Autism spectrum disorder (ASD) is a neurodevelopmental condition characterized by early-onset social-communication challenges, restricted and repetitive behaviors, or unusual sensory-motor behaviors. A lack of specific biomarkers hinders the early diagnosis and treatment of this disease in many children. This study analyzes and validates potential urinary biomarkers using mass spectrometry proteomics. Global proteomics profiles of urine from 19 ASD patients and 19 healthy control subjects were compared to identify significantly changed proteins. These proteins were validated with targeted proteomics using parallel reaction monitoring (PRM) in an independent validation set consisting of samples from 40 ASD patients and 38 healthy controls. A total of 34 significantly changed proteins were found in the discovery set, among which seven proteins were identified as potential biomarkers for ASD through PRM assays in the validation set. Of these seven proteins, immunoglobulin kappa variable 4-1, immunoglobulin kappa variable 3-20, and immunoglobulin lambda variable 1-51 were up-regulated, while ATP synthase F1 subunit alpha, 10 kDa heat shock protein, apolipoprotein C-III, and arylsulfatase F were down-regulated. Six of these seven proteins support previous findings that ASD is accompanied by altered immune response and lipid metabolism, as well as mitochondrial dysfunction. This study lays the groundwork for additional research using biomarkers to clinically diagnose ASD. The proteomics and PRM raw data of this study have been deposited under the accession number IPX0002592000 at iProX. SIGNIFICANCE: This study identified 34 proteins in urine of ASD patients that were significantly changed from the urinary proteins of healthy subjects using LC-MS/MS-based proteomics in a discovery set. Seven of these proteins were validated by PRM analysis in an independent validation set. This report represents the first description of combined label-free quantitative proteomics and PRM analysis of targeted proteins for discovery of ASD urinary biomarkers. The results will be helpful for early diagnosis and can provide additional insight into the molecular mechanisms of ASD.

Redox-based post-translational modifications (PTMs) involving protein cysteine residues as redox sensors are important to various physiological processes. However, little is known about redox-sensitive proteins in guard cells and their functions in stomatal immunity. In this study, we applied an integrative protein labeling method cysTMTRAQ, and identified guard cell proteins that were altered by thiol redox PTMs in response to a bacterial flagellin peptide flg22. In total, eight, seven and 20 potential redox-responsive proteins were identified in guard cells treated with flg22 for 15, 30 and 60 min, respectively. The proteins fall into several functional groups including photosynthesis, lipid binding, oxidation-reduction, and defense. Among the proteins, a lipid transfer protein (LTP)-II was confirmed to be redox-responsive and involved in plant resistance to Pseudomonas syringe pv. tomato DC3000. This study not only creates an inventory of potential redox-sensitive proteins in flg22 signal transduction in guard cells, but also highlights the biological relevance of the lipid transfer protein in plant defense against bacterial pathogens. SIGNIFICANCE: Protein redox modifications play important roles in many physiological processes. However, redox proteomics has rarely been studied in plant single cell-types. In this study, isobaric tandem mass tag-based redox proteomics technology was applied to discover redox-sensitive proteins and corresponding cysteine residues in guard cell response to a bacterial flagellin peptide flg22. Many redox-responsive proteins related to photosynthesis, lipid binding, oxidation-reduction, and defense were identified. Using reverse genetics and biochemical analyses, a lipid transfer protein was functionally characterized to be involved in plant defense against pathogens. The study highlights the utility of redox proteomics in discovering new proteins and redox modifications in important stomatal guard cell functions. Furthermore, detailed functional characterization demonstrates the biological relevance of the redox-responsive lipid transfer protein in plant pathogen defense.

TGFβ1 is a profibrotic mediator that contributes to a broad spectrum of pathologies, including systemic sclerosis-associated pulmonary fibrosis (SSc-PF). However, the secretome of TGFβ1-stimulated primary human normal lung (NL) fibroblasts has not been well characterized. Using fluorescent 2-dimensional gel electrophoresis (2D-PAGE) and differential gel electrophoresis (DIGE) followed by Mass Spectrometry, we identified 37 differentially secreted proteins in the conditioned media of TGFβ1-activated NL fibroblasts and generated a protein-protein association network of the TGFβ1 secretome using STRING. Functional enrichment revealed that several biological processes and pathways characteristic of PF were enriched. Additionally, by comparing the TGFβ1 secretome of NL fibroblasts to proteomic biomarkers from biological fluids of systemic sclerosis (SSc) patients, we identified 11 overlapping proteins. Together our data validate the TGFβ1-induced secretome of NL fibroblasts as a valid in vitro model that reflects SSc biomarkers and identify potential therapeutic targets for SSc-PF. SIGNIFICANCE: All proteins secreted by fibroblasts into the extracellular space, representing the secretome, promote cell-to-cell communication as well as tissue homeostasis, immune mechanisms, developmental regulation, proteolysis, development of the extracellular matrix (ECM) and cell adhesion. Therefore, it is crucial to understand how TGFβ1, a well-known profibrotic cytokine, modulates the secretome of pulmonary fibroblasts, and how the TGFβ1-induced secretome resembles biomarkers in SSc. Using functional enrichment analysis, key pathways and hub proteins can be identified and studied as potential therapeutic targets for pulmonary fibrosis.

