For centuries, crop plants have represented the basis of the daily human diet. Among them, cereals and legumes, accumulating oils, proteins and carbohydrates in their seeds, distinctly dominate modern agronomic practice. Indeed, these plants play an essential role in the food industry and fuel production. Therefore, the seeds of crop plants are intensively studied by food chemists, biologists, biochemists, and nutritional physiologists. Accordingly, not only seed development and germination, but also age- and stress-related alterations in seed vigor, longevity, nutritional value and safety can be addressed by a broad panel of analytical, biochemical and physiological methods. Currently, functional genomics is one of the most powerful tools, giving direct access to characteristic metabolic changes, accompanying plant development, senescence and response to biotic or environmental stress. Among individual methodological platforms, proteomics represents one of the most effective ones, giving access to cellular metabolism at the level of proteins. Here we discuss the main methodological approaches employed by seed proteomics in the context of physiological changes related to seed development, ageing and response to environmental stress.

Type 2 Diabetes Mellitus is a chronic metabolic disease that poses a significant long-term health risk due to its debilitating complications. While the diagnosis and monitoring of diabetes are well-established, the monitoring of its numerous complications presents a challenge, which can compromise patients' prognosis and quality of life. We analyzed the proteome of ten saliva samples, five from healthy individuals and five from patients with Type 2 Diabetes Mellitus. The aim was to identify potential biomarkers of Type 2 Diabetes Mellitus and its complications. We used PANTHER and FUNRICH tools to conduct a functional analysis and enrichment approach. Our findings revealed 622 proteins involved in 16 disrupted biological processes, which are linked to Type 2 Diabetes Mellitus. Metabolic processes, response to stimuli, processes of the immune system, and signaling are notable due to their known relation with Type 2 Diabetes Mellitus. Enrichment analysis has revealed that there are eleven biological processes that have been deregulated. These processes involve twenty different salivary proteins that are associated with multiple complications in diabetes. Our findings demonstrate that saliva is a dependable source of possible biomarkers that can be used to monitor diabetes and its complications.

Cryptosporidiosis is a widespread disease caused by the parasitic protozoan Cryptosporidium spp., which infects various vertebrate species, including humans. Once unknown as a gastroenteritis-causing agent, Cryptosporidium spp. is now recognized as a pathogen causing life-threatening disease, especially in immunocompromised individuals such as AIDS patients. Advances in diagnostic methods and increased awareness have led to a significant shift in the perception of Cryptosporidium spp. as a pathogen. Nowadays, genomic and proteomic studies have played a main role in understanding the molecular biology of this complex-life-cycle parasite. Genomics has enabled the identification of numerous genes involved in the parasite's development and interaction with hosts. Proteomics has allowed for the identification of protein interactions, their function, structure, and cellular activity. The combination of these two approaches has significantly contributed to the development of new diagnostic tools, vaccines, and drugs for cryptosporidiosis. In this review, we summarize the major accomplishments in the field of Cryptosporidium research using genomics and proteomics.

In 1984, Susumu Ohno hypothesized that the nylon-degrading enzyme NylB arose de novo via a frameshift mutation within a hypothetical precursor protein (PR.C). However, Ohno never tested his hypothesis or provided supporting biological evidence. For decades, Ohno’s famous frame-shift hypothesis has been uncritically accepted as the correct explanation for the origin of NylB and has been used to illustrate how simple it is for a totally new enzyme to arise spontaneously. In this paper we test Ohno’s hypothesis in light of data not available in 1984. We searched multiple protein databases and found that the NylB protein is widely occurring, has thousands of homologs, and is found in diverse organisms and diverse habitats. Conserved domain searches showed that the NylB sequence is homologous to beta lactamases - a family of highly conserved enzymes. However, our searches showed that there is no evidence for the existence of Ohno’s hypothetical PR.C protein, nor any credible homolog. Our results effectively falsify Ohno's frameshift hypothesis. We extended this analysis to other nylonases and found all the nylonases we examined had large numbers of homologs throughout the biosphere. This falsifies the long-held assumption that all nylonases evolved after the invention of nylon in 1935.

Recently we have seen a relaxation of the historic restrictions on the use and subsequent research on the Cannabis plants, generally classified as Cannabis sativa and Cannabis indica. What research has been performed to date has centered on chemical analysis of plant flower products, namely cannabinoids and various terpenes that directly contribute to phenotypic characteristics of the female flowers. In addition, we have seen many groups recently completing genetic profiles of various plants of commercial value. To date, no comprehensive attempt has been made to profile the proteomes of these plants. We report herein our progress on constructing a comprehensive draft map of the Cannabis proteome. To date we have identified over 17,000 potential protein sequences. Unfortunately, no annotated genome of Cannabis plants currently exists. We present a method by which “next generation” DNA sequencing output and shotgun proteomics data can be combined to produce annotated FASTA files, bypassing the need for annotated genetic information altogether in traditional proteomics workflows. The resulting material represents the first comprehensive annotated protein FASTA for any Cannabis plant. Using this annotated database as reference we can refine our protein identifications, resulting in the confident identification of 13,000 proteins with putative function. Furthermore, we demonstrate that post-translational modifications play an important role in the proteomes of Cannabis flower, particularly lysine acetylation and protein glycosylation. To facilitate the evolution of analytical investigations into these plant materials, we have created a portal to host resources we have developed from proteomic and metabolomic analysis of Cannabis plant material as well as our results integrating these resources. All data for this project is available to view or download at www.CannabisDraftMap.Org

We report on the state of the art of proteins recognized as potential targets for the development of leishmania treatments through the search of biologically active chemical species, either from experimental in vitro, in vivo, or in silico sources. We classify the gathered information, in several ways: vector taxonomy and geographical distribution, leishmania parasite taxonomic and geographical distribution and enzymatic function (oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases and cytokines). Our aim is to provide a much needed reference layout for research efforts aimed to understand the background of ligand-protein activation/inactivation processes, in this specific case, related with enzymes known to be part of biochemical cascade reactions initiated following a leishmania infectious episode.

There is evidence that spaceflight poses acute and late risks on the central nervous system. To explore possible mechanisms, the proteomic changes following spaceflight in mouse brain were characterized. Space Shuttle Atlantis (STS-135) was launched at the Kennedy Space Center on a 13-day mission. Within 3–5 hours after landing, brain tissue was collected to evaluate protein expression profiles using quantitative proteomic analysis. Our results showed that there were 26 proteins that were significantly altered after spaceflight in the grey and/or white matter. While there was no overlap between the white and grey matter in terms of individual proteins, there was overlap in terms of function, synaptic plasticity, vesical activity, protein/organelle transport, and metabolism. Our data demonstrate that exposure to the spaceflight environment induces significant changes in protein expression related to neuronal structure and metabolic function. This might lead to a significant impact on brain structural and functional integrity that could affect the outcome of space missions.

Seafood is considered one of the main food allergen sources by the European Food Safety Authority (EFSA). It comprises several distinct groups of edible aquatic animals including fish and shellfish such as crustacean and mollusks. Recently the EFSA recognized the high risk of food allergy over the world and established the necessity of developing new methodologies for its control. Consequently, accurate, sensitive and fast detection methods for seafood allergy control and detection in food products are highly recommendable. In this work, we present a comprehensive review of the applications of the proteomics methodologies for the detection and quantification of seafood allergens. For that, two consecutive proteomics strategies (Discovery and Targeted Proteomics) applied for the study and control of seafood allergy are reviewed in detail. In addition, future directions and new perspectives were also provided.

Preeclampsia (PE) is a serious complication of pregnancy with a pathogenesis that is not fully understood, though it involves the impaired invasion of extravillous trophoblasts (EVTs) into the decidual layer during implantation. Recently, others have found that diverse cell types, including EVTs, produce exosomes filled with molecular cargo (mainly proteins and RNAs) that can be transported to other cells and organs both locally and long-range. The cargo delivered by exosomes can signal to and modify the receiving cells and their environment, and EVT-derived exosomes may influence the pathogenesis of PE. Because the risk of PE is actually decreased by cigarette smoking, we considered the possibility that nicotine, a critical component of tobacco smoke, might protect against PE by modifying the content of exosomes from EVTs. In this study, we applied nicotine stimulation to cultured EVTs and subjected their secreted exosomes to proteomic analysis. We identified many proteins whose abundance in exosomes was modified by nicotine treatment of the donor EVTs, and we used bioinformatic annotation and network analysis to select five key hub proteins with potential roles in the pathogenesis or prevention of PE.

Matrix metalloproteases (MMPS) are a family of well-known enzymes which operate prevalently in the extracellular domain, where they fulfil the function of remodeling the extracellular matrix. Within the about 26 family members, encoded by 24 genes in humans, MMP-2 and MMP-9, have been regarded as the primary responsibility for the basement membrane and pericellular ECM rearrangement. In cases of infiltrating carcinomas, which arise from the epithelial tissues of a gland or of an internal organ, a marked alteration of the expression and the activity levels of both MMPs is known to occur. Present investigation represents the continuation and upgrading of our previous studies, now focusing on the occurrence and intensity levels of MMP-2 and -9, and their proteomic correlations, in a cohort of 80 breast cancer surgical tissues

Meningiomas are the most prevalent primary intracranial tumors. The majority are benign but can undergo dedifferentiation in grades classified from I to III. Meningiomas tremendous variability in tumor behavior and slow growth rates complicate their diagnosis and treatment. A deeper comprehension of the molecular pathways and cellular microenvironment factors implicated in meningioma survival and pathology is needed. This review summarizes the known genetic and epigenetic aberrations involved in meningioma, with a focus on Neurofibromatosis type 2 (NF2) and non-NF2 mutations. Novel potential biomarkers for meningioma diagnosis and prognosis are also discussed, including epigenetic-, RNA-, and protein-based markers. Finally, the landscape of available meningioma-specific animal models is overviewed. Use of these animal models can enable planning of adjuvant treatment, potentially assisting in preoperative and postoperative decision-making. Discovery of novel biomarkers will allow more precise meningioma grading, including meningioma identification, subtype determination, and prediction of metastasis, recurrence, and response to therapy. Moreover, these biomarkers may be exploited in the development of personalized targeted therapies that can distinguish between the 15 diverse meningioma subtypes.

