Metabolome and proteome profiling of biofluids, e.g., urine, plasma, has generated vast and ever-increasing amounts of knowledge over the last few decades. Paradoxically, omics analyses of sweat, one of the most readily available human biofluids, have lagged behind. This review capitalizes on the current knowledge and state of the art analytical advances of sweat metabolomics and proteomics. Moreover, current applications of sweat omics such as the discovery of disease biomarkers and monitoring athletic performance are also presented in this review. Another area of emerging knowledge that has been highlighted herein lies in the role of skin host-microbiome interactions in shaping the sweat metabolite-protein profiles. Discussion of future research directions describes the need to have a better grasp of sweat chemicals and to better understand how they function as aided by advances in omics tools. Overall, the role of sweat as an information-rich biofluid that could complement the exploration of the skin metabolome/proteome is emphasized.

Gliadin is a major wheat seed storage protein that affects the extensibility of flour dough. Multiple genes encode gliadin, and there are numerous isoforms encoded by these genes, some of which might be related to flour quality. In this study, gliadin isoforms encoded by 30 α-gliadin genes from the wheat cultivar "Chinese Spring" (CS) were identified using 2-DE and MS/MS. The chromosomes where these isoform genes are located were determined using Gli-2 locus-deficient lines. A quantitative analysis by 2-DE revealed differences in expression levels among α-gliadin isoforms. We also separated the polymer and monomer fractions of the total protein by SEC. We found that an α-gliadin isoform with 7 cysteine residues was present at relatively higher levels in the polymer fraction than an α-gliadin isoform with 6 cysteine residues. The present study results can help in understanding the relationship between the properties of α-gliadin isoforms and the physical properties of dough in the future. SIGNIFICANCE: For investigating the relationship between isoforms and dough extensibility, we identified α-gliadin isoforms encoded by 30 genes among the 50 genes cloned until date. Moreover, the polymer and monomer fractions of the total protein were separated by SEC. We found that an α-gliadin isoform with 7 cysteine residues was present at relatively higher levels in the polymer fraction than an α-gliadin isoform with 6 cysteine residues. This study provided useful information for elucidating the relationship between the properties of α-gliadin isoforms and the physical properties of dough.

Among cancers, prostate cancer (PCa) is frequently detected solid tumor and a growing problem for the male population, globally. Newer treatment modalities with specific targets are required for management. Plant-derived agents/drugs have historically been useful in cancer therapeutics. Natural metabolite i.e. plectranthoic acid (PA), inhibits the proliferation of PCa cells and has potent anti-cancer potential. Herein, we aim to identify the molecular signatures of PA. Proteins from control and PA-treated PCa cells were analysed using high-throughput labeled free proteomics approach. Data was processed with the SIEVE software and thoroughly analysed by using Ingenuity pathway analysis (IPA) and PANTHER. A total of 98 unique peptides, showing >2 fold change, were identified. Results indicated that PA modulates oncogenic pro-survival and pro-apoptotic signaling pathways in PCa cells. mTOR was the major canonical pathway targeted by PA, the inhibition of which was likely to induce PA mediated apoptosis. Moreover, PA interacts with the rapamycin binding domain of mTOR, demonstrated by the molecular dynamic (MD) simulation and binding free energy calculations. Furthermore, the biological process moderated by PA with a high percentage was a metabolic process. Taken together, PA appears to have pleiotropic effects, as it modulates multiple key signaling pathways, supporting the potential usefulness. SIGNIFICANCE: Studies on the mechanism of action of therapeutic agents are crucial for drug development. These studies support the selection of a therapeutic agent, appropriate models of its efficacy, and designing of further experiments. Furthermore, information on mechanism of action may suggest strategies for combination therapies. In this regard Proteomics provide the platform for comprehensive understanding of the molecular action mechanisms of newly discovered therapeutic agents. Current research highlights the new insights into mode of action of novel therapeutic metabolite i.e. Plectranthoic acid (PA). Using labeled free proteomics approach we extracted the underlying mechanisms for the anticancer activity of PA using prostate cancer model. The result of the study will pay the way for further investigations on this potent natural compound in different cancers and will provide a root for its development as a lead.