Millipedes are among the most diverse and abundant arthropods in terrestrial environments. However, little is known about their innate immune response against invading pathogenic microorganisms, which is very intriguing considering that the evolutionary success of millipedes is largely due to this complex and primitive defense system, since it allowed them to colonize a wide variety of microhabitats characterized by their high microbial proliferation. Accordingly, the aim of the present work was to determine the presence of antimicrobial peptides in the hemolymph of the millipede Rhinocricus sp. In total, four native peptides with potent antimicrobial activity against different microorganisms, lack of cytotoxicity against Vero cells and lack of hemolytic effects against human erythrocytes were isolated and named RP40-16, RP40-19, RP40-20/1 and RP40-20/2. The analysis with bioinformatics tools suggested that these peptides may be encrypted in large proteins present in the plasma: Hemocyanin and thioester-containing protein. Considering these results, it can be said that millipede hemolymph represents a promising source of molecules with potential for the development of non-conventional antibiotics. Therefore, in order to have a clearer notion of the biotechnological potential and the role of these peptides in the innate immune response of Rhinocricus sp., future studies should focus on elucidating their mechanisms of action, as well as additional biological properties.

Germination is a critical process in the reproduction and propagation of flowering plants, and is also the key stage of industrial grain malting. Germination commences when seeds are steeped in water, followed by degradation of the endosperm cell walls, enzymatic digestion of starch and proteins to provide nutrients for the growing plant, and emergence of the radicle from the seed. Dormancy is a state where seeds fail to germinate upon steeping, but which prevents inappropriate premature germination of the seeds before harvest from the field. This can result in inefficiencies in industrial malting. We used Sequential Window Acquisition of all THeoretical ions Mass Spectrometry (SWATH-MS) proteomics to measure changes in the barley seed proteome throughout germination. We found a large number of proteins involved in desiccation tolerance and germination inhibition rapidly decreased in abundance after imbibition. This was followed by a decrease in proteins involved in lipid, protein and nutrient reservoir storage, consistent with induction and activation of systems for nutrient mobilisation to provide nutrients to the growing embryo. Dormant seeds that failed to germinate showed substantial biochemical activity distinct from that of seeds undergoing germination, with differences in sulfur metabolic enzymes, endogenous alpha-amylase/trypsin inhibitors, and histone proteins. We verified our findings with analysis of germinating barley seeds from two commercial malting facilities, demonstrating that key features of the dynamic proteome of germinating barley seeds were conserved between laboratory and industrial scales. The results provide a more detailed understanding of the changes in the barley proteome during germination and give possible target proteins for testing or to inform selective breeding to enhance germination or control dormancy. SIGNIFICANCE: Germination is critical to the reproduction and propagation of flowering plants, and in industrial malting. Dormancy, where seeds fail to germinate upon steeping, can result in inefficiencies in industrial malting. Our DIA/SWATH-MS proteomics analyses identified key changes during germination, including an initial loss of proteins involved in desiccation tolerance and germination inhibition, followed by decreases in lipid, protein and nutrient reservoir storage. These changes were consistent between laboratory and industrial malting scales, and therefore demonstrate the utility of laboratory-scale barley germination as a model system for industrial malt house processes. We also showed that dormant seeds that failed to germinate showed substantial biochemical activity distinct from that of seeds undergoing germination, consistent with dormancy being an actively regulated state. Our results provide a more detailed understanding of the changes in the barley proteome during germination and give possible target proteins for testing or to inform selective breeding to enhance germination or control dormancy.

Peritoneal dialysis (PD) is a replacement therapy for end-stage renal disease patients. In the first 4-8 weeks of PD, the patients were given an empirical dialysis prescription due to unknown peritoneal transport characteristics. Proteomic analysis could be used to identify serum biomarkers. In a discovery set, patients were divided into three groups according to the peritoneal equilibration test (PET) results: high (H), high average (HA), low average and low (LA&L) groups. A total of 1051 identified proteins were screened by Nano HPLC-MS/MS. The top two proteins among different peritoneal transport characteristics were Orosomucoid 2 (ORM2) and C-reactive protein (CRP). In a validation set, CRP was significantly elevated in H group than LA&L group, consistent with proteomic analysis. Serum ORM2 was enhanced in LA&L group compared with H and HA group. The expression of ORM2 in peritoneum was also enriched in LA&L group. At last, supplying exogenous ORM could reduce peritoneal proteins loss, without causing a pro-inflammatory response in mice. ORM2 and CRP could be used as biomarkers to predict the baseline peritoneal transport characteristics, and guide the early PD treatment. ORM may serve as a novel therapeutic target for decreasing peritoneal proteins loss in PD patients. SIGNIFICANCE: Peritoneal dialysis (PD) is associated with the functional alterations of the peritoneum. PD patients were often given an empirical dialysis prescription due to the unknown peritoneal transport characteristics in the first 4-8 weeks since PD started. Therefore, it is urgently needed to find biomarkers to predict the baseline peritoneal transport characteristics. In this study, we employed a proteomic analysis to identify serum biomarkers in a training set and verified the screened biomarkers in a validation set. We also found that Orosomucoid (ORM) has the potential to decrease peritoneal proteins loss in PD therapy.