Tandem mass tag (TMT)-based proteomics facilitate multiplexing in mass spectrometry (MS)-based quantification and identification of proteins and their post-translational modifications. The use of TMT isobaric tags can enable multiplexing of up to 18 samples using commercially available kits. A single TMT experiment can quantify proteome, serine, threonine phosphorylation, and tyrosine phosphorylation. Of note, tyrosine phosphorylation is of low abundance, and identification/quantification can be improved using two complementary strategies. First, by employing SH2 superbinder which increases the number of identified sites. The SH2 Superbinder is more cost-effective than the commonly used phosphotyrosine antibodies. Second, by employing phosphotyrosine booster strategy, a pervanadate-treated channel to boost the signal of low-abundant phosphotyrosine. Noteworthy, pervanadate boost increases the likelihood of low abundant peptide to be selected for MS2, and facilitating the detection of > 6000 proteins, 10,000 unique pS/T and 1000 unique pY sites.

Cancer is a complex systemic disease that changes the entire proteome. The analysis of this transformation makes it possible to determine tumor markers, that is, the most characteristic biomacromolecules produced by tumor cells. Here, the question of finding ideal tumor markers, which should be sensitive, specific, and reliable, is an acute issue. Unfortunately, none of the tumor markers, even those used in the clinic, has all these characteristics. Despite this, many tumor markers have demonstrated excellent clinical relevance for monitoring the effectiveness of different treatments for cancer patients. The use of markers also aids in the early detection of cancer recurrence and prognosis. Therefore, the situation in this area can be improved in two ways – by attempting to find an ideal single tumor marker or generating panels of different markers. In both cases, proteomics certainly plays a major role. Human plasma is one of the most popular samples as it is commonly collected in the clinic and provides noninvasive, rapid analysis for any type of disease including cancer. Many efforts have been applied in searching for “ideal” tumor markers digging very deep plasma proteome. There is a line of evidence that the most abundant, so-called “classical plasma proteins”, may be used to generate a tumor biomarker profile. To be comprehensive these profiles should have information not only about protein levels but proteoform distribution for each protein. Initially, the profile of these proteins in norm should be generated. Here, we present data about these profiles generated by two-dimensional electrophoresis with the following mass-spectrometry and immunodetection.

The deadliest disease in the world, cancer, kills many people every year. The early detection is the only hope for the survival of malignant cancer patients. As a result, in the preliminary stages of , the diagnosis of cancer biomarkers at the cellular level is critical for improving cancer patient survival rates. For decades, scientists have focused their efforts on the invention of biosensors. Biosensors, in addition to being employed in other practical scenarios, can essentially function as cost effective and highly efficient devices for this purpose. Traditional cancer screening procedures are expensive, time-consuming, and inconvenient for repeat screenings. Biomarker-based cancer diagnosis, on the other hand, is rising as one of the most potential tools for early detection, disease progression monitoring, and eventual cancer treatment. As Biosensor is an analytical device, it allows the selected analyte to bind to the biomolecules being studied (– for example RNA, DNA, tissue, proteins, cells). They can be divided based on the kind of biorecognition or transducer elements on the sensor. Most biosensor analyses necessitate the analyte being labeled with a specific marker. In this review article, the application of distinct variants of biosensors against cancer has been described.

In the longtime challenge of identifying specific, easily-detectable and reliable biomarkers of Idiopathic Pulmonary Fibrosis (IPF), bronchoalveolar lavage fluid (BALF) proteomics is providing interesting new insights into its pathogenesis. To the best of our knowledge, the present study is the first shotgun proteomic investigation of EVs isolated from BALF of IPF patients. Our main aim was to characterize the proteome of the vesicular component of BALF and to explore its individual impact on the pathogenesis of IPF. To this purpose, ultracentrifugation was chosen as EVs isolation technique and their purification was assessed by TEM, 2DE and LC-MS/MS. Our 2DE data and scatter plots showed considerable differences between the proteome of EVs and that of whole BALF and of its fluid component. Analysis of protein content and protein functions evidenced that EV proteins are predominantly involved in cytoskeleton remodeling, adenosine signaling, adrenergic signaling, C-peptide signaling and lipid metabolism. Our findings may suggest a wider system involvement in the disease pathogenesis and support the importance of pre-fractioning of complex samples, like BALF, in order to let low-abundant proteins-mediated pathways to emerge.

As lung cancer shows the highest mortality in cancer related death, serum biomarkers are demanded for the lung cancer diagnosis and its treatment. To discover lung cancer protein biomarkers, secreted proteins from primary cultured lung cancer and adjacent normal tissues from patients were subjected to LC/MS-MS proteomic analysis. Quescin sulfhydryl oxidase(QSOX1) was selected as a biomarker candidate from the proteins enriched in the secretion of lung cancer cells. QSOX1levels were higher in 82% (51 of 62 tissues) of lung cancer tissues compared to adjacent normal tissues. Importantly, QSOX1 serum levels were significantly higher in cancer patients (p<0.05, AUC=0.89), when measured by multiple reaction monitoring(MRM). Higher levels of QSOX1 are also uniquely detected in lung cancer tissues among several other solid cancers by immunohistochemistry. QSOX1 knock-downed Lewis lung cancer (LLC) cells was less viable from oxidative stress and had reduced migration and invasion. In addition, LLC mouse models with QSOX1 knock-down also proved that QSOX1 functions in promoting cancer metastasis. In conclusion, QSOX1 might be a lung cancer tissue-derived biomarker and involved in the promotion of lung cancers, and thus can be a therapeutic target for lung cancers.

Due to low culturing costs and high seed protein contents, legumes represent the main global source of food protein. Pea (Pisum sativum L.) is one of the major economically important legume crops, impacting both animal feed and human nutrition. Therefore, the quality of pea seeds needs to be ensured in the context of sustainable crop production and nutritional efficiency. Obviously, changes in seed protein patterns might directly affect both of these aspects. Thus, here we address the pea seed proteome in more detail and provide, to the best of our knowledge, the most comprehensive annotation of the functions and intracellular localization of pea seed proteins. Accordingly, 1938 and 1989 non-redundant proteins were identified in yellow and green pea seeds, in total. Only 35 and 44 proteins, respectively, could be additionally identified after protamine sulfate precipitation (PSP) potentially indicating the high efficiency of our experimental workflow. In total 981 protein groups could be assigned to 34 functional classes, which were to a large extent differentially represented in yellow and green seeds. Closer analysis of these differences by processing of the data in KEGG and String databases revealed their possible relation to a higher metabolic status and reduced longevity of green seeds.

Synthesis and antitumour activity of a palladium compound, [Bis (quinoline) palladium (II) chloride] (coded as NH1) alone and in combination along with its proposed mechanism of action has been described in the present study. Antitumour activity has been performed in seven different cancer cell lines using colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The investigated palladium compound demonstrated superiority over clinical standard cisplatin against drug resistant ovarian cancer cell lines. NH1 also demonstrates 4.4 times greater activity than cisplatin against breast cancer model. Binary sequenced combination study showed that NH1 in combination with curcumin is mostly additive to antagonistic. However, combination of NH1 with 6-shogaol demonstrated synergism depending on dose and sequence. Proteomic study revealed that changes in expression of 14 proteins were significantly associated with anticancer mechanism of NH1. In vivo anticancer activity of NH1 at a dose of 0.75 mg/kg was also evaluated using Erlich Ascites Carcinoma (EAC) cell lines in Swiss-albino mice model. Preliminary toxicity study in Swiss-albino mice proved that NH1 is comparatively less toxic at both of the administered doses, either 2.5 mg/kg or 5 mg/kg. However, cisplatin demonstrated significant toxicity at both of the administered doses. Biochemical investigations also supported that NH1 demonstrated lesser toxicity profile towards liver or kidney compared to clinical standard cisplatin.

Omics technologies, viz., genomics, transcriptomics, proteomics, metabolomics, and phenomics, are becoming an integral part of virtually every commercial cereal breeding program because they provide substantial dividends per unit time in both pre-breeding and breeding phases. Continuous advances in cereal-omics promise—in combination with time efficiency—the cost benefits. In this review, we provide a comprehensive overview of the established cereal-omics methods in five major cereals, viz., rice, sorghum, maize, barley, and bread wheat. We cover the evolution of technologies in each omics section independently and concentrate on their use to improve economically important agronomic as well as biotic and abiotic stress-related traits. Advancements in the (1) identification, mapping, and sequencing of molecular/structural variants, (2) high-density transcriptomics data to study gene expression patterns, (3) global and targeted proteome profiling to study protein structure and interaction, (4) metabolomic profiling to quantify organ level small-density metabolites and their composition, and (5) high-resolution high-throughput image-based phenomics approaches are surveyed in this review.

Cotton fiber development transition from elongation to secondary cell wall biosynthesis is a critical growth shifting phase that affects cotton fiber final length, strength and other properties. Morphological dynamic analysis indicates that an asynchronous fiber developmental pattern between two cotton species. The critical time point for Gh and Gb fiber elongation termination is, respectively, 23 and 27 days post-anthesis (dpa). The temporal changes of protein expression at three representative development periods (15–19, 19–23, 23–27 dpa) were examined in both species with iTRAQ technics. Strikingly, a large proportion of differentially expressed proteins (DEPs) was identified at 19–23 dpa in Gh or at 23–27 dpa in Gb, corresponding to their fiber developmental transition timing from elongation to secondary cell wall biosynthesis. To better understand fibers transitional development, we comparatively analyzed those DEPs in 19–23 dpa of Gh vs. in 23–27 dpa of Gb, and noted that these cotton species indeed share fundamentally similar fiber development features under the biological processes. It also showed that there have limited overlaps in both specific upregulated and downregulated proteins between the two species, suggesting specie-specific protein regulations in development. Proteomic profiling revealed dynamic changes of several key proteins and biological processes that potentially correlate with fiber development transition. During the transition, upregulated proteins mainly involved in carbohydrate/energy metabolism, oxidation-reduction, cytoskeleton, protein turnover, Ca²⁺ signaling etc, whereas important downregulated proteins mostly concentrated in phenylpropanoid and flavonoid secondary metabolism pathways. Several changed proteins in this key stage were also validated by qRT-PCR. Overall, the present study provides accurate pictures of the regulatory networks of functional proteins during the fiber developmental transition.

