to dissect different aspects of symbiotic plant interactions. The application of proteomics, metaproteomics, and secretomics as enabling approaches for the functional analysis of plant-associated microbial communities is also discussed., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)