Hepatic steatosis is a very common response to liver injury and often attributed to metabolic disorders. Prior studies have demonstrated the efficacy of a biotechnologically produced oyster mushroom (Pleurotus sajor-caju, PSC) in alleviating hepatic steatosis in obese Zucker rats. This study aims to elucidate molecular events underlying the anti-steatotic effects of PSC. Tandem mass tag (TMT) peptide labeling coupled with LC-MS/MS/MS was used to quantify and compare proteins in the livers of lean Zucker rats fed a control diet (LC), obese Zucker rats fed the same control diet (OC) and obese Zucker rats fed the control diet supplemented with 5% PSC (OPSC) for 4 weeks. Using this technique 3128 proteins could be quantified, out of which 108 were differentially abundant between the OPSC and OC group. Functional enrichment analysis of the up-regulated proteins showed that these proteins were mainly involved in metabolic processes, while the down-regulated proteins were involved in inflammatory processes. Results from proteomic analysis were successfully validated for two up-regulated (carbonic anhydrase 3, regucalcin) and two down-regulated (cadherin-17, ceruloplasmin) proteins by means of immunoblotting. SIGNIFICANCE: Valorization of low-grade agricultural waste by edible fungi, such as the mushroom Pleurotus sajor-caju (PSC), represents a promising strategy for the production of protein rich biomass since they boast of a unique enzyme system that has the ability to recover nutrients and energy from biodegradable waste. Herein, we describe the metabolic effects of PSC feeding using a combined quantitative proteomics and bioinformatics approach. In total, 108 proteins were identified to be regulated by PSC feeding in the liver of the obese rats. Complementary usage of a bioinformatics approach allowed us to decipher the mechanisms underlying the recently observed lipid-lowering and anti-inflammatory activity of PSC feeding in obese Zucker rats, namely a reduction of fatty acid synthesis, an improvement of hepatoprotective mechanisms and an enhancement of anti-inflammatory effects.

Interpopulation venom variation has been widely documented in snakes across large geographical distances. This variability is known to markedly influence the effectiveness of snakebite therapy, as antivenoms manufactured against one population may not be effective against others. In contrast, the extent of intrapopulation venom variability, especially at finer geographical scales, remains largely uninvestigated. Moreover, given the historical focus on the 'big four' Indian snakes, our understanding of venom variation in medically important yet neglected snakes, such as the monocellate cobra (Naja kaouthia), remains unclear. To address this shortcoming, we investigated N. kaouthia venoms sampled across a small spatial scale (<50 km) in Eastern India. An interdisciplinary approach employed in this study unveiled considerable intrapopulation differences in the venom proteomic composition, pharmacological and biochemical activities, and toxicity profiles. Documentation of stark differences in venoms at such a finer geographical scale, despite the influence of similar ecological and environmental conditions, is intriguing. Furthermore, evaluation of in vitro and in vivo venom recognition and neutralisation potential of Indian polyvalent 'big four' antivenoms and Thai monovalent N. kaouthia antivenom revealed concerning deficiencies. These results highlight the negative impact of phylogenetic divergence and intrapopulation snake venom variation on the effectiveness of conventional antivenom therapy. SIGNIFICANCE: In contrast to our understanding of snake venom variation across large distances, which is theorised to be shaped by disparities in ecology and environment, intrapopulation variation at finer geographic scales remains scarcely investigated. Assessment of intrapopulation venom variability in Naja kaouthia at a small spatial scale (<50 km) in Eastern India unravelled considerable differences in venom compositions, activities and potencies. While the influence of subtle differences in prey preference and local adaptations cannot be ruled out, these findings, perhaps, also emphasise the role of accelerated molecular evolutionary regimes that rapidly introduce variations in evolutionarily younger lineages, such as advanced snakes. The inability of 'big four' Indian antivenoms and Thai N. kaouthia monovalent antivenom in countering these variations highlights the importance of phylogenetic considerations for the development of efficacious snakebite therapy. Thus, we provide valuable insights into the venoms of one of the most medically important yet neglected Indian snakes.