SAA is an acute phase protein that elevates under inflammatory circumstance. The serum level of SAA was associated with the progression of inflammation in numerous diseases. However, little attention was paid to the correlation between SAA and ONFH. In this study, SAA was found risen in the femoral head of osteonecrosis patients through proteomics analysis and further confirmed by ELISA. Furthermore, SAA was proven affecting bone metabolism in rBMSCs. It facilitated the proliferation of rBMSCs whereas it suppressed the osteogenic differentiation of rBMSCs and accelerated the adipogenic differentiation of rBMSCs. Thus, we deem that serum amyloid A, which is a vital acute phase protein in inflammation, affected bone metabolis and plays an imperative role in the pathophysiological process of ONFH.

In this review we extensively describe the main post-translational modifications that give rise to the multiple proteoforms characterized to date in the human salivary proteome and their potential role. Most of the data reported were obtained by our group in over twenty-five years of research carried out on human saliva mainly by applying a top-down strategy. At the beginning we describe the products generated by proteolytic cleavages, which can occur before and after secretion. In this section the most relevant families of salivary proteins are also described. Next, we report the current information concerning the human salivary phospho-proteome and the limited news available on sulfo-proteome. Three sections are dedicated to the description of the glycation and enzymatic glycosylation. Citrullination and N- and C- terminal PTMs and a miscellaneous of other modifications are described in the last two sections. Results highlighting the variation in the level of some proteoforms in local or systemic pathologies are also reviewed along the sections of manuscript to underline the impact and relevance of this information for the development of new diagnostic biomarkers useful in clinical practice.

Mesenchymal stem cells (MSCs) are an attractive therapeutic tool for tissue engineering and regenerative medicine owing to their regenerative and trophic properties. The best-known and most widely used are bone marrow MSCs which are currently being harvested and developed from a wide range of adult and perinatal tissues. MSCs from different sources are believed to have different secretion potentials and production which may influence their therapeutic effects. To confirm it, we performed a quantitative proteomic analysis based on the TMT technique of MSCs from three different sources: Wharton’s jelly (WJ), dental pulp (DP) and bone marrow (BM). Our analysis focused on MSC biological properties of interest for tissue engineering. We identified a total of 611 differentially expressed human proteins. WJ-MSCs showed the greatest variation compared with the other sources. WJ produced more extracellular matrix (ECM) proteins and ECM-affiliated proteins and appeared more able to modulate the inflammatory and immune response. BM-MSCs displayed enhanced differentiation and paracrine communication capabilities. DP-MSC appeared to promote exosome production. The results obtained confirm the existence of differences between WJ, DP and BM-MSC and the need to select the MSC origin according to the therapeutic objective sought.

Our understanding of the long-term consequences of chronic ionising radiation for living organisms remains scarce. Modern molecular biology techniques are helpful tools for researching pollutant effects on biota. To reveal the molecular phenotype of plants growing under chronic radiation exposure, we sampled Vicia cracca L. plants in the Chernobyl Exclusion Zone and in areas with normal radiation backgrounds. We performed a detailed analysis of soil and gene expression patterns, and made coordinated multi-omics analyses of plant samples, including transcriptomics, proteomics, and metabolomics. Chronic exposure to ionising radiation induced complex and multidirectional changes, including significant alterations in the metabolism and gene expression patterns of irradiated plants. We revealed profound changes in carbon metabolism, nitrogen reallocation, and photosynthesis. These plants were characterised by increased DNA damage, redox imbalance, and stress response reactions. The upregulation of histones, chaperones, peroxidases, and secondary metabolism were noted.

Legume seed protein is an important source of nutrition, but it is less digestible than animal protein. Poor protein digestibility in legume seeds and seedlings may partly reflect defences against herbivores. Protein changes during germination typically increase proteolysis and digestibility, by lowering the levels of anti-nutrient protease inhibitors, activating proteases, and breaking down storage proteins (including allergens). Germinating legume sprouts also show striking increases in free amino acids (especially asparagine), but their roles in host defence or other processes are not known. While the net effect of germination is generally to increase the digestibility of legume seed proteins, the extent of improvement in digestibility is species and strain dependent. Further research is needed to highlight which changes contribute the most to improved digestibility of sprouted seeds. Such knowledge could guide the selection of varieties that are more digestible, and also guide the development of food preparations that are more digestible, potentially combining germination with other factors altering digestibility, such as heating and fermentation. Techniques to characterize the shifts in protein make-up, activity and degradation during germination need to draw on traditional analytical approaches, complemented by proteomic and peptidomic analysis of mass spectrometry identified peptide breakdown products.

Left Ventricular Outflow Tract (LVOT) obstruction occurs in approximately 70% of Hypertrophic Cardiomyopathy (HCM) patients and currently requires imaging or invasive testing for diagnosis, sometimes in conjunction with provocative physiological or pharmaceutical stimuli. To identify potential biomarkers of LVOT obstruction, we performed proteomics profiling of 1305 plasma proteins in 12 HCM patients with documented LVOT obstruction referred for surgical myectomy. Plasma was collected at the surgical preoperative visit approximately one month prior to surgery and then at the post surgical visit approximately 3 months later. Proteomic profiles were generated using the aptamer-based SOMAscan assay. Principal Component Analysis using the highest statistically significant proteins separated all preoperative samples from all postoperative samples. Further analysis revealed a set of 25 proteins that distinguished the preoperative and postoperative states with a paired t-test p value of <0.01. Ingenuity Pathway analysis facilitated the generation of protein interaction networks and the elucidation of key upstream regulators of the differentially expressed proteins such as interferon-, TGF-1 and TNF. Biological pathways affected by the surgery included organ inflammation, migration and motility of leukocytes, fibrosis, vasculogenesis, angiogenesis, acute coronary events, endothelial proliferation, eicosanoid metabolism, calcium flux, apoptosis and morphology of the cardiovascular system. Our results indicate that surgical relief of dynamic outflow tract obstruction in HCM patients is associated with unique alterations in plasma proteomic profiles that likely reflect improvement in organ inflammation and physiological function.

Chronic heat stress (HS), aggravated by global warming, reduces the production efficiency of the buffalo dairy industry. Here, we conducted a proteomic analysis to investigate the adaptation strategies used by buffalo in response to heat stress. Seventeen differentially abundant proteins with known functions were detected using label-free quantification (LFQ), and five of these differentially expressed proteins were validated with parallel reaction monitoring (PRM). These five proteins were associated with various aspects of heat stress, including decreased heat production, increased blood oxygen delivery, and enhanced natural disease resistance. Lipase (LPL), glutathione peroxidase 3 (GPX3), cathelicidin-2 (CATHL2, LL-37), ceruloplasmin (CP), and hemoglobin subunit alpha 1 (HBA1) were shown to play cooperative roles in the tolerance of chronic HS in dairy buffalo. We found that high levels of HBA1 increased blood oxygen transport capacity. Our results increase our understanding of the adaptation of dairy buffalo to chronic heat stress.

Leaf variegation pale yellowing is observed in the Calcineurin B-Like-Interacting Protein Kinase14 (CIPK14) overexpression line (oeCIPK14) and double knockout WHIRLY1/WHIRLY3 (why1/3) lines of Arabidopsis, the distribution of WHIRLY1 (WHY1) protein between plastids and the nucleus are affected by the phosphorylation of WHY1 by CIPK14. To elucidate the coregulation of CIPK14 and WHIRLY1/WHIRLY3 mediated leaf pale yellowing, a differential proteomic analysis is conducted between the oeCIPK14 variegated (oeCIPK14-var) line, why1/3 variegated (why1/3-var) line and wild type (WT). More than 800 protein spots are distinguished on each gel, 67 differential abundance proteins (DAPs) are identified by matrix-assisted laser desorption ionization-time of flight/time of flight mass spectrometry (MALDI-TOF/TOF-MS), of which, 34 DAPs are in the oeCIPK14-var, 33 DAPs are in the why1/3-var compared to WT. Five overlapping proteins differentially change both in the oeCIPK14-var and in the why1/3-var. They are ATP-dependent Clp protease proteolytic subunit-related protein 3 (ClpR3), Ribulose bisphosphate carboxylase large chain (RBL), Beta-amylase 3 (BAM3), Ribosome-recycling factor (RRF), Ribulose bisphosphate carboxylase small chain (RBS). Bioinformatics analysis show that most of DAPs are involved in photosynthesis, defense and antioxidation pathway, protein metabolism, amino acid metabolism, energy metabolism, malate biosynthesis, lipid metabolism and transcription. Thus, the photosystem parameters are measured that the content of chlorophyll, the photochemical efficiency of PSⅡ (Fv/Fm), and electron transport rates (ETR) decrease in the why1/3-var and oeCIPK14-var, but the non-photochemical quenching (NPQ) increases. Both mutants show high sensitivity to strong light. Based on the annotation of DAPs from both why1/3-var and oeCIPK14-var lines, we conclude that CIPK14 phosphorylation mediated WHY1 deficiency in plastids is related to impairment of protein metabolism leading to chloroplast dysfunction.

Cancer is a complex and heterogeneous disease, influenced by various factors that affect its progression and response to treatment. Although histopathological diagnosis is crucial for identifying and classifying cancer, it may not accurately predict the disease's development and evolution in all cases. To address this limitation, liquid biopsy has emerged as a valuable tool, enabling a more precise and non-invasive analysis of cancer. Liquid biopsy offers the ability to detect tumor DNA fragments, circulating tumor cells, and exosomes released by cancer cells into the bloodstream. Exosomes, in particular, have attracted significant attention in cancer research due to their specific protein composition, which can provide valuable insights into the disease. The protein profile of exosomes often differs from that of normal cells, reflecting the unique molecular characteristics of cancer. Analyzing these proteins can help identify cancer-associated markers that play critical roles in tumor progression, invasion, and metastasis. Ongoing research and clinical validation are essential to advance and effectively utilize protein biomarkers in cancer. Nevertheless, their potential to improve diagnosis and treatment is highly promising. This review discuss several exosome proteins of interest in breast cancer, particularly focusing on studies conducted in mammary tissue and cell lines in humans and experimental animals. Unfortunately, the studies conducted in the canine species are scarce. This emphasis sheds light on the limited research available in this field. Additionally, we present a curated selection of studies that have explored exosomal proteins as potential biomarkers, aiming to achieve the aforementioned benefits in breast cancer diagnosis, prognosis, monitoring, and treatment.