In cyanobacteria, it is known that the excitation ratios of photosystem (PS) I and PSII changes with the wavelength of irradiated light due to mobile phycobilisome (PBS) and spillover, affecting the photosynthetic ATP/NADPH synthesis ratio and metabolic flux state. However, the mechanisms by which these changes are controlled have not been well studied. In this study, we performed a targeted proteomic analysis of Synechocystis sp. PCC 6803 under different spectral light conditions to clarify the regulation mechanisms of mobile PBS, spillover and metabolisms under different light qualities at the protein level. The results showed an increase in the amount of proteins mainly involved in CO2 fixation under Red1 light conditions with a high specific growth rate, suggesting that the rate of intracellular metabolism is controlled by the rate of carbon uptake, not by changes in the amount of each enzyme. Correlation analysis between protein levels and PSI/PSII excitation ratios revealed that PsbQUY showed high correlations and significantly increased under Blue and Red2 light conditions, where the PSI/PSII excitation ratio was higher due to spillover. In the strains lacking the genes encoding these proteins, a decrease in the PSI/PSII excitation ratio was observed, suggesting that PsbQUY contribute to spillover occurrence. SIGNIFICANCE: In cyanobacteria, the photosynthetic apparatus's responses, such as state transition [mobile PBS and spillover], occur due to the intensity and wavelength of irradiated light, resulting in changes in photosynthetic electron transport and metabolic flux states. Previous studies have analyzed the response of Synechocystis sp. PCC 6803 to light intensity from various directions, but only spectroscopic analysis of the photosynthetic apparatus has been done on the response to changes in the wavelength of irradiated light. This study analyzed the response mechanisms of mobile PBS, spillover, photosynthetic, and metabolic systems in Synechocystis sp. PCC 6803 under six different spectral light conditions by a targeted proteomic analysis. As a result, many proteins were successfully quantified, and the metabolic enzymes and photosynthetic apparatus were analyzed using an integrated approach. Principal component and correlation analyses and volcano plots revealed that the PSII subunits PsbQ, PsbU, and PsbY have a strong correlation with the PSI/PSII excitation ratio and contribute to spillover occurrence. Thus, statistical analysis based on proteome data revealed that PsbQ, PsbU, and PsbY are involved in spillover, as revealed by spectroscopic analysis.

Wheat intolerance has various systemic manifestations that can affect people's quality of life, and few studies have focused on the mechanism of wheat intolerance and the signaling pathways involved in wheat intolerance have not been fully identified. We compared the protein profiles of patients with wheat intolerance with those of healthy controls using LASSO (least absolute shrinkage and selection operator) and PLS (partial least squares regression) to obtain DEPs (differentially expressed proteins) for GO (Gene Ontology) analysis, KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis, and PPI (protein-protein interaction) network analysis. Internal validation and external validation were conducted for target proteomics testing. The correlation between differently expressed protein and the wheat-specific IgG antibody concentration was analyzed. Then ROC curve (receiver operating characteristic curve) was generated to validate the differentially expressed proteins. We identified 33 DEPs as significant candidate proteins of wheat intolerance. These proteins were mainly enriched in complement and coagulation cascade pathways, immune activation, and immune response-related pathways. After internal and external target proteomics validation, CFHR3 (complement factor H-related protein 3) was identified as a key protein that may have an important role in wheat intolerance. We found CFHR3 protein expression abundance and the wheat-specific IgG antibody concentration were significantly negatively correlated (P = 0.035; Spearman correlation coefficient r = -0.565). The AUC (median area under the ROC curve) of CFHR3 is 0.857 in external verification data. This study provides insights into wheat intolerance that can be used to further explore the pathogenesis of this condition. SIGNIFICANCE: Proteomics has performed important potential in food allergy research and is conducive to improving our comprehension on molecular mechanisms of food allergy. The present study identified significant signaling pathways and differentially expressed proteins in patients with wheat intolerance by means of bioinformatics from the viewpoint of mass spectrometry-based proteomics, which provided insights into further research on the pathogenesis and timely diagnosis of wheat intolerance.

Negative energy balance (NEB) is associated with metabolic disorders in early lactation dairy cows. Therefore, our objective was to characterize the liver proteome in cows experiencing either NEB or positive energy balance (PEB). Forty-two multiparous Holstein dairy cows were milked either 2 or 3 times daily for the first 30 days in milk (DIM) to alter EB, and were classified retrospectively as NEB (n = 18) or PEB (n = 22). Liver biopsies were collected from 10 cows (n = 5 from each milking frequency) at 17 ± 3 DIM (NEB, n = 6; PEB, n = 4). The liver proteome was characterized using label-free quantitative shotgun proteomics and Ingenuity Pathway Analysis used to identify key affected canonical pathways. Overall, 2741 proteins were identified, and 68 of those were differentially abundant (P ≤ 0.05 and FC ± 1.5). ENO3 (FC = 10.3, P < 0.01) and FABP5 (FC = -12.5, P = 0.045) were the most dramatically upregulated and downregulated proteins, respectively, in NEB cows. Numerous mitochondrial proteins (NDUFA5, NDUFS3, NDUFA6, COX7A2L, COX6C, and COA5) were differentially abundant. Canonical pathways associated with NEB were LPS/IL-1 mediated inhibition of RXR function, oxidative phosphorylation, and mitochondrial dysfunction. Additionally, cows experiencing NEB had less hepatic IL10 transcript abundance than PEB. Together, NEB was associated with altered hepatic inflammatory status, likely due to oxidative stress from mitochondrial dysfunction. SIGNIFICANCE: Our manuscript describes the associations of negative energy balance with the liver proteome in early lactation dairy cows, when metabolic stress and the incidence of diseases is increased. Specifically, we found associations of negative energy balance with shifts in hepatic protein abundance involved in fatty acid uptake, impaired anti-inflammatory responses, and mitochondrial dysfunction. Moving forward, differentially abundant proteins found in this study may be useful as either biological markers for disease or therapeutic targets to improve metabolic adaptations to lactation in postpartum dairy cattle.