Recent studies reported that JH-regulated phosphorylation status of the JH-receptor complex contributes to its transcription activity in Aedes aegypti. However, phosphorylation sites of these proteins have not yet been identified. In this study, we found that the fusion of an EGFP tag to Ae. aegypti Kr-h1 (AaKr-h1) and Met (AaMet) improved their stability in mosquito Aag-2 cells, which allowed their purification. The liquid chromatography and tandem mass spectrometry analysis of the purified AaKr-h1 showed that the phosphoserine residue at position 694, located in the evolutionarily conserved SVIQ motif, is dephosphorylated when the cells are exposed to JH. The AaKr-h1 dephosphorylation mutant (S694V) showed significantly higher activity in inducing the luciferase gene regulated by JH response elements. The phosphorylation profile of Met also changed after exposing Aag-2 cells to JH III. The Ser-77 and Ser-710 residues of Met were phosphorylated after JH III treatment. In contrast, the two phosphoserine residues at positions 73 and 747 were dephosphorylated after JH III treatment. JH exposure also induced transient and reversible phosphorylation of Thr-664 and Ser-723 residues. Overall, these data show that JH induces changes in post-translational modifications of AaMet and AaKr-h1. SIGNIFICANCE: Female Aedes aegypti mosquitoes are known to vector many disease agents, including Zika virus, dengue virus chikungunya virus, and Mayaro and yellow fever virus. In the present study, we developed an efficient method to prepare Ae. aegypti Met and Kr-h1, which are typically difficult to produce and purify, using a mosquito cell line expression system. A liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approaches were utilized to map the phosphorylation profiles of the isolated proteins. We then monitored the changes induced by JH activation in the phosphorylation profiles to check if the JH modulates post-translation modification of its key transcription factors. We found that the JH induced alterations in the phosphorylation profiles of the multiple residues of AaMet. In contrast, activation of the JH signaling pathway was accompanied by dephosphorylation of AaKr-h1 at phosphoserine-694, increasing its transcriptional activity. In addition, S694 of AaKr-h1 was located in the RMSSVIQYA motif highly conserved in orthologous proteins from other insect species. These results can help us further understand how JH modulates its key transcription factors and provide a basis for the development of novel insect control strategies.

Corticosteroid-releasing hormone (CRH) is a crucial neuroendocrine-immune factor regulating the immune response of Scylla paramamosain. To understand the regulatory mechanisms of CRH in S. paramamosain, the hemolymph of S. paramamosain with injection of CRH (1.5 ng/crab) at 24 h were chosen to perform proteomic analysis in this study. Furthermore, quantitative real-time PCR (RT-PCR) method was used to validate the accuracy of proteomic data at 24 h after CRH injection. The proteomic data showed that 255 DEPs were identified, in which 231 and 24 were up- or down-regulated, respectively. Besides, the results of enriched pathways showed that the DEPs were involved in signaling pathways, cellular immunity, humoral immunity and the response of immune related processes. These results revealed that CRH promoted the activation of signal transduction, regulated immune systems and antioxidation, and enhanced the immune related processes (such as protein synthesis, protein transport, carbohydrate mobilization and energy redistribution). These findings will benefit to foster the understanding on the effects of glucocorticoids on neuroendocrine-immune (NEI) networks of crustacean, and supply a substantial material and foundation for further researching of the NEI response. SIGNIFICANCE: Corticotrophin-releasing hormone (CRH) is a 41-amino acid neuropeptide and has been preliminarily studied in aquatic animals. CRH can regulate many important physiological activities comprising protein synthesis, energy metabolism, growth, breeding and behavior in fish, which play an important roles in neuroendocrine-immune (NEI) regulatory network of fish. The neuroendocrine system of crustacean has a primary research, that inspired by fish NEI network. Despite the research on the neuroendocrine system in crustacean has rapidly increased in recent years, our understanding of the regulation between neuroendocrine system and immune system in crustacean is still limited. The research on the strategy of NEI network in crustaceans becomes a significant issue. In the present study, the isobaric tags for relative and absolute quantification (iTRAQ) technology approach were applied to examine the NEI network of Scylla Paramamosain. control group and treatment group (CRH: 1.5 ng/crab) were settled for the iTRAQ experiment, and sampled at 24 h after CRH injection. The study aimed to gain knowledge on the immune response in Scylla Paramamosain after CRH injection and identify related differentially expressed proteins (DEPs) of the crab. The results of this study provide a preliminary resource for analysis the immune mechanism for crustaceans. In general, our work represents the first report of the utilization of the iTRAQ proteomics method for the study of NEI regulatory network in Scylla Paramamosain after CRH injection. We identified a number of DEPs involved in diverse pathways including immune signaling pathways, cellular immunity, humoral immunity, immune related process. These results demonstrated a very complex network involving immune and multiple related metabolic pathways in hemocytes of Scylla Paramamosain and will be of great value in understanding the crab neuroendocrine-immune immune mechanism.