Information regarding physiological and molecular plant responses to the treatment with new biofertilizers is limited. In this study a fast-composting soil amendment (FCA), obtained from solid waste by means of a Fenton reaction, was tested to evaluate the effects on the growth of Lactuca sativa L. var. longifolia seedlings. Growth rate, leaves and roots biomass, chlorophyll concentration and total soluble proteins of seedlings treated with the 2% FCA soil amendment showed significant increase in comparison with control seedlings. Leaf proteomic analysis revealed that the FCA soil amendment induced the up-regulation of proteins belonging to photosynthesis machinery, carbohydrate metabolism and promoted the up-regulation of the water stress response. Root proteomics indicated that FCA strongly induced the organs morphogenesis and developments; root cap development, lateral root formation, post-embryonic root morphogenesis were the main biological processes enriched by the treatment. Overall, our data suggest that addition of FCA formulation to the base soils might ameliorate plant growth by inducing carbohydrates primary metabolism and the differentiation of a robust root system.

High blood cholesterol levels are one of the main risks for atherosclerotic disease. A purified aqueous extract of Fucus vesiculosus, characterized as rich in phlorotannins and peptides, was described as having the capacity to inhibit cholesterol biosynthesis and intestinal absorption. In this work, the effect of this extract on intestinal cells metabolites and proteins was analysed, aiming to expand the knowledge about its mode of action targeting cholesterol metabolism, in particular exogenous cholesterol absorption and transport. Caco-2 cells differentiated into enterocytes were exposed to the purified aqueous extract of F. vesiculosus and analysed by undirected metabolomics and proteomics. The results of the metabolomic analysis showed only statistically significant differences in glutathione content of the cells exposed to the extract relatively to the control cells, with a decreased glutathione expression in exposed cells. The proteomic analysis showed an increased expression for cells exposed to the extract of NPC1, an important protein known to be involved in cholesterol transport. To extent of our knowledge this is the first study using untargeted metabolomics and proteomic analysis to study the effect of F. vesiculosus on differentiated Caco-2 cells, which aims to provide some insight about the molecular mechanism of extract compounds on intestinal cells.

Age-related macular degeneration (AMD) is a common ocular disease characterized by the de-generation of the central area of the retina in elderly population. Progression and response to treatment is influenced by genetic and non-genetic factors. Proteomics is a powerful tool to study, at the molecular level, the mechanisms underlaying the progression of the diseases, to identify new therapeutical targets and to establish biomarkers to monitor progression and treatment ef-fectiveness. In this work we pursue to systematically review the use of proteomic-based ap-proaches for the study of the molecular mechanisms underlying the development of AMD, as well as the progression of the disease and the on-treatment patient monitoring. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) reporting guidelines were followed. Proteomic approaches have identified key players on the onset of the disease, such as proteins involved in lipid metabolism and oxidative stress, but also in the progression to advanced stages, including factors related to extracellular matrix integrity and angiogenesis. Although an-ti-vascular endothelial growth factor (anti-VEGF)-based therapy has been crucial in the treatment of neovascular AMD it is necessary to get deeper into the underlying disease mechanisms to move forward to next-generation therapies of the later-stage forms of this multifactorial disease.

Proteomic technology has improved at a staggering pace in recent years, with even practitioners challenged to keep up with new methods and hardware. The most common metric used for method performance is the number of peptides and proteins identified. While this metric may be helpful for proteomics researchers shopping for new hardware, this is often not the most biologically relevant metric. Biologists often utilize proteomics in the search for protein regulators that are of lower relative copy number in the cell. In this review, I re-evaluate untargeted proteomics data using a simple graphical representation of the absolute copy number of proteins present in a single cancer cell as a metric. By comparing single shot proteomics data to the coverage of the most in-depth proteomic analysis of that cell line acquired to-date we can obtain a rapid metric of method performance. Using a simple copy number metric allows visualization of how proteomics has developed in both sensitivity and overall dynamic range when using both relatively long and short acquisition times. To enable reanalysis beyond what is presented here, two available web applications have been developed for single and multi-experiment comparisons with reference protein copy number data for multiple cell lines and organisms.

The early detection of cutaneous melanoma, a potentially lethal cancer with rising incidence, is key to increase survival and therapeutic adjustment. Especially in stages II-IV biomarkers are urgently needed for adjuvant therapy purposes after resection and for treatment of metastatic patients. We here investigated if fatty acid (FA) and protein composition of small extracellular vesicles (sEV) deriving from plasma of 0-I, II, and III-IV stage melanoma patients (n=38) could reflect disease stage and thus function as biomarker. The subpopulation of sEV expressing CD81 (CD81sEV) was isolated by an ad hoc immune affinity technique from microvesicle-depleted plasma. Biological macromolecules were investigated by gas chromatography and mass spectrometry in CD81sEV. A higher content of FA and a decrease in Saturation Index (C18:0/C18:1), already detectable in early stages, distinguished patients’ from healthy donor CD81sEV. Proteomics (identifier PXD024434) detected an exclusive and significant increase of CD14, PON1, PON3 and APOA5 in stage II and a significant decrease of Rap1b in stage III-IV CD81sEV. The FA and proteomic stage dependent CD81sEV signature strengthens the potential of circulating sEV studies in providing discriminatory information for early diagnosis, prediction of metastatic behavior and follow up of melanoma patients.

1) Background: Fetal Growth Restriction (FGR) has been associated with adverse perinatal outcomes and epigenetic modifications that impact gene expression leading to permanent changes of fetal metabolic pathways and thereby influence development of disease in childhood and adult life. Both clinical and experimental studies showed that maternal nutrition during pregnancy is critical since malnutrition adversely affects fetal growth and physiology. In this study, we investigated the result of maternal food restriction on liver protein expression in Wistar male newborn pups. (2) Materials & methods: Pups born to food restricted mothers were subdivided to FGR and non-FGR groups. Livers of control, FGR and non-FGR groups were analyzed using quantitative proteomics. (3) Results: In total 6665 proteins were profiled. Of these, 451 and 751 were differentially expressed in FGR and non-FGR vs. control respectively, whereas 229 were common between the two groups. Bioinformatics analysis of the differentially expressed proteins (DEPs) in FGR vs. control revealed: induction of the super-pathway of cholesterol biosynthesis and inhibition of thyroid hormone metabolism, fatty acid beta oxidation and apelin liver signaling pathway. In the DEPs of non-FGR vs. control groups there was inhibition of thyroid hormone metabolism, fatty acid beta oxidation and apelin liver signaling pathway as well. (4) Conclusion: This study demonstrates the impact of prenatal food restriction on the proteomic liver profile of FGR and non-FGR offspring underlying the importance of both prenatal adversities and birth weight on liver dependent postnatal disease.

Photosynthetic function, photoprotection, and the response of related proteomics of mulberry (Morus alba L.) seedling leaves under NaCl and NaHCO3 stress with the same Na+ concentration (100 mmol•L-1) were studied by using photosynthetic gas exchange and chlorophyll fluorescence techniques combined with TMT proteomics. The results showed that NaCl stress had no significant effect on photosystem II (PSII) activity in mulberry seedling leaves, and the expressions of the related proteins, OEE3-1 and PPD4, of the PSII oxygen-evolving complex (OEC) and the antenna proteins, CP24 10A, CP26, and CP29, of LHCII in the leaves also increased to varying degrees. The photosystem I (PSI) activity in the leaves of mulberry seedling also increased, and the expressions of some proteins, PsaF, PsaG, PsaH, PsaL, PsaN, and Ycf4, in PSI increased significantly under NaCl stress. Under NaHCO3 stress, the activity of PSII and PSI and the expression of their protein complexes and the electron transfer-related proteins significantly decreased. NaCl stress had little effect on RuBP regeneration during dark reaction in the leaves and the expressions of glucose synthesis related proteins and net photosynthetic rate (Pn) did not decrease significantly. The leaves could adapt to NaCl stress by reducing stomatal conductance (Gs) to increase water use efficiency (WUE). Under NaHCO3 stress, the expression of dark reaction-related proteins was mostly down-regulated, and Gs was significantly reduced, which indicated that non-stomatal factors were important reasons for the significant inhibition of carbon assimilation. In the photoprotective mechanism under NaCl stress, the expression of cyclic electron flow (CEF) related proteins, ndhH, ndhI, ndhK, and ndhM, involved in NAD(P)H dehydrogenase (NDH) and the key enzyme of the xanthophyll cycle, violaxanthin de-epoxidase (VDE) were up-regulated. In addition, the ratio of xanthophyll cycle components (A+Z)/(V+A+Z) was increased. The expressions of proteins FTR and Fd-NiR, which are related to Fd-dependent ROS metabolism and nitrogen metabolism, were also significant up-regulated under NaCl stress, which can effectively reduce the electronic pressure on Fd. Under NaHCO3 stress, the expressions of CEF-related proteins, VDE, ZE, FTR, Fd-NiR, Fd-GOGAT, SGAT, and GGAT, were significant down-regulated, and the photoprotective mechanism, like the xanthophyll cycle, CEF, and photorespiration, might be damaged, resulting in the inhibition of PSII activity and carbon assimilation in leaves of mulberry seedling under NaHCO3 stress.