The proteomic, enzymatic, toxicological, and immunogenic profiles of the venom of C. d. pifanorum were studied. It was found that venom of C. d. pifanorum is composed of 63% phospholipases A2 (PLA2s), 13% serine proteinases (SVSPs), 8% bradykinin-potentiating peptides (BPPs), 4% L-amino acid oxidases (LAAOs), 3% metalloproteinases (SVMPs), and other minor components. This composition allows the venom to exert lethal, PLA2, myotoxic, coagulant and defibrinogenating activities, but no azocaseinolytic or hemorrhagic activities. The addition of C. d. pifanorum venom to the group of venoms used as immunogens to produce the Central American antivenom PoliVal-ICP (i.e., venoms of Bothrops asper, Crotalus simus and Lachesis stenophrys) resulted in 1) the expansion of the neutralization scope of the antivenom to cover the venom of C. d. pifanorum and other antigenically related venom (i.e., C. s. scutulatus venom), 2) improvement of the neutralizing potency towards the venom of C. simus, and 3) no significant reduction of the neutralization of venoms of B. asper and L. stenophrys. It was concluded that supplementation of the immunogens used to produce PoliVal-ICP with the venom of C. d. pifanorum is a viable alternative to expand the neutralization scope of the antivenom. BIOLOGICAL SIGNIFICANCE: The venom of Crotalus durissus pifanorum from Venezuela has a proteomic profile like those of other subspecies of the South American rattlesnake C. durissus, with predominance of phospholipases A2 (especially crotoxin) and serine proteinases. This explains a toxicological profile characterized by neurotoxicity, myotoxicity, and coagulopathies, but being devoid of hemorrhagic activity. The antivenom used in Central America (PoliVal-ICP) includes the venom of C. simus, which has a different composition, in the immunizing strategy. Accordingly, this antivenom does not neutralize C. d. pifanorum venom. The addition of C. d. pifanorum venom to the immunizing mixture of PoliVal-ICP expands the neutralizing scope of this antivenom, to cover additional rattlesnake venoms, while not affecting the response to C. simus, Bothrops asper and Lachesis stenophrys venoms. This represents an improvement of the current PoliVal-ICP.

Peptides present in the seminal fluid of Drosophila melanogaster can function as antimicrobial agents, enzyme inhibitors and as pheromones that elicit physiological and behavioural responses in the post-mated female. Understanding the molecular interactions by which these peptides influence reproduction requires detailed knowledge of their molecular structures. However, this information is often lacking and cannot be gleaned from just gene sequences and standard proteomic data. We now report the native structures of four seminal fluid peptides (andropin, CG42782, Met75C and Acp54A1) from the ejaculatory duct of male D. melanogaster. The mature CG42782, Met75C and Acp54A1 peptides each have a cyclic structure formed by a disulfide bond, which will reduce conformational freedom and enhance metabolic stability. In addition, the presence of a penultimate Pro in CG42782 and Met75C will help prevent degradation by carboxypeptidases. Met75C has undergone more extensive post-translational modifications with the formation of an N-terminal pyroglutamyl residue and the attachment of a mucin-like O-glycan to the side chain of Thr4. Both of these modifications are expected to further enhance the stability of the secreted peptide. The glycan has a rare zwitterionic structure comprising an O-linked N-acetyl hexosamine, a hexose and, unusually, phosphoethanolamine. A survey of various genomes showed that andropin, CG42782, and Acp54A1 are relatively recent genes and are restricted to the melanogaster subgroup. Met75C, however, was also found in members of the obscura species groups and in Scaptodrosophila lebanonensis. Andropin is related to the cecropin gene family and probably arose by tandem gene duplication, whereas CG42782, Met75C and Acp54A1 possibly emerged de novo. We speculate that the post-translational modifications that we report for these gene products will be important not only for a biological function, but also for metabolic stability and might also facilitate transport across tissue barriers, such as the blood-brain barrier of the female insect. BIOLOGICAL SIGNIFICANCE: Seminal fluid peptides of D. melanogaster function as antimicrobials, enzyme inhibitors and as pheromones, eliciting physiological and behavioural responses in the post-mated female. A fuller understanding of how these peptides influence reproduction requires knowledge not only of their primary structure, but also of their post-translational modification. However, this information is often lacking and difficult to glean from standard proteomic data. The reported modifications, including the unusual glycosylation, adds much to our knowledge of this important class of peptides in this model organism, par excellence.

Erratum in J Proteomics. 2022 Mar 20;255:104484.