Intramuscular fat (IMF) content is an important factor in porcine meat quality. Previous studies have screened multiple candidate genes related to IMF deposition, but the lipids that affect IMF deposition and their lipid-protein network remain unknown. In this study, we performed proteomic and lipidomic analyses of the longissimus dorsi (LD) muscle from high-IMF (IMFH) and low-IMF (IMF-L) groups of Xidu black pigs. Eighty-eight proteins and 143 lipids were differentially abundant between the groups. The differentially abundant proteins were found to be involved in cholesterol metabolism, the PPAR signaling pathway, and ferroptosis. The triacylglycerols (TAGs) upregulated in the IMF-H group were mainly shown to be synthesized by saturated fatty acids (SFAs), while the downregulated TAGs were mainly synthesized by polyunsaturated fatty acids (PUFAs). All differentially abundant phosphatidylinositols (PIs) and phosphatidylserines (PSs) were found to be upregulated in the IMF-H group. A correlation analysis of the proteomic and lipidomic revealed candidate proteins (APOA4, VDAC3, PRNP, CTSB, GSPT1) related to TAG, PI, and PS lipids. These results revealed differences in proteins and lipids between the IMF-H and IMF-L groups, which represent new candidate proteins and lipids that should be investigated to determine the molecular mechanisms controlling IMF deposition in pigs. SIGNIFICANCE: Intramuscular fat (IMF) is a key factor affecting meat quality, and meat with a higher IMF content can have a better flavor. In this study, proteomic results show that the ferroptosis pathway, including the PRNP, VDAC3 and CP proteins, affects IMF deposition. Lipid composition is the key factor affecting IMF deposition, but there are few reports on this. In this study, through lipidomic analysis, we suggest that saturated fatty acid (SFA), phosphatidylinositol (PI), and phosphatidylserine (PS) may contribute to IMF deposition. A correlation analysis reveals the potential regulatory network between lipids and proteins. This study clarifies the difference in protein and lipid compositions in longissimus dorsi (LD) muscle with high and low IMF contents. This information suggests that it would be beneficial to increase the intramuscular fat content of pork not only from a genetic perspective but also from a nutritional perspective.

Animal production and health are of significant economic importance, particularly regarding the world food supply. Animal and veterinary sciences have evolved immensely in the past six decades, particularly in genetics, nutrition, housing, management and health. To address major challenges such as those posed by climate change or metabolic disorders, it is of utmost importance to use state-of-the-art research tools. Proteomics and the other post-genomic tools (transcriptomics or metabolomics) are among them. Proteomics has experienced a considerable development over the last decades. This brought developments to different scientific fields. The use and adoption of proteomics tools in animal and veterinary sciences has some limitations (database availability or access to proteomics platforms and funding). As a result, proteomics' use by animal science researchers varies across the globe. In this viewpoint article, we focus on the developments of domestic animal proteomics over the last decade in different regions of the globe and how the researchers have coped with such challenges. In the second part of the article, we provide examples of funding, educational and laboratory establishment initiatives designed to foster the development of (animal-based) proteomics. International scientific collaboration is a definitive and key feature in the development and advancement of domestic animal proteomics. SIGNIFICANCE: Animal production and health are very important for food supply worldwide particularly as a source of proteinaceous foods. Animal and veterinary sciences have evolved immensely in the last decades. In order to address the major contemporary challenges facing animal and veterinary sciences, it is of utmost importance to use state-of-the-art research tools such as Proteomics and other Omics. Herein, we focus on the major developments in domestic animal proteomics worldwide during the last decade and how different regions of the world have used the technology in this specific research field. We address also major international efforts aiming to increase the research output in this area and highlight the importance of international cooperation to address specific problems inherent to domestic animal proteomics.

Soft tissue sarcomas (STS) are a group of rare and heterogeneous cancers. While large-scale genomic and epigenomic profiling of STS have been undertaken, proteomic analysis has thus far been limited. Here we utilise sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS) for proteomic profiling of formalin fixed paraffin embedded (FFPE) specimens from a cohort of STS patients (n = 36) across four histological subtypes (leiomyosarcoma, synovial sarcoma, undifferentiated pleomorphic sarcoma and dedifferentiated liposarcoma). We quantified 2951 proteins across all cases and show that there is a significant enrichment of gene sets associated with smooth muscle contraction in leiomyosarcoma, RNA splicing regulation in synovial sarcoma and leukocyte activation in undifferentiated pleomorphic sarcoma. We further identified a subgroup of STS cases that have a distinct expression profile in a panel of proteins, with worse survival outcomes when compared to the rest of the cohort. Our study highlights the value of comprehensive proteomic characterisation as a means to identify histotype-specific STS profiles that describe key biological pathways of clinical and therapeutic relevance; as well as for discovering new prognostic biomarkers in this group of rare and difficult-to-treat diseases.