Significance: Cellular redox processes are highly interconnected, yet not in equilibrium, and governed by a wide range of biochemical parameters. Technological advances continue refining how specific redox processes are regulated, but broad understanding of the dynamic interconnectivity between cellular redox modules remains limited. Systems biology investigates multiple components in complex environments and can provide integrative insights into the multi-faceted cellular redox state. This review describes the state of the art in redox systems biology as well as provides an updated perspective and practical guide for harnessing thousands of cysteine sensors in the redoxome for multi-parameter characterization of cellular redox networks. Recent Advances: Redox systems biology has been applied to genome-scale models and large public datasets, challenged common conceptions and provided new insights that complement reductionist approaches. Advances in public knowledge and user-friendly tools for proteome-wide annotation of cysteine sentinels can now leverage cysteine redox proteomics datasets to provide spatial, functional, and protein structural information. Critical Issues: Careful consideration of the analytical approaches is needed to broadly characterize the systems level properties of redox signaling networks and be experimentally feasible. The cysteine redoxome is an informative focal point since it integrates many aspects of redox biology. The mechanisms and redox modules governing cysteine redox regulation, cysteine oxidation assays, proteome-wide annotation of the biophysical and biochemical properties of individual cysteines, and their clinical application are discussed. Future Directions: Investigating the cysteine redoxome at a systems level will uncover new insights into the mechanisms of selectivity and context-dependence of redox signaling networks.

A balanced chromosomal translocation disrupting DISC1 (Disrupted in Schizophrenia 1) gene has been linked to psychiatric diseases, such as major depression, bipolar disorder and schizophrenia. Since the discovery of this translocation, many studies have focused on understating the role of the truncated isoform of DISC1, hypothesizing that the gain of function of this protein could be behind the neurobiology of mental conditions, but not so many studies have focused in the mechanisms impaired due to its loss of function. For that reason, we perform an analysis on the cellular proteome of primary neurons in which DISC1 was knocked down with the goal of identifying relevant pathways directly affected by DISC1 loss of function. Using an unbiased proteomic approach, we found that the expression of 31 proteins related to neurodevelopment (e.g. CRMP-2, stathmin) and synaptic function (e.g. MUNC-18, NCS-1) is regulated by DISC1 in primary mouse neurons. Hence, this study reinforces the idea that DISC1 is a unifying regulator of both neurodevelopment and synaptic function, thereby providing a link between these two key anatomical and cellular circuitries.

The relationship between Parkinson's disease (PD), the second most common neurodegenerative disease after Alzheimer's disease, and palmitoylation, a post-translational lipid modification, is not well understood. In this study, we have analyzed the differential palmitome present in the cerebral cortex of PD patients compared to controls (n=4 per group) to better understand the role of protein palmitoylation in PD and the pathways altered in this disease. Data-mining of the cortical palmitome from PD patients and controls has allowed to: i) detect a set of 150 proteins with altered palmitoylation in PD subjects in comparison with controls, ii) describe the biological pathways and targets predicted to be altered by these palmitoylation changes, and iii) depict the overlap between the differential palmitome identified in our study with protein interactomes of the PD-linked proteins α-synuclein, LRRK2, DJ-1, PINK1, GBA and UCHL1. In summary, we have partially characterized the altered palmitome in the cortex of PD patients which is predicted to impact cytoskeleton, mitochondrial and fibrinogen functions, as well as cell survival. Our study points out that protein palmitoylation could have a role in the pathophysiology of PD, and that comprehensive palmitoyl-proteomics offers a powerful approach for elucidating novel cellular pathways modulated in this neurodegenerative disease.

Mimosa acutistipula is endemic to Brazil and grows in ferruginous outcrops (canga) in Serra dos Carajás, eastern Amazon, where one of the largest iron ore deposits in the world is located. Plants that develop in these ecosystems are subject to severe environmental conditions and must have adaptive mechanisms to grow and thrive in cangas. Mimosa acutistipula is a native species used to restore biodiversity in post-mining areas in canga. Understanding the molecular mechanisms involved in the adaptation of M. acutistipula in canga is essential to deduce the ability of native species to adapt to possible stressors in rehabilitating minelands over time. In this study, the root proteomic profiles of M. acutistipula grown in a native canga ecosystem and rehabilitating minelands were compared to identify essential proteins involved in the adaptation of this species in its native environment and that should enable its establishment in rehabilitating minelands. The results showed differentially abundant proteins, where 436 proteins with significant values (p < 0.05) and fold change ≥ 2 were more abundant in canga and 145 in roots from the rehabilitating minelands. Among them, a representative amount and diversity of proteins were related to responses to water deficit, heat, and responses to metal ions. Other identified proteins are involved in biocontrol activity against phytopathogens and symbiosis. This research provides insights into proteins involved in M. acutistipula responses to environmental stimuli, suggesting critical mechanisms to support the establishment of native canga plants in rehabilitating minelands over time.

If one considers chemical-biology toolsets that have had the greatest impact on numerous fields of life sciences over the most recent years, proximity-labeling tools, such as APEX, and Bio-ID arguably lead the way. This article reflects upon the current state-of-the-art and discusses key limitations underlying these emerging approaches, in particular, the limited functional knowledge they provide in understanding local proteomes / interactomes. This limitation is directly linked to the use of non-biologically- or non-pharmaceutically-relevant reactive intermediates in the course of covalently labeling the local proteomes. As such, these methods cannot report on specific functions of localized protein players, nor can they scrutinize whether the specific functions of such proteins/interactomes can be directly manipulated by pharmacologically-relevant small-molecule ligands. The latest data hint that precision localized electrophile delivery concept ushers a means to address this limitation with high spatiotemporal resolution, and ultimately, in relevant live animals.

Extremely sensitive food-allergic patients may react to very small amounts of allergenic foods. Precautionary allergen labelling (PAL) warns from possible allergenic contaminations. We explored the ability of proteomic methods to identify minute amounts of milk/egg allergens in a brand of PAL-labelled milk- and egg-free biscuits. We evaluated the reactivity of children with severe milk and egg allergy, by oral food challenge. Traces of milk and/or egg allergens in biscuits were measured by two different liquid-chromatography-mass spectrometry methods. The binding of patient's serum with egg/milk proteins was assessed at immunoblotting. None of the patients reacted to biscuits. Egg and milk proteins were found under the limit of detection of 0.6 µg/g for milk and egg (method A), and of 0.1 and 0.3 µg /g for milk and egg, respectively (method B). The immunoblots did not show milk/egg proteins in the studied biscuits. Our biscuits did not contain allergens of clinical significance. Their milk/egg content is far lower than 4 µg of milk or egg protein per gram of product, the minimal doses considered theoretically capable of causing reactions. With high sensitivity, proteomic assessments predict the harmlessness of very small amount of allergenic foods and can be used to avoid unnecessary PAL.

Protein translational modifications (PTMs) generate an enormous, but as yet undetermined, expansion of the expressed proteoforms. In this Viewpoint, we firstly differentiate the concepts of proteoform and peptidoform by reviewing and discussing previous literature. We show that the current PTM biological investigation and annotation largely follow a PTM site-specific rather than proteoform-specific approach. We further illustrate a potentially useful matching strategy in which a particular “modified peptidoform” is matched to the corresponding “unmodified peptidoform” as a reference for the quantitative analysis between samples and conditions. We suggest this strategy could provide directly relevant information for learning the PTM site-specific biological functions. Accordingly, we advocate for the wider use of the nomenclature “peptidoform” in the future bottom-up proteomic studies.

The most common form of mixed dementia (MixD) is constituted by abnormal protein deposits associated with Alzheimer´s disease (AD) that coexist with vascular disease. Although olfactory dysfunction is considered a clinical sign of AD-related de-mentias, little is known about the impact of this sensorial impairment in MixD at molecular level. To address this gap in knowledge, we have assessed olfactory bulb (OB) proteome-wide expression in MixD subjects (n=6) respect to neurologically intact controls (n=7). Around 9% of the quantified proteins were differentially expressed, pinpointing aberrant proteostasis involved in synaptic transmission, nucleoside monophosphate and carbohydrate metabolisms and neuron projection regeneration. In addition, net-work-driven proteomics revealed a modulation in cell-survival related pathways such as ERK, AKT and PDK1-PKC axis. Part of the differential OB protein set was not specific of MixD, being also deregulated across different tauopathies, synucleinopathies and tardopathies. However, the comparative functional analysis of OB proteome data between MixD and pure AD pathologies deciphered commonalities and differences between both related phenotypes. Finally, olfactory proteomics allowed to propose serum Prolow-density lipoprotein receptor-related protein 1 (LRP1) as a candidate marker to differentiate AD from MixD phe-notypes.

Proteins are the primary effectors of function in biology, and thus complete knowledge of their structure and properties is fundamental to deciphering function in basic and translational research. The chemical diversity of proteins is expressed in their many proteoforms, which result from combinations of genetic polymorphisms, RNA splice variants and post-translational modifications. This knowledge is foundational for the biological complexes and networks that control biology, yet remains largely unknown. We propose here an ambitious initiative to define the human proteome; that is to generate a definitive reference set of the proteoforms produced from the genome. Several examples of the power and importance of proteoform-level knowledge in disease-based research are presented, along with a call for improved technologies in a two-pronged strategy to accomplish the Human Proteoform Project.

Piscirickettsia salmonis is an intracellular bacterial fish pathogen that causes piscirickettsiosis, a disease with numerous negative impacts in the Chilean salmon farming industry. Although transcriptomic studies of P. salmonis and its host have been performed, dual host-pathogen proteomic approaches during infection are still missing. Considering that gene expression not always corresponds with observed phenotype, and bacteriological culture studies inadequately reflect infection conditions, to improve the existing knowledge for the pathogenicity of P. salmonis we present here a global proteomic profiling of Salmon salar macrophage-like cell cultures infected with P. salmonis LF-89. The proteomic analyses identified several P. salmonis proteins from two temporally different stages of macrophages infection; some of them related to key functions for bacterial survival in other intracellular pathogens. Metabolic differences were observed in early-stage infection bacteria, compared to late-stage infections. Virulence factors related to membrane, LPS and surface component modifications, cell motility, toxins and secretion systems also varied between the infection stages. Pilus proteins, beta-hemolysin and the T6SS were characteristic of the early-infection stage, while fimbria, upregulation of 10 toxins or effector proteins, and the Dot/Icm T4SS were representative of the late-infection stage bacteria. Previously described virulence-related genes in P. salmonis plasmids were identified by proteomic assays during infection in SHK-1 cells, accompanied by an increase of mobile-related elements. By comparing the infected and un-infected proteome of SHK-1 cells, we observed changes in cellular and ROS homeostasis, innate immune response, microtubules and actin cytoskeleton organization and dynamics, alteration in phagosome components, iron transport and metabolism, and amino acids, nucleoside and nucleotide metabolism, together with an overall energy and ATP production alteration. Our global proteomic profiling and the current knowledge of the P. salmonis infection process allowed us to propose a model of the macrophage-P. salmonis interaction.