Plant viruses trigger numerous responses in their insect vectors. Using iTRAQ-based quantitative proteomics analysis, early responses of the insect vector, the small brown planthopper (Laodelphax striatellus Fallén, SBPH), after acquiring Rice black-streaked dwarf virus (RBSDV) at 3 days and 5 days post first access to diseased plants (padp) were revealed. A total of 582 differentially abundant proteins (DAPs) in SBPH with a fold change >1.500 or <0.667 (p-value < 0.05) were identified. The proteomic analysis in SBPH at 3 days padp revealed 106 highly abundant proteins and 193 of low abundance, while 5 days padp revealed 214 highly abundant proteins and 182 of low abundance. Among them, 51 highly abundant proteins and 42 of low abundance were shown consistently at both 3 days and 5 days padp. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis mapping and Gene Ontology (GO) term classification suggested impairment of mitochondria in SBPH after RBSDV acquisition, and the 77 out of 582 differentially abundant SBPH proteins analyzed by the STRING program revealed the interaction network of the mitochondrial DAPs, showing an overall down-regulation of mitochondrial proteins including the electron transport chain proteins and mitochondrial ribosome proteins. The high abundance of Parkin at 5 days padp suggests that activation of mitophagy induced degradation of mitochondria occurred. Further verification of autophagy/mitophagy-related genes by reverse-transcription quantitative RT-PCR (RT-qPCR) in SBPH after RBSDV acquisition showed up-regulation of the autophagy receptors Optineurin (OPTN), Sequestosome-1 (SQSTM1, also known as p62) and Tax1-binding protein 1 (TAX1BP1) which targets ubiquitinated damaged mitochondria during mitophagy. The phosphorylation of the three autophagy receptors may be up-regulated through an increase of transcription level TRAF-associated NFκB activator (TANK)-binding kinase 1 (TBK1). As a result, an overall reduction in the abundance of mitochondrial proteins was observed and the selective autophagic degradation was up-regulated through increased transcription level of OPTN, p62/SQSTM1, TAX1BP1 and TBK1. Therefore, acquisition of RBSDV associated with up-regulated autophagy and selective mitochondrial degradation in SBPH suggest prevention of mitochondrial-mediated apoptosis and extension of the vector life span. BIOLOGICAL SIGNIFICANCE: RBSDV causes severe yield loss in rice plants. RBSDV is transmitted efficiently only through SBPH. It is important to understand how RBSDV infects SBPH in a persistent, circulative and propagative manner. However, there has been no study on the interaction between RBSDV and SBPH at the early acquisition stage using a proteomics approach. In this study, we combined iTRAQ technique and LC-MS/MS to analyze the vector proteomics at both the initial and latent infection stages after RBSDV acquisition and verified the results by RT-qPCR. Our results revealed that significantly low DAPs were involved in various pathways, including biosynthesis of secondary metabolites, ribosomes, carbon metabolism, biosynthesis of amino acids and TCA cycle. Further clustering of the DAPs revealed significant changes in SBPH mitochondria, including decreased proteins in mitochondrial ribosomes and electron transport chain complex I, II and V. On the other hand, there was a high abundance of Parkin, suggesting the occurrence of mitochondria damage and subsequent Parkin-mediated mitophagy for clearance of impaired mitochondria. Moreover, the decreased level of PMPCB in terms of gene expression and protein abundance suggested decreased PINK1 turnover, promoting Parkin/PINK1-mediated mitophagy. Further analysis on autophagy/mitophagy-related gene transcription level indicated up-regulation of OPTN, p62/SQSTM1, TAX1BP1 and TBK1, promoting selective autophagy in SBPH after RBSDV acquisition. These findings provided new insights into the effects of RBSDV on SBPH after early acquisition by selective degradation of mitochondria, especially on reprogramming of energy metabolism and decreased mitochondria biogenesis, to prevent apoptosis and prolong the life span of SBPH post virus acquisition.

Meningiomas are brain tumors that originate from the meninges and has been primarily classified into three grades by the current WHO guidelines. Although widely prevalent and can be managed by surgery there are instances when the tumors are located in difficult regions. This results in considerable challenges for complete surgical resection and further clinical management. While the genetic signature of the skull base tumors is now known to be different from the non-skull base tumors, there is a lack of information at the functional aspects of these tumors at the proteomic level. Thus, the current study thereby aims to obtain mechanistic insights between the two radiologically distinct groups of meningiomas, namely the skull base & supratentorial (non-skull base-NSB) regions. We have employed a comprehensive mass spectrometry-based label-free quantitative proteomic analysis in Skull base and supratentorial meningiomas. Further, we have used an Artificial Neural Networking employing a sparse Multilayer perceptron (MLP) architecture to predict protein concordance. A patient-derived spectral library has been employed for a novel peptide-level validation of proteins that are specific to the radiological regions using the SRM assay based targeted proteomics approach. The comprehensive proteomics enabled the identification of nearly 4000 proteins with high confidence (1%FDR ≥ 2 unique peptides) among which 170 proteins were differentially abundant in Skull base vs Supratentorial tumors (p-value ≤0.05). In silico analysis enabled mapping of the major alterations and hinted towards an overall perturbation of extracellular matrix and collagen biosynthesis components in the non-skull base meningiomas and a prominent perturbation of molecular trafficking in the skull base meningiomas. Therefore, this study has yielded novel insights into the functional association of the proteins that are differentially abundant in the two radiological subgroups. SIGNIFICANCE: In the current study, we have performed label-free proteomic analysis on fresh frozen tissue of 14 Supratentorial (NSB) and 7 Skull base meningiomas to assess perturbations in the global proteome, we have further employed an in-depth in silico analysis to map the pathways that have enabled functional mapping of the differentially abundant proteins in the Skull base and Supratentorial tumors. The findings from the above were also subjected to a machine learning-based neural networking to find out the proteins that have the most concordance of occurrence to determine the most influential proteins of the network. We further validated the differential abundance of identified protein markers in a larger patient cohort of Skull base and Supratentorial employing targeted proteomics approach to validate key protein candidates emerging from ours and other recent studies. The previous studies that have explored the skull base and convexity meningiomas have been able to reveal alterations in the genetic mutations in these tumor types. However, there are not many studies that have explored the functional aspects of these tumors, especially at the proteome level. We have attempted for the first time to map the functional modules associated with altered proteins in these tumors and have been able to identify that there is a possibility that the Skull base meningiomas to be considerably different from the Non-skull base (NSB) tumors in terms of the perturbed pathways. Our study employed global as well as targeted proteomics to examine the proteomic alterations in these two tumor groups. The study indicates that proteins that were more abundant in Skull base tumors were part of molecular transport components, non-skull base proteins majorly mapped to the components of extracellular matrix remodeling pathways. In conclusion, this study substantiates the distinction in the proteomic signatures in the skull base and supratentorial meningiomas paving way for further investigation of the identified markers for determining if some of these proteins can be used for therapeutic interventions for cases that pose considerable challenges for complete resection.