Mass spectrometry-based proteomics relies on dedicated software for peptide and protein identification. These software include open-source or commercial-based search engines; wherein, they employ different algorithms to establish their scoring and identified proteins. Although previous comparative studies have differentiated the proteomics results from different software, there are still yet studies specifically been conducted to compare and evaluate the search engine in the field of halal analysis. This is important because the halal analysis is often using commercial meat samples that have been subjected to various processing, further complicating its analysis. Thus, this study aimed to assess three open-source search engines (Comet, X! Tandem, and ProteinProspector) and a commercial-based search engine (ProteinPilot™) against 135 raw tandem mass spectrometry data files from 15 types of pork-based food products for halal analysis. Each database search engine contained high false-discovery rate (FDR); however, a post-searching algorithm called PeptideProphet managed to reduce the FDR, except for ProteinProspector and ProteinPilot™. From this study, the combined database search engine (executed by iProphet) reveals a thorough protein list for pork-based food products; wherein the most abundant proteins are myofibrillar proteins. Thus, this proteomics study will aid the identification of potential peptide and protein biomarkers for future precision halal analysis. SIGNIFICANCE: A critical challenge of halal proteomics is the availability of a database to confirm the inferential peptides as well as proteins. Currently, the established database such as UniProtKB is related to animal proteome; however, the halal proteomics is related to the highly processed meat-based food products. This study highlights the use of different database search engines (Comet, X! Tandem, ProteinProspector, and ProteinPilot™) and their respective algorithms to analyse 135 raw tandem mass spectrometry data files from 15 types of pork-based food products. This is the first attempt that has compared different database search engines in the context of halal proteomics to ensure the effectiveness of controlling the FDR. Previous studies were just focused on the advantages of a certain algorithm over another. Moreover, other previous studies also have mainly reported the use of mass spectrometry-based shotgun proteomics for meat authentication (the most similar field to halal analysis), but none of the studies were reported on halal aspects that used samples originated from highly processed food products. Hence, a systematic comparative study is duly needed for a more comprehensive and thorough proteomics analysis for such samples. In this study, our combinatorial approach for halal proteomics results from the different search engines used (Comet, X! Tandem, and ProteinProspector) has successfully generated a comprehensive spectral library for the pork-based meat products. This combined spectral library is freely available at https://data.mendeley.com/datasets/6dmm8659rm/3. Thus far, this is the first and new attempt at establishing a spectral library for halal proteomics. We also believe this study is a pioneer for halal proteomics that aimed at non-conventional and non-model organism proteomics, protein analytics, protein bioinformatics, and potential biomarker discovery.

Iron prawn" is a condition of severe growth retardation that fishers call. The giant river prawn (Macrobrachium rosenbergii) is a commercially important species contains high protein content and functional nutrients. However, no proteomic information is available for this species. We performed the shotgun 2DLC-MS/MS proteomic analysis of the total protein from "iron prawn". Total 19,758 peptides corresponding to 2613 high-confidence proteins were identified. These proteins range in size from 40 to 70 kDa. KEGG analysis revealed that the largest group consisting total 102 KEGG pathway proteins comparing the "iron prawn" with the normal prawn. Additionally, 7, 11, 1, 6, and 5 commercially important enzymes were found in the eyestalk, liver, muscle, ovary, and testis, respectively. The functions of these differently expressed enzymes include immune system action against pathogens, muscle contraction, digestive system metabolism, cell differentiation, migration, and apoptosis in the severe growth retardation of "iron prawn". Our work provides insight into the understanding of the formation mechanism of "iron prawn".

Nicotine is a prominent active compound in tobacco and many smoking cessation products. Some of the biological effects of nicotine are well documented in in vitro and in vivo systems; however, data are scarce concerning the time-dependent changes on protein and phosphorylation events in response to nicotine. Here, we profiled the proteomes of SH-SY5Y and A549 cell lines subjected to acute (15 min, 1 h and 4 h) or chronic (24 h, 48 h) nicotine exposures. We used sample multiplexing (TMTpro16) and quantified more than 9000 proteins and over 7000 phosphorylation events per cell line. Among our findings, we determined a decrease in mitochondrial protein abundance for SH-SY5Y, while we detected alterations in several immune pathways, such as the complement system, for A549 following nicotine treatment. We also explored the proposed association between smoking (specifically nicotine) and SARS-CoV2. Here, we found several host proteins known to interact with viral proteins that were affected by nicotine in a time dependent manner. This dataset can be mined further to investigate the potential role of nicotine in different biological contexts. SIGNIFICANCE: Smoking is a major public health issue that is associated with several serious chronic, yet preventable diseases, including stroke, heart disease, type 2 diabetes, cancer, and susceptibility to infection. Tobacco smoke is a complex mixture of thousands of different compounds, among which nicotine is the main addictive compound. The biological effects of nicotine have been reported in several models, however very little data are available concerning the temporal proteomic and phosphoproteomic changes in response to nicotine. Here, we provide a dataset exploring the potential role of nicotine on different biological processes over time, including implications in the study of SARS-CoV2.

Selenium (Se) is an essential mineral nutrient for animals. Se deprivation can lead to many disorders and even death. This study investigated the response of Przewalski's gazelle (P. przewalskii) to Se-deprived environment. We found that Se deprivation in soil and forage not only influenced the mineral contents of the blood and hair in P. przewalskii, but also severely disrupted their blood parameters. We identified significant changes in the abundance of 146 proteins and 25 metabolites (P < 0.05) in serum, including the selenoproteins L8IG93 (glutathione peroxidase) and F4YD09 (Cu/Zn superoxide dismutase). Furthermore, the major known proteins and metabolites associated with the Se stress response in P. przewalskii were Cu/Zn superoxide dismutase, the vitamin K-dependent protein C, the C4b-binding protein alpha chain, complement component C7, lipase linoleic acid, peptidase D, thymidine, pseudo-uridine, L-phenylalanine, L-glutamine, PGA1, and 15-deoxy-delta-12,14-PGJ2. The main signaling pathways involved included complement and coagulation cascades, metabolic pathways, and stress granule formation. Our results indicate that the intake of Se-deficient forage elicited an oxidative stress response in P. przewalskii. These findings provide insights into the response mechanisms of this threatened gazelle to Se stress, and enable the development of conservation strategies to protect populations on the Qinghai-Tibetan Plateau. SIGNIFICANCE: This study is the first to point out the presence of oxidative stress in P. przewalskii in selenium-deficient areas through proteomics and metabolomics studies. These findings should prove helpful for conservation efforts aimed at P. przewalskii populations and maintenance of the integrity of their ecological environment.