In the last two decades, extracellular vesicles (EVs) from the three domains of life, archae, bacteria and eukaryota, have gained increasing scientific attention. As such, the role of EVs in host-pathogen communication and immune modulation are being intensely investigated. Pivotal to EV research is the determination of how and where EVs are taken up by recipient cells and organs in vivo, which requires suitable tracking strategies including labelling. Labelling of EVs is often performed post-isolation which increases risks of non-specific labelling and the introduction of labelling artefacts. Here we exploited the inability of helminths to de novo synthesise fatty acids to enable labelling of EVs by whole organism uptake of fluorescent lipid analogues and the subsequent incorporation in EVs. We showed uptake of DOPE-Rhodamine in Anisakis spp. and Trichuris suis larvae. EVs isolated from supernatant of Anisakis spp. labelled with DOPE-Rhodamine were characterised to assess effects of labelling on size, structure and fluorescence of EVs. Fluorescent EVs were successfully taken up by the human macrophage cell line THP-1. This study therefore presents a novel staining method that can be utilized by the EV field in parasitology and potentially across multiple species.

We analyzed the affinity-proteomics data of saliva absorbed to plate-bound Spike protein of SARS-CoV-2, and identified major virus-binding proteins as MUC7, MUC5B, DMBT1, and neutrophil defensins. Furthermore, we found that saliva from healthy donors inhibited the binding of Spike-protein-specific polyclonal antibodies to Spike antigen. These data suggest that the Spike protein’s glycoprotein-binding domains (GBD) may be targeted to block virus adherence or entry of SARS-CoV-2.

Accumulation of damage is generally considered the cause of aging. Interventions that delay aging mobilize mechanisms that protect and repair cellular components. Consequently, research has been focused on studying the protective and homeostatic mechanisms within cells. However, in humans and other multicellular organisms, cells are surrounded by extracellular matrices (ECM), which are important for tissue structure, function and intercellular communication. During aging, components of the ECM become damaged through fragmentation, glycation, crosslinking, and accumulation of protein aggregation, all of which contribute to age-related pathologies. Interestingly, placing senescent cells into a young ECM rejuvenates them and we found that many longevity-assurances pathways re-activate de-novosynthesis of ECM proteins during aging. This raises the question of what constitutes a young ECM to reverse aging or maintain health? In order to make inroads to answering this question, I suggest a systems-level approach of quantifying the matrisome or ECM compositions reflecting health, pathology, or phenotype and propose a novel term, the “matreotype”, to describe this. The matreotype is defined as the composition and modification of ECM or matrisome proteins associated with or caused by a phenotype, such as longevity, or a distinct and acute physiological state, as observed during aging or disease. Every cell type produces its unique ECM. Interestingly, cancer-cell types can even be identified based on their unique ECM composition. Thus, the matreotype reflects cellular identity and physiological status. Defined matreotypes could be used as biomarkers or prognostic factors for disease or health status during aging with potential relevance for personalized medicine. Treatment with biologics that alter ECM-to-cell mechanotransduction might be a strategy to reverse age-associated pathologies. An understanding of how to reverse from an old to a young matreotype might point towards novel strategies to rejuvenate cells and help maintain tissue homeostasis to promote health during aging.

The proteomic profile of extracellular vesicles (EVs) has been of increasing interest, particularly in understanding cancer growth, drug resistance, and metastatic behavior. Emerging data suggests that cancer-derived EVs may carry an array of oncogenic cargo, including certain integrin proteins that may, in turn, promote cell detachment, migration, and selection of future metastatic sites. We previously reported a large comparison of secreted vesicle protein cargo across sixty diverse human cancer cell lines. Here, we analyze the distinct integrin profiles of these cancer EVs. We further demonstrate the enrichment of integrin receptors in breast cancer EVs compared to vesicles secreted from benign breast epithelial cells. Total EV integrin levels, including the quantity of integrins α2, αv, β4, and β5 correlate with breast tumor stage. In particular, integrin α2 also largely reflects progenitor cell expression, highlighting the utility of this integrin protein as a potential circulating biomarker of primary tumors. This study provides preliminary evidence of the value of vesicle-associated integrin proteins in cancer diagnosis and prediction of tumor stage. Differential expression of integrins across cancer cells, and selective packaging of integrins into EVs may contribute to further understanding the development and progression of tumor growth and metastasis across a variety of cancer types.

An understanding of the mechanism underlying fruit ripening is critical for fruit quality improvement. Although post-harvest ethylene application is known to enhance the onset of fruit ripening, exact mechanisms remain unclear. To characterize the fruit ripening process and mechanism, we investigated the effects of exposing kiwifruit cultivars ‘Hayward’ and ‘Gamrok’ to exogenous ethylene treatment post-harvest using comprehensive proteomic analyses. Comparative two-dimensional gel electrophoresis showed that most of the proteins aggregated in ethylene-treated samples compared to the control (non-treated). We observed that among all ethylene treatments, 95 proteins from ‘Hayward’ and 106 from ‘Gamrok’ were differentially expressed. Interestingly, among the elicited protein successfully identified by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry, 50% from “Hayward’ and 60% from ‘Gamrok’ are associated with fruit ripening. Furthermore, 18% and 10% of proteins, respectively, are associated with defense response, whereas other major proteins are related to protein biosynthesis and photosynthesis/Calvin cycle. Interactions between identified proteins were demonstrated by bioinformatic analysis, providing insights into biological pathways and molecular functions in post-harvest kiwifruit ripening elicited by ethylene application. The present proteomic study in accordance with physiological analysis provides a quantitative evaluation of fruit ripening in response to exogenous ethylene in post-harvest kiwifruit.

Dawn of the omics revolution in biological sciences meant that we seek to understand more and in greater detail the molecular constituents of cells and biological systems. While we have gained significant insights from conventional omics tools, we now seek to understand the spatial dimensions of the data where subcellular localisation may impact on cellular physiology and phenotype. This review paper seeks to address current questions in the new field of image-based spatial proteomics as well as outline future challenges of the field. At first glance, spatial proteomics offers enormous potential to expand our understanding of different cell types in different disease and cell states. But limitations in types of fluorophores and issues with spectral overlap significantly hampers the practical implementation of the technique. On the other hand, while we have super-resolution microscopy techniques such as STED, PALM and STORM able to achieve 10 to 20 nm spatial resolution in single molecule localisation, problems with slow image acquisition limits high temporal resolution tracking of multiple protein targets in live cell imaging. Hence, the field of spatial proteomics is a mix of promises and challenges where we could image, in multi-colour, upwards of 10 well-chosen proteins that could inform on the molecular mechanisms of selected biological processes, but, at present, the method could not tackle larger scale questions. In essence, current implementation of image-based spatial proteomics is useful, but it is unable to fulfil the mission of large-scale projects such as the Human Protein Atlas or Human Cell Atlas. Future challenges in the field includes the development of more fluorophores (especially photoswitchable and photoactivable ones) for single molecule localisation microscopy, as well as seeking to improve temporal resolutions to the sub-millisecond range.

The ever-growing repertoire of genomic techniques continues to expand our understanding of true diversity and richness of prokaryotic genomes. Riboproteogenomics laid the foundation for dynamic studies of previously overlooked genomic elements. Most strikingly, bacterial genomes were revealed to harbour robust repertoires of small open reading frames (sORFs) encoding a diverse and broadly expressed range of small proteins, or sORF-encoded polypeptides (SEPs). In recent years, continuous efforts led to great improvements in annotation and characterization of such proteins, yet many challenges remain to fully understand the pervasive nature of small proteins and their impact on bacterial biology. In this work we review recent developments in the dynamic field of bacterial genome reannotation, catalogue important biological roles carried out by small proteins and identify challenges obstructing the way to full understanding of these elusive proteins.

Hyperaccumulator plant species growing on metal-rich soils can accumulate high quantity of metals and metalloids in aerial tissues and several proteomic studies on the molecular mechanisms at the basis of metals resistance and hyperaccumulation have been published. Hyperaccumulator are also at the basis of phytoremediation strategy to remove more efficiently metals from polluted soils or water. Arabidopsis halleri and Noccea caerulescens are both hyperaccumulators of metals and nano-metals. In this study it was assessed the change in some proteins in A. halleri and N. caer-ulescens after the growth in soil with cadmium and zinc, provided as sulphate salts (CdSO4 and ZnSO4) or sulfide quantum dots (CdS QDs and ZnS QDs). The protein extracts obtained from plants after 30 days of growth were analyzed by 2D-gel electrophoresis (2D SDS-PAGE) and identified by MALDI-TOF/TOF mass spectrometry. A bioinformatics analysis was carried out on quantitative protein differences between control and treated plants. In total, 43 proteins resulted significatively modulated in A. halleri, while 61 resulted modulated in N. caerulescens. Though these two plants are hyperaccumulator of both metals and nano-metals, at protein levels the mechanisms involved do not proceed in the same way but at the end bring to a similar physiological result.

Proteases or peptidases are hydrolases that catalyze the breakdown of polypeptide chains into smaller peptide subunits. Proteases exist in all life forms, including archaea, bacteria, protozoa, insects, animals, and plants, due to their vital functions in cellular processing and regulation. There are several classes of proteases in the MEROPS database based on their catalytic mecha-nisms. This review focuses on the post-proline cleaving enzymes (PPCEs), especially the prolyl endoprotease/oligopeptidase (PEP/POP). To date, most PPCEs studied are of microbial and ani-mal origins. Recently, there are reports of new plant PPCEs. The most common PEP/POP are members of the S9 family that comprise two conserved domains. The substrate-limiting β-propeller domain prevents unwanted digestion, while the α/β hydrolase catalyzes reaction at the carboxyl-terminal of proline residues. PPCEs have diverse applications, are widely used in the beer brewing industry, and have potential as therapeutic agents for Alzheimer’s disease and celiac disease by targeting proline-rich substrates. Protein engineering via mutagenesis has been performed to improve heat resistance, pepsin-resistant capability, specificity, and protein turno-ver of PPCEs for pharmacological applications. This is the first comprehensive review to cover the biotechnological applications of PPCEs and discuss the unique prolyl cleaving activity of dif-ferent enzymes based on the recent structure-function studies from diverse taxa.