Sleep deprivation (SD) has been linked to impaired mental and physical health, obesity, and various diseases. However, the molecular mechanism underlying the effects of SD in the liver is still unclear. To investigate the metabolome and proteome alterations in the liver, an in vivo model of SD was established based on automated random motion platform techniques by applying a strategy of 10 consecutive days of 20 h of sleep deprivation +4 h of resting. The liver's altered metabolites and proteins were detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and data analyses were performed with MetaboAnalyst 5.0. This study found 15 differential metabolites, including 12 upregulated- metabolites and 3 downregulated- metabolites. A total of 493 proteins were differentially regulated, including 377 upregulated- proteins and 116 downregulated- proteins. The glutathione metabolism, fructose and mannose metabolism, and pyruvate metabolism pathways had significant effects on the sleep-deprived mouse livers. These three active pathways cause energy metabolism disorder and may induce obesity. In conclusion, this study demonstrates that SD could change the metabolism of glucose, and specific fatty acids, amino acids, and critical enzymes in the liver, providing a reference for the health effects of insufficient sleep. SIGNIFICANCE STATEMENT: So far, little is known about the changes in metabolites and proteins in the liver of individuals who suffer from SD. Metabolites and proteins in serum, urine and hypothalamus do not entirely reflect the effects of sleep deprivation on the whole body. In addition, many SD-induced models used the multiplatform water environment method, which causes mice to fall into the water frequently. Under this condition, the physical exertion of mice is extremely high, and it is not suitable for long-term sleep deprivation. The SD induction process has caused some influence on the model. Finally, few studies have elucidated the imbalance of energy metabolism caused by SD to induce obesity from the molecular mechanism. This study used a rotary table deprivation apparatus to trigger SD. This method will not cause excessive consumption and stimulation of mice. Furthermore, this study analyzed the metabolic and proteomic changes in the liver and enriched the range and means of metabolic and proteomic changes in sleep deprived mice. Finally, this research provides reference for elucidating the molecular mechanism of sleep deprivation causing energy metabolism disorders in the liver of mice.

In this compilation we collect information about the main protein components in hemolymph and stress the continued interest in their study. The reasons for such an attention span several areas of biological, veterinarian and medical applications: from the notions for better dealing with the species - belonging to phylum Arthropoda, subphylum Crustacea, and to phylum Mollusca - of economic interest, to the development of 'marine drugs' from the peptides that, in invertebrates, act as antimicrobial, antifungal, antiprotozoal, and/or antiviral agents. Overall, the topic most often on focus is that of innate immunity operated by classes of pattern-recognition proteins. SIGNIFICANCE: The immune response in invertebrates relies on innate rather than on adaptive/acquired effectors. At a difference from the soluble and membrane-bound immunoglobulins and receptors in vertebrates, the antimicrobial, antifungal, antiprotozoal and/or antiviral agents in invertebrates interact with non-self material by targeting some common (rather than some highly specific) structural motifs. Developing this paradigm into (semi) synthetic pharmaceuticals, possibly optimized through the modeling opportunities offered by computational biochemistry, is one of the lessons today's science may learn from the study of marine invertebrates, and specifically of the proteins and peptides in their hemolymph.

There are important challenges when investigating individual post-translational modifications (PTMs) or protein interaction network and delineating if PTMs or their changes and cross-talks are involved during infection, disease initiation or as a result of disease progression. Proteomics and in silico approaches now offer the possibility to complement each other to further understand the regulatory involvement of these modifications in parasites and infection biology. Accordingly, the current review highlights key expressed or altered proteins and PTMs are invisible switches that turn on and off the function of most of the proteins. PTMs include phosphorylation, glycosylation, ubiquitylation, palmitoylation, myristoylation, prenylation, acetylation, methylation, and epigenetic PTMs in P. falciparum which have been recently identified. But also other low-abundant or overlooked PTMs that might be important for the parasite's survival, infectivity, antigenicity, immunomodulation and pathogenesis. We here emphasize the PTMs as regulatory pathways playing major roles in the biology, pathogenicity, metabolic pathways, survival, host-parasite interactions and the life cycle of P. falciparum. Further validations and functional characterizations of such proteins might confirm the discovery of therapeutic targets and might most likely provide valuable data for the treatment of P. falciparum, the main cause of severe malaria in human.