A comparative proteomic analysis between two near-isogenic rice lines, displaying a resistant and susceptible phenotype upon infection with Magnaporthe oryzae was performed. We identified and validated factors associated with rice disease susceptibility, representing a flourishing source toward a more resolute rice-blast resistance. Proteome profiles were remarkably different during early infection (12 h post-inoculation), revealing several proteins with increased abundance in the compatible interaction. Potential players of rice susceptibility were selected and gene expression was evaluated by RT-qPCR. Gene Ontology analysis disclosed susceptibility gene-encoded proteins claimed to be involved in fungus sustenance and suppression of plant immunity, such as sucrose synthase 4-like, serpin-ZXA-like, nudix hydrolase15, and DjA2 chaperone protein. Two other candidate genes, picked from a previous transcriptome study, were added into our downstream analysis including pyrabactin resistant-like 5 (OsPYL5), and rice ethylene-responsive factor 104 (OsERF104). Further, we validated their role in susceptibility by Transient-Induced Gene Silencing (TIGS) using short antisense oligodeoxyribonucleotides that resulted in a remarkable reduction of foliar disease symptoms in the compatible interaction. Therefore, we successfully employed shotgun proteomics and antisense-based gene silencing to prospect and functionally validate rice potential susceptibility factors, which could be further explored to build rice-blast resistance. SIGNIFICANCE: R gene-mediated disease resistance is race-specific and often not durable in the field. More recently, advancements in new breeding techniques (NBTs) have made plant disease susceptibility genes (S-genes) a new target to build a broad spectrum and more durable resistance, hence an alternative source to R-genes in breeding programs. We successfully coupled shotgun proteomics and gene silencing tools to prospect and validate new rice-bast susceptibility genes that can be further exploited toward a more resolute blast disease resistance.

Genetic selection for meat production performance of broilers concomitantly causes excessive abdominal fat deposition, accompanied by several adverse effects, such as the reduction of feed conversion efficiency and reproduction performance. Our previous studies have identified important genes regulating chicken fat deposition, using the Northeast Agricultural University broiler lines divergently selected for abdominal fat content (NEAUHLF) as an animal model. However, the molecular mechanism underlying fat deposition differences between fat and lean broilers remains largely unknown. Here, we integrated the transcriptome (RNA-Seq) and quantitative proteome (isobaric tags for relative and absolute quantitation, iTRAQ) profiling analyses on abdominal fat tissues from NEAUHLF chicken lines. Differentially expressed genes (2167 DEGs, corrected p-value < 0.01) and differentially abundant proteins (199 DAPs, corrected p-value < 0.05) were identified in lean line compared to fat line. Down-regulated DEGs and DAPs mainly enriched in pathways related to fatty acid metabolism, fatty acid biosynthesis, and PPAR signaling, and interestingly, up-regulated DEGs and DAPs enriched both in lysosome pathway. Moreover, numerous key DEGs and DAPs involved in long-chain fatty acid uptake, in situ lipogenesis (fatty acid and cholesterol synthesis), and lipid droplet accumulation were discovered after integrated transcriptome and proteome analysis. SIGNIFICANCE: Excessive abdominal fat deposition critically affects the health of broilers and causes economic loss to broiler producers, but the molecular mechanism of abdominal fat deposition is still unclear in chicken. We identified key DEGs/DAPs and potential pathways through an integration of chicken abdominal fat tissues transcriptome and proteome analyses. Our findings will facilitate a better revealing the mechanism and provide a novel insight into abdominal fat content discrepancy between the fat and lean chicken lines.