Abstract: Autism Spectrum Disorder (ASD) is a complex neurodevelopmental disorder with extensive genetic and aetiological heterogeneity. While the underlying molecular mechanisms involved remain unclear, significant progress has been facilitated by recent advances in high-throughput transcriptomic, epigenomic and proteomic technologies. Here, we review recently published ASD proteomic data and compare proteomic func-tional enrichment signatures to those of transcriptomic and epigenomic data. We iden-tify canonical pathways that are consistently implicated in ASD molecular data and find an enrichment of pathways involved in mitochondrial metabolism and neurogenesis. We identify a subset of differentially expressed proteins that are supported by ASD tran-scriptomic and DNA methylation data. Furthermore, these differentially expressed proteins are enriched for disease phenotype pathways associated with ASD aetiology. These proteins converge on protein-protein interaction networks that regulate cell pro-liferation and differentiation, metabolism and inflammation which demonstrates a link between canonical pathways, biological processes and the ASD phenotype. This review highlights how proteomics can uncover potential molecular mechanisms to explain a link between mitochondrial dysfunction and neurodevelopmental pathology.

In comparison to other human cancer types, malignant melanoma exhibits the greatest amount of heterogeneity. After DNA-based detection of the BRAF V600E mutation in melanoma patients, targeted inhibitor treatment is the current recommendation. This approach, however, does not take the abundance of the therapeutic target, i.e., the B-raf V600E protein, into consideration. As shown by immunohistochemistry, the protein expression profiles of metastatic melanomas do clearly reveal the existence of inter- and intra-tumor variability. Nevertheless, the technique is only semi-quantitative. To quantitate the mutant protein there is a fundamental need for more precise techniques that are aimed at defining the currently non-existent link between the levels of the target protein and subsequent drug efficacy. Using cutting-edge mass spectrometry combined with DNA and mRNA sequencing, the mutated B-raf protein within metastatic tumors was quantitated for the first time. B-raf V600E protein analysis revealed a subjacent layer of heterogeneity for mutation-positive metastatic melanomas. These were characterized into two distinct groups with different tumor morphologies, protein profiles and patient clinical outcomes. This study provides evidence that a higher level of expression for the mutated protein is associated with a more aggressive tumor progression. Our study design that is comprised of surgical isolation of tumors, histopathological characterization, tissue biobanking, and protein analysis may enable the eventual delineation of patient responders/non-responders and the subsequent therapy of malignant melanoma.

Water stress (WS) and heat stress (HS) have a negative effect on soybean plant growth and crop productivity. During WS, soybean plants opt for survival through ion homeostasis and the conformations of proteins are disconcerted as plant cells lose water while HS leads to difficulties in flowering and fruiting. Some of these changes include oxidative stress leading to the destruction of photosynthetic apparatus, macromolecules within cells and the onset of complex signaling cascades. Changes in the physiological characteristics, proteome, and certain metabolites investigated on molecular and cellular functions were studied in two soybean cultivars exposed to different heat and water stress conditions independently and in combination. Leaf protein composition was studied using 2-DE and complemented with MALDI TOF mass spectrometry. While two cultivars displayed genetic variation in response to water and heat stress, thirty-nine proteins were significantly altered in their relative abundance in response to WS, HS and combined WS+HS in both cultivars; a majority of them involved in metabolism, response to heat and photosynthesis showing significant cross-tolerance mechanisms. Functional analysis revealing a majority of heat responsive-proteins were more abundant during HS and combined stress (WS+HS) whereas these proteins were low to WS in cultivar PI 471938 and heat shock proteins were in low abundance to water, heat and combined stresses in cultivar R95-1705. Most protein abundances were not correlated with their expression at mRNA levels in PI cultivar, however, in cultivar R 95, the expression levels of transcript follow their relative abundance in proteins. Our systems bioinformatics analyses revealed that MED37C, a probable mediator of RNA polymerase transcription II protein showed potential interacting partners in Arabidopsis and our studies signifies the marked impact of this protein in PI cultivar. Elevated activities in antioxidant enzymes indicate that the PI-371938 cultivar has the ability to restore the oxidation levels and sustain the plant during the stress. Our study hypothesizes the plant’s development of cross-stress tolerance which will help foster the ongoing ventures in genetic modifications in stress tolerance.

The objective of this study was to determine the effects of supplementing L-isoleucine (L-Ile) on milk protein synthesis, using an immortalized bovine mammary epithelial (MAC-T) cell line. In this case, the cells were treated with 0, 0.3, 0.6, 0.9, 1.2 and 1.5 mM of supplemental Isoleucine (Ile), and the most efficient time for protein synthesis for each amino acid was determined by measuring the cell, medium and total protein at 0, 24, 48, 72 and 96 h. Confirmatory tests showed that 48h incubation time and 0.6 mM dosage of L-Ile are considered as the optimal time and dosage. The mechanism of milk protein synthesis was elucidated through proteomics analysis to clarify the metabolic pathway. When the L-Ile was supplemented, extracellular protein (medium protein) reached a peak at 48h, whereas in the case of the intracellular cell protein, it was shown to have reached to its peak at 24h in all L-Ile dosage treatments. In total, it is noted that there were 63 upregulated and 52 downregulated proteins. The results of the protein pathway analysis showed that the L-Ile group stimulated insulin/IGF pathway-mitogen activated protein kinase kinase/MAP kinase cascade, insulin/IGF pathway-protein kinase B signaling cascade, p53 pathway, de novo purine biosynthesis, Wnt signaling pathway, glycolysis, pentose phosphate pathway, and ATP synthesis which are pathways involved and related to protein and energy metabolism. Together, these results demonstrate that L-Ile supplementation was effective in stimulating β-casein synthesis by stimulating genes and pathways which are significantly related to protein and energy metabolism.

In metastatic pancreatic cancer patients non eligible to surgery, signal-targeted therapies so far failed to show a significant amelioration of survival. These therapeutic options were tested in Phase II/III clinical trials mostly in combination with the reference treatment Gemcitabine. These innovative therapies aim at annihilating the oncogene dependency; they also aim at renormalizing the tumoral stroma to allow immune cell function or re-vascularisation. Transcriptomics and genomics large scale analysis show the great heterogeneity of pancreatic cancers and failed to clearly delineate specific oncogene dependency besides oncogenic Kras. In this review, we will describe the most recent proteomic data in pancreatic tumors and its metastasis, which could help at identifying their major signalling dependencies, as well as explain why they are intrinsically resistant to signal-targeted therapies. We will also discuss why PI3K signalling, as a paradigm of pro-tumorigenic cell signalling and of tumoral adaptative resistance to drugs, is a relevant target in this context.

In metastatic pancreatic cancer patients non eligible to surgery, signal-targeted therapies so far failed to show a significant amelioration of survival. These therapeutic options were tested in Phase II/III clinical trials mostly in combination with the reference treatment Gemcitabine. These innovative therapies aim at annihilating the oncogene dependency; they also aim at renormalizing the tumoral stroma to allow immune cell function or re-vascularisation. Transcriptomics and genomics large scale analysis show the great heterogeneity of pancreatic cancers and failed to clearly delineate specific oncogene dependency besides oncogenic Kras. In this review, we will describe the most recent proteomic data in pancreatic tumors and its metastasis, which could help at identifying their major signalling dependencies, as well as explain why they are intrinsically resistant to signal-targeted therapies. We will also discuss why PI3K signalling, as a paradigm of pro-tumorigenic cell signalling and of tumoral adaptative resistance to drugs, is a relevant target in this context.

Complex proteomic and physiological approaches to study cold and heat stress responses in plant mitochondria are still limited. Variations in the mitochondrial proteome of cauliflower (Brassica oleracea var. botrytis) curds after cold and heat and after stress recovery were assayed by 2D PAGE in relation to respiratory parameters. Quantitative analysis of the mitochondrial proteome revealed numerous stress-affected protein spots. In cold alternative oxidase isoforms were extensively upregulated; major downregulations in the level of photorespiratory enzymes, porine isoforms, oxidative phosphorylation (OXPHOS) and some low-abundant proteins were observed. On the contrary, distinct proteins, including carbohydrate metabolism enzymes, heat-shock proteins, translation, protein import, and OXPHOS components were involved in heat response and recovery. Few metabolic regulations were suggested. Cauliflower plants appeared less susceptible to heat; closed stomata in heat stress resulted in moderate photosynthetic, but only minor respiratory impairments, however photosystem II performance was unaffected. Decreased photorespiration corresponded with proteomic alterations in cold. Our results show that cold and heat stress not only operate in diverse mode (exemplified by cold-specific accumulation of some heat shock proteins), but exert some associations on molecular and physiological levels. This implies more complex model of action of investigated stresses on plant mitochondria.

The present investigation has been conducted on one hundred tissue fragments of breast cancer, collected and immediately cryopreserved following the surgical resection. The fragments were selected from patients with invasive ductal carcinoma of the breast, the most common and potentially aggressive type of mammary cancer, with the objective to increase the knowledge of breast cancer molecular markers, useful for diagnostic and prognostic categorization of patients, in assessing post-surgical therapeutic regimes. The proteomic screening, by 2D-IPG and mass spectrometry, allowed us to identify two main classes of protein clusters: proteins expressed ubiquitously at high levels in all patients, and proteins expressed sporadically among the same patients. Within the group of ubiquitous proteins, glycolytic enzymes and proteins with anti-apoptotic activity were predominant. Among the sporadic ones, proteins involved in cell motility, molecular chaperones and proteins involved in the detoxification appeared prevalent. The data of the present study indicates that the primary tumor growth is generally supported by two concurrent pathways: the inhibition of apoptosis and the stimulation of cellular proliferation. The second phase of the evolution of the tumor can be prematurely scheduled by the occasional presence of proteins involved in cell motility and in the defenses of the oxidative stress. To our knowledge this report on large-scales proteomics of breast cancer is currently a unique approach in the literature that offers the opportunity to evaluate the presence and recurrence of proteins to be used as prognostic indicators and susceptibility to metastasis in patients operated on for invasive ductal carcinoma of the breast.