Wheat is one of the most widely grown and important food crops in the world, providing approximately 20% of the food energy and protein produced for human consumption. The progress of wheat breeding is seriously restricted by the narrow genetic basis of common wheat germplasms. Dasypyrum villosum, a wild grass species that is commonly used in wheat improvement, has many excellent traits such as disease resistance, drought resistance, cold resistance, strong tillering ability, and processing quality. In this study, we compared and analyzed the cultivated wheat variety Chinese Spring (CS) and D. villosum using comparative proteomics. A total of 883 different abundant proteins (DAPs) were identified. Some of these different abundant proteins are associated with defense and stress, such as the Gα subunit, zinc finger protein family, PR1, HSP family, LEA protein, and serpin family. And a total of 24 different abundant proteins are gluten proteins. There are also 24 different abundant proteins associated with starch and sucrose metabolism. These results will provide potential candidate genes and a foundation for further research on resistance and quality for wheat genetics and breeding. SIGNIFICANCE: Proteins are the direct functional molecules of living organisms. It is of great significance to study the function of plant related genes from the perspective of protein. In this study, proteomics methods based on iTRAQ were used to compare the proteomic differences between wheat varieties Chinese Spring (CS) and D. villosum. The results provide novel insight into improving the quality of wheat. It is helpful to search for potential candidate genes for improving wheat quality and elucidate the molecular mechanisms associated with these genes.

In proteomics, the identification of peptides from mass spectral data can be mathematically described as the partitioning of mass spectra into clusters (i.e., groups of spectra derived from the same peptide). The way partitions are validated is just as important, having evolved side by side with the clustering algorithms themselves and given rise to many partition assessment measures. An assessment measure is said to have a selection bias if, and only if, the probability that a randomly chosen partition scoring a high value depends on the number of clusters in the partition. In the context of clustering mass spectra, this might mislead the validation process to favor clustering algorithms that generate too many (or few) spectral clusters, regardless of the underlying peptide sequence. A selection bias toward the number of peptides is desirable for proteomics as it estimates the number of peptides in a complex protein mixture. Here, we introduce an assessment measure that is purposely biased toward the number of peptide ion species. We also introduce a partition assessment framework for proteomics, called the Partition Assessment Tool, and demonstrate its importance by evaluating the performance of eight clustering algorithms on seven proteomics datasets while discussing the trade-offs involved. SIGNIFICANCE: Clustering algorithms are widely adopted in proteomics for undertaking several tasks such as speeding up search engines, generating consensus mass spectra, and to aid in the classification of proteomic profiles. Choosing which algorithm is most fit for the task at hand is not simple as each algorithm has advantages and disadvantages; furthermore, specifying clustering parameters is also a necessary and fundamental step. For example, deciding on whether to generate "pure clusters" or fewer clusters but accepting noise. With this as motivation, we verify the performance of several widely adopted algorithms on proteomic datasets and introduce a theoretical framework for drawing conclusions on which approach is suitable for the task at hand.

Background The study of molecular profiling of dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) contributes to understanding the high proliferation ability and multi-lineage differentiation potential. Objectives The aim of the study was to compare the protein abundance and specific markers of DPSCs and PDLSCs by protein profiles. Material and methods The DPSCs and PDLSCs extracted from the same tooth were lysed with 3 biological replicates and the protein was collected. Two-dimensional electrophoresis technology and TMT proteomics were used to separate and identify proteins. The data are available via ProteomeXchange with identifier PXD021997. The RT-qPCR detection of mRNA expression revealed a special marker for distinguishing two kinds of dental stem cells. Results Compared with PDLSCs, 962 differential proteins (DAPs) were up-regulated, and 127 were down-regulated in DPSCs. In the up-regulated DAPs, two high-scoring sub-networks were detected for neural-related molecules, which encode cell vesicle transport and mitochondrial energy transfer to regulate cell proliferation and secretion factors. A large number of cell adhesion molecules were distinguished among the highly expressed molecules of PDLSCs, supporting that stem cells provide cell attachment functions. It was interpreted ENPL, HS90A and HS90B were highly expressed in DPSCs, while CKB was highly abundant in PDLSCs. Another cell group confirmed that these molecules can be used as special biomarkers to identify and distinguish between DPSCs and PDLSCs. Conclusions This study can promote the basic research and clinical application of dental stem cells. Significance The high-throughput protein profiles were tested by combining two-dimensional gel proteomics and TMT-based proteomics. The proteomics of DPSCs and PDLSCs without individual difference demonstrated an accurate and comprehensive molecular expression profiles and interpretation of neural application potential, this study promotes the basic research of dental stem cells and clinical application.