Proteasome inhibitors are an important class of chemotherapeutic drugs. In this study, we performed a large-scale ubiquitylome analysis of the three proteasome inhibitors MG132, bortezomib and carfilzomib. Although carfilzomib is currently being used for the treatment of multiple myeloma, it has not yet been subjected to a global ubiquitylome analysis. In this study, we identified more than 14,000 unique sites of ubiquitylation in more than 4400 protein groups. We introduced stringent criteria to determine the correct ubiquitylation site ratios and used five biological replicates to achieve increased statistical power. With the vast amount of data acquired, we made proteome-wide comparisons between the proteasome inhibitors and indicate candidate proteins that will benefit from further study. We find that in addition to the expected increase in ubiquitylation in the majority of proteins, unexpectedly a select few are specifically and significantly decreased in ubiquitylation at specific sites after treatment with proteasome inhibitors. We chose to follow-up on Mortality factor 4-like 1 (MORF4L1), which was significantly decreased in ubiquitylation at lysine 187 and lysine 104 upon proteasome inhibition, but increased in protein abundance by approximately two-fold. We demonstrate that the endogenous protein level of MORF4L1 is highly regulated by the ubiquitin proteasome system. SIGNIFICANCE: This study provides a highly curated dataset of more than 14,000 unique sites of ubiquitylation in more than 4400 protein groups. For the proper quantification of ubiquitylation sites, we introduced a higher standard by quantifying only those ubiquitylation sites that are not flanked by neighboring ubiquitylation, thereby avoiding the report of incorrect ratios. The sites identified will serve to identify important targets of the ubiquitin proteasome system and aid to better understand the repertoire of proteins that are affected by inhibiting the proteasome with MG132, bortezomib, and carfilzomib. In addition, we investigated the unusual observation that ubiquitylation of the tumor suppressor Mortality factor 4-like (MORF4L1) protein decreases rather than increases upon proteasome inhibition, which may contribute to an additional anti-tumor effect of bortezomib and carfilzomib.

As an important perennial warm-season turfgrass species, bermudagrass (Cynodon dactylon L.) forms underground-growing rhizomes and aboveground-growing stolons simultaneously, making it a fast propagating clonal plant with strong regeneration ability. In the current study, we compared the internode proteomes of rhizomes and stolons at the same developmental stage in the bermudagrass cultivar Yangjiang using iTRAQ. The results indicated that 228 protein species were differentially accumulated in the two specialized stems. In agreement with the different contents of starch, chlorophyll, anthocyanin and H2O2 in the two types of stems, photosynthesis and flavonoid biosynthesis were enriched with differentially accumulated protein species (DAPs) in stolons, whereas starch and sucrose metabolism, glycolysis, and H2O2 metabolism were enriched with DAPs in rhizomes. Burying stolons in the soil resulted in the gradual degradation of chlorophyll and anthocyanin, accumulation of starch, and increment of H2O2, which is similar to the physiological characteristics of rhizomes. These results collectively revealed that stolons and rhizomes of bermudagrass have significant differences at the proteome level and light might play important regulatory roles in the discrepancy of the proteome profiles and specialization of the two stems, providing new insights into the adaptation of plant stems to aboveground and underground growth. BIOLOGICAL SIGNIFICANCE: As two types of specialized stems that grow underground and aboveground respectively, rhizomes and stolons play important roles in overwintering and ecological invasion of many perennial and clonal plants. However, because rhizomes and stolons rarely coexist in single plant species, the differences between the two stems remain unclear at the molecular level. In this study, through an iTRAQ comparative proteomic analysis, we reported the identification of 228 differentially accumulated protein species (DAPs) in rhizomes and stolons of bermudagrass for the first time. We found that the 228 DAPs were interconnected to form protein networks in regulating diverse cellular activities and biochemical reactions. We also observed that stolons growing underground showed similar physiological activities and DAP expression as those of underground-growing rhizomes, suggesting that light might play important regulatory roles in the specialization of stolons and rhizomes. These results expanded our understanding of the mysterious adaption of plant stems to different growth conditions.

Reproduction and growth follow a seasonal pattern in many fish species involving changes in gonadal development, growth, and metabolism. Significant metabolic energy is needed during gametogenesis in both female and male to produce hundreds of eggs and billions of sperms. Seasonal variations are controlled by the hormones of brain-pituitary-peripheral axis and are accompanied by significant metabolic changes. There is evidence that GnRH and GnIH are among the key neurohormones that regulate the reciprocal control of growth and reproduction. The objective of this study was to investigate changes in metabolic profile and energy allocation patterns at different stages of reproduction, using goldfish as a model organism and LC-MS as analytical platform for metabolic analysis. Goldfish undergoes a clear seasonal cycle of growth and reproduction. In vivo experiments were conducted at three different time point of the annual cycle: regressed gonadal phase (peak growth phase), mid gametogenesis and late gametogenesis. Emphasis is placed on changes in liver metabolic pathways to energetically sustain the physiological processes related to growth and reproduction. Moreover, we tested the hypothesis that GnRH and GnIH may play a role in the regulation of metabolism by investigating acute effects of these peptides at different stages of reproductive cycle. SIGNIFICANCE: The findings in this paper provide novel information on the seasonal changes in basal metabolism during different stages of reproductive cycle, and evidence for differential allocation of energy during reciprocal control of reproduction and growth in goldfish. Chemometrics combined with pathway-driven bioinformatics elucidated a shift in the metabolic profile, indicating distinct patterns of energy allocation in the reproductive and growth seasons. Furthermore, to our knowledge this is the first study to provide evidence for a possible regulatory role of GnRH and GnIH in liver metabolism and energy allocation patterns associated with growth and reproductive processes. Together our findings present a framework for better understanding of the hormonally induced changes in metabolism to energetically sustain growth and reproduction in fish and other oviparous species undergoing seasonal cycle.