Human milk is the first source of nutrition for infants, which delivers an array of unique bioactive components to offspring. Modern bovine-milk-based infant formulas are good substitutes when mother's milk is not available. As the third most abundant component in human milk, human free oligosaccharides (HMOs) may interference the analysis of total N-glycans on the glycoproteins in human milk. Herein, we combined acetone precipitation protein with the filter aided sample preparation method (FASP) to thoroughly remove HMOs and purify N-glycans. Furthermore, we also compared both N-glycosylation and glycoproteins between human and bovine milk, which may provide new ideas for the composition adjustment of infant formula in the food industry. SIGNIFICANCE: We described a new method, which can successfully remove HMOs, further extract and purify the N-glycans on glycoproteins from pooled human milk for MALDI-TOF MS analysis by applying acetone precipitation and FASP together. We applied the new method to purify the N-glycans from whey proteins in pooled bovine milk and compared the N-glycosylation differences between pooled human and bovine milk by MALDI-TOF MS. We first reported the difference of N-glycan pattern of glycoproteins between pooled bovine and human milk by lectin blotting, and found significant differences in types and abundance of glycoproteins between the two sourced milk.

Metabolomics databases contain crucial information collected from various biological systems and experiments. Developers and scientists performed massive efforts to make the database public and accessible. The diversity of the metabolomics databases arises from the different data types included within the database originating from various sources and experiments can be confusing for biologists and researchers who need further manual investigation for the retrieved data. Xconnector is a software package designed to easily retrieve and visualize metabolomics data from different databases. Xconnector can parse information from Human Metabolome Database (HMDB), Livestock Metabolome Database (LMDB), Yeast Metabolome Database (YMDB), Toxin and Toxin Target Database (T3DB), ReSpect Phytochemicals Database (ReSpectDB), The Blood Exposome Database, Phenol-Explorer Database, Kyoto Encyclopedia of Genes and Genomes (KEGG), and Small Molecule Pathway Database (SMPDB). Using Python language, Xconnector connects the targeted databases, recover requested metabolites from single or different database sources, reformat and repack the data to generate a single Excel CSV file containing all information from the databases, in an application programming interface (API)/ Python dependent manner seamlessly. In addition, Xconnector automatically generates graphical outputs in a time-saving approach ready for publication. SIGNIFICANCE: The powerful ability of Xconnector to summarize metabolomics information from different sources would enable researchers to get a closer glimpse on the nature of potential molecules of interest toward medical diagnostics, better biomarker discovery, and personalized medicine. The software is available as an executable application and as a python package compatible for different operating systems.

Duck enteritis virus (DEV), the causative agent of duck viral enteritis, causes a contagious, lethal viral disease in Anseriformes (waterfowls). In virus infection, host-virus interaction plays a crucial role in virus replication and pathogenesis. In our previous study, mRFP was fused with the C-terminus of DEV glycoprotein C (gC) to construct a fluorescent-tag DEV virus rgCRFP. In the current study, fluorescent fusion protein (gC-mRFP) was used as the proteomic probe. Co-immunoprecipitation and mass spectrometric analysis of proteins from rgCRFP-infected chicken embryo fibroblasts using commercial anti-RFP antibody led to the identification of a total of 21 gC interacting host proteins. Out of these 21 proteins, the interaction of seven host proteins (GNG2, AR1H1, PPP2CA, UBE2I, MCM5, NUBP1, HN1) with DEV gC protein was validated using membrane-bound split-ubiquitin yeast two-hybrid system (MbYTH) and bimolecular fluorescence complementation (BiFC) analyses. It indicated direct interaction between these proteins with DEV gC protein. This study has furthered the current understanding of DEV virus infection and pathogenesis. SIGNIFICANCE: gC is an crucial glycoprotein of duck enteritis virus that plays an important role in the viral life cycle. Uncovering the interaction between virus-host is very important to elucidate the pathogenic mechanism of the virus. In this study, host factors interacting with DEV gC have been discerned. And seven host proteins (GNG2, AR1H1, PPP2CA, UBE2I, MCM5, NUBP1, HN1) have been further validated to interact with DEV gC using MbYTH and BiFC analyses. These outcomes could shed light on how DEV manipulates the cellular machinery, which could further our understanding of DEV pathogenesis.

Immunoglobulin G (IgG) glycosylation corresponds well with immune system changes, so it can potentially be used as a biomarker for the consequences of chronic stress such as low-grade inflammation and enhanced immunosenescence in older animals. Here we present a high-throughput glycoproteomic workflow, including IgG enrichment, HILIC glycopeptide purification, and nano-LC-MS analysis of tryptic glycopeptides applied for the analysis of rat IgG. A cohort of 80 animals was exposed to seven stressors in a customized chronic stress protocol with blood and tissue sampling in three timepoints. Young female rats experienced an increase in agalactosylated glycoforms on IgG2a and IgG2c accompanied by a decrease in monogalactosylation. Among old females, increased galactosylation was observed in the IgG2b subclass, pointing to an anti-inflammatory activity of IgG. Additionally, IgG Fc N-glycosylation patterns in Sprague Dawley rats were analyzed, quantified, and reported for the first time. Our findings emphasize age-, sex- and subclass-dependent differences in IgG glycosylation related to chronic stress exposure, confirming the relevance of newly developed methods for further research in glycobiology of rodent immune response. SIGNIFICANCE: In this study, we showed that a high-throughput streamlined methodology based on protein L 96-well monolithic plates for efficient rat IgG immunoaffinity enrichment from blood plasma, paired with appropriate tryptic glycopeptide preparation, HILIC-SPE enrichment, and nano-LC-MS methods was suitable for quick processing of large sample sets. We report a subclass-specific profiling and changes in rat IgG Fc galactosylation and adrenal gland immunohistochemistry of male and female animals exposed to a customized chronic stress protocol.

Pharmaceutical compounds have been found in rivers and treated wastewaters. They often contaminate irrigation waters and consequently accumulate in edible vegetables, causing changes in plants metabolism. The main objective of this work is to understand how lettuce plants cope with the contamination from three selected pharmaceuticals using a label free proteomic analysis. A lettuce hydroponic culture, grown for 36 days, was exposed to metformin, acetaminophen and carbamazepine (at 1 mg/L), during 8 days, after which roots and leaves were sampled and analysed using a liquid chromatography-mass spectrometry proteomics-based approach. In roots, a total of 612 proteins showed differentially accumulation while in leaves 237 proteins were identified with significant differences over controls. Carbamazepine was the contaminant that most affected protein abundance in roots, while in leaves the highest number of differentially accumulated proteins was observed for acetaminophen. In roots under carbamazepine, stress related protein species such as catalase, superoxide dismutase and peroxidases presented higher abundance. Ascorbate peroxidase increased in roots under metformin. Cell respiration protein species were affected by the presence of the three pharmaceuticals suggesting possible dysregulation of the Krebs cycle. Acetaminophen caused the main differences in respiration pathways, with more emphasis in leaves. Lettuce plants revealed different tolerance levels when contaminants were compared, being more tolerant to metformin presence and less tolerant to carbamazepine. SIGNIFICANCE: The significant increase of emerging contaminants in ecosystems makes essential to understand how these compounds may affect the metabolism of different organisms. Our study contributes with a detailed approach of the main interactions that may occur in plant metabolism when subjected to the stress induced by three different pharmaceuticals (acetaminophen, carbamazepine and metformin).

Alcoholic liver disease (ALD) and nonalcoholic fatty liver disease (NAFLD) are typically asymptomatic and slow-progressing but potentially fatal diseases that are common causes of liver cirrhosis and related complications. Exosomes are nano-sized extracellular vesicles that have been linked to various intercellular communication processes and can carry biological materials reflecting the state and severity of disease. In this study, shotgun proteomic analysis of the protein expression profiles of extracellular vesicles, including exosomes and microvesicles, enriched from human serum samples of 24 patients diagnosed with various fatty liver diseases was performed using liquid chromatography tandem mass spectrometry (LC-MS/MS) followed by protein identification and label-free quantification using the MaxQuant platform. A total of 329 proteins, including 190 previously reported exosome-specific proteins, were identified from four types of liver disease, where significant differences in protein expression were found in apolipoproteins, immunoglobulins, and other previously reported markers of liver disease. Principal component analysis of 61 proteins identified from MaxQuant analysis of the LC-MS/MS data provided a confident differentiation between ALD and NAFLD. SIGNIFICANCE: The current investigation revealed the difference among various types of liver disease using LC-MS/MS of exosomes enriched from human serum samples of 24 patients where the most significantly up-regulation proteins were alpha-2-macroglobulin for alcoholic hepatitis and apolipoprotein C3 for nonalcoholic fatty liver disease.

The opportunistic fungal pathogen Trichosporon asahii (T. asahii) is an important causal agent of mortality in immunocompromised patients and associated with frequent relapses, even with sufficient antifungal treatment. Investigating the proteomes of initial and recurrent isolates may help to identify within-host adaptive changes. In this study, using tandem mass tag (TMT)-labeling combined with liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) technology, we analyzed the proteomes of two T. asahii strains that were isolated 15 years apart from the same patient who suffered initial and recurrent episodes of systemic disseminated trichosporonosis. A total of 597 differentially expressed proteins were identified. Functional analysis showed that the increased proteins were primarily concentrated on peptide/protein/energy/drug metabolism and translation. Most of the results were determined to be consistent with the findings of phenotypic assays, such as tests for drug susceptibility, temperature growth, biofilm formation, melanization and paromomycin assays. Moreover, we performed multiple reaction monitoring (MRM) mass spectrometry to verify 27 candidate proteins, and the results of this experiment were also highly consistent with the results of the TMT analysis. Therefore, to the best of our knowledge, these data provide the first molecular evidence of how the T. asahii proteome changes related to host-specific adaptation during human infection. SIGNIFICANCE: Systemic infection with Trichosporon asahii (T. asahii) has recently been recognized as an important causal agent of mortality in immunocompromised patients. Although triazole treatment usually works efficiently in the early phase of infection, many patients relapse. Hence, comparative analyses of the proteomics of initial and recurrent isolates may reveal evidence of adaptive changes within the host. Our study demonstrates that the recurrent strain has undergone proteomic changes using tandem mass tag (TMT)-labeling combined with liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). Moreover, the results of phenotypic assays, including drug susceptibility, temperature growth, biofilm formation, melanization and paromomycin assays, were highly consistent with the proteomic changes, and multiple reaction monitoring (MRM) verification also showed similar trends to the TMT results. In summary, our study is the first to investigate the adaptation of T. asahii under pressure from antifungal chemotherapy and host immune responses.