Demonstrating biosimilarity entails comprehensive analytical evaluations, clinical pharmacolo-gy profiling, and efficacy testing for at least one medical indication in patients. These require-ments are stipulated by the U.S. Biologics Price Competition and Innovation Act (BPCIA). The costliest element—efficacy testing—can be waived if other compliance benchmarks are satisfied, including comparing functional pharmacodynamic (PD) biomarkers, even when they do not di-rectly correlate with clinical outcomes. Most biological drugs, such as monoclonal antibodies (mAbs), lack identifiable PD biomarkers. The FDA has employed various 'omics' technologies to identify potential PD biomarkers, including proteomics, glycomics, transcriptomics, genomics, epigenomics, and metabolomics. Although these efforts provide a robust scientific basis for estab-lishing biosimilarity, they are neither practical nor necessarily superior to existing functional biomarkers, such as receptor binding and mode-of-action outcomes. As we report for the first time, these functional biomarkers can effectively serve as PD indicators for all FDA-licensed bio-logical drugs. We recommend that the FDA consider officially listing these functional biomarkers to expedite and reduce the cost of biosimilar development, thereby increasing the accessibility of biological drugs. PD surrogates, like the receptor binding and pharmacokinetic profiles, are more robust and offer a rational solution to finding PD markers to compare for establishing biosimi-larity.

During meat processing, lactic acid bacteria (LAB) have to competitively adapt to the hostile en-vironment produced by curing additives (CA). The objective of this study was to investigate the ability of Latilactobacillus curvatus CRL 705, a bioprotective strain of meat origin, to adapt to CA. A physiological and proteomic approach was performed. CRL 705 was grown in a chemically de-fined medium (CDM) containing specific concentrations of CA (NaCl, nitrite, sucrose and ascor-bic acid). The results showed minor differences in growth kinetics in the presence of CA. Glucose consumption, present in CDM, and production of lactic acid and bacteriocins were not signifi-cantly affected. Proteomic analyses indicated that most of the identified proteins (36 out of 39) mainly related to carbohydrate metabolism (18%), posttranslational modifications (15.6%), energy production and conversion (11.1%), translation (11.1%) and nucleotide metabolism (8.9%), were under expressed. In response to the studied CA, CRL 705 slowed down its general metabolism, achieving slight changes in physiological and proteomic parameters. The observed performance is another characteristic that extends the well-known competitive profile of CRL 705 as a meat starter- and -bioprotective culture. This is the first report dealing with the impact of CA on LAB proteomics.

Recent reports suggest that dysregulation of lipid metabolism is a key feature of the most invasive cancers. To identify potential biomarkers of tumor aggressiveness, we compared the proteomes of two experimental models of malignant mesothelioma in rats exhibiting different invasive properties. Quantitative changes between the most invasive, M5-T1, versus the least invasive, F4-T2, first led to a list of 424 proteins. A second step, cross-comparing this list with 433 proteins distinguishing invasive vs non-invasive tumors, led to identifying 88 proteins that specifically increased and 157 that decreased, respectively, characterizing the most aggressive M5-T1 tumor. Among the 15 mitochondrial proteins found in these lists, the very long-chain specific acyl-CoA dehydrogenase, encoded by the Acadvl gene and involved in fatty acid beta oxidation, appeared to play an important role in the metabolic reprogramming of the tumor microenvironment. Immunohistochemical staining of tumor sections confirmed increased expression of Acadvl in the M5-T1 tumor. Finally, the dramatic increase and decrease, observed in 25 and 17 proteins, respectively, suggested the existence of a strong link between mitochondrial events and modifications of the extracellular matrix, immune cell components or other subcellular compartments. These findings highlight some important aspects of the tumor microenvironment changes linked to aggressiveness.

In the era of multi-omic sciences, dogma on singular cause-effect in physio-pathological processes is overcome and system biology approaches have been providing new perspectives to see through. In this context, extracellular vesicles (EVs) are offering a new level of complexity, given their role in cellular communication and their activity as mediators of specific signals to target cells or tissues. Indeed, their heterogeneity in terms of content, function, origin and potentiality contribute to the cross-interaction of almost every molecular process occurring in a complex system. Such features make EVs proper biological systems being, therefore, optimal targets of omic sciences. Currently, most studies focus on dissecting EVs content in order to either characterize it or to explore its role in various pathogenic processes at transcriptomic, proteomic, metabolomic, lipidomic and genomic levels. Despite valuable results are being provided by individual omic studies, the categorization of EVs biological data might represent a limit to be overcome. For this reason, a multi-omic integrative approach might contribute to explore EVs function, their tissue-specific origin and their potentiality. This review summarizes the state-of-the-art of EVs omic studies, addressing recent research on the integration of EVs multi-level biological data and challenging developments in EVs origin.

In proteomics, it is essential to quantify proteins in absolute terms if we wish compare results among studies and integrate high-throughput biological data into genome-scale metabolic models. While labeling target peptides with stable isotopes allows protein abundance to be accurately quantified, the utility of this technique is constrained by the low number of quantifiable proteins that it yields. Recently, label-free shotgun proteomics has become the “gold standard” for carrying out global assessments of biological samples containing thousands of proteins. However, this tool must be further improved if we wish to accurately quantify absolute levels of proteins. Here, we used different label-free quantification techniques to estimate absolute protein abundance in the model yeast Saccharomyces cerevisiae. More specifically, we evaluated the performance of seven different quantification methods, based either on spectral counting (SC) or extracted-ion chromatogram (XIC), which were applied to samples from five different proteome backgrounds. We also compared the accuracy and reproducibility of two strategies for transforming relative abundance into absolute abundance: a UPS2-based strategy and the total protein approach (TPA). This study mentions technical challenges related to UPS2 use and proposes ways of addressing them, including utilizing a smaller, more highly optimized amount of UPS2. Overall, three SC-based methods (PAI, SAF, and NSAF) yielded the best results because they struck a good balance between experimental performance and protein quantification.

Oxidative phosphorylation (OxPhos) is the basic function of mitochondria although the land-scape of mitochondrial functions is continuously growing to include more aspects of cellular homeostasis. Thanks to the application of -omics technologies to the study of the OxPhos system, novel features emerge from the cataloging of novel proteins as mitochondrial thus adding de-tails to the mitochondrial proteome and defining novel metabolic cellular interrelations, espe-cially in the human brain. We focussed on the diversity of bioenergetics demand and different aspects of mitochondrial structure, functions, and dysfunction in the brain. Definition as ‘mitoexome’, ‘mitoproteome’ and ‘mitointeractome’ have entered the field of ‘mitochondrial medicine’. In this context, we reviewed several genetic defects that hamper the last step of aerobic metabolism mostly involving the nervous tissue as one of the most prominent energy-dependent tissues and, as consequence, as a primary target of mitochondrial dysfunction. The dual genetic determination of the OxPhos complexes is one of the reasons for the complexity of the geno-type-phenotype correlation when facing human diseases associated with mitochondria defects; clinically, are characterized by extremely heterogeneous symptoms, ranging from organ-specific to multisystemic dysfunction with different clinical courses. Finally, we briefly discuss the fu-ture directions of the multi-omics study of human brain disorders.

Pseudomonas aeruginosa is a critical healthcare challenge due to its ability to cause persistent infections and the acquisition of antibiotic resistance mechanisms. Lack of preventive vaccines and rampant drug resistance phenomenon has rendered patients vulnerable. As new antimicrobials are in the preclinical stages of development, mining for the unexploited drug targets is also crucial. Here, we designed a chimeric vaccine against P. aeruginosa using a subtractive proteomics approach and identified nine unique enzymes as novel drug targets in PAO1 proteome. A total of five unique proteins were selected as potential vaccine candidates based on essentiality, extracellular localization, virulence, antigenicity, pathway association, protein-protein interaction analysis, hydrophilicity, and low molecular weight. These include two outer membrane porins OprF (P13794) and OprD (P32722), a protein activator precursor pra (G3XDA9), a probable outer membrane protein precursor PA1288 (Q9I456), and a conserved hypothetical protein PA4874 (Q9HUT9). These proteins were further analyzed using a reverse vaccinology approach to identify immunogenic and antigenic T cell and B cell epitopes. The best scoring epitopes qualifying for all set criteria were then further subjected to the construction of a polypeptide multi-epitope vaccine construct with cholera toxin B (CtxB) subunit as an adjuvant. The identified drug targets qualifying the screening criteria were: UDP-2-acetamido-2-deoxy-d-glucuronic acid 3-dehydrogenase WbpB (G3XD23), aspartate semialdehyde dehydrogenase (Q51344), 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase (Q9HV71), 3-deoxy-D-manno-octulosonic-acid transferase (Q9HUH7), glycyl-tRNA synthetase alpha chain (Q9I7B7), riboflavin kinase/FAD synthase (Q9HVM3), aconitate hydratase 2 (Q9I2V5), probable glycosyltransferase WbpH (G3XD85) and UDP-3-O-[3-hydroxylauroyl] glucosamine N-acyltransferase (Q9HXY6). For druggability and pocketome analysis crystal and homology structures of these proteins were retrieved and developed. A sequence-based search was performed in different databases (ChEMBL, Drug Bank, PubChem and Pseudomonas database) for the availability of reported ligands and tested drugs for the screened targets. These predicted targets may provide a basis for the development of reliable antibacterial preventive and therapeutic options against P. aeruginosa.

The mangrove plant Acanthus ilicifolius and its relative, A. mollis, have been previously proved to possess diverse pharmacological effects. Therefore, evaluating the differentially expressed proteins of these species under tidal flooding stress is essential to fully exploit and benefit from their medicinal values. The roots of A. ilicifolius and A. mollis were exposed to 6 h of flooding stress per day for 10 days. The dry weight, hydrogen peroxide (H2O2) content, anatomical characteristics, carbon and energy levels, and two-dimensional electrophoresis coupled with MALDI-TOF/TOF MS technology were used to reveal the divergent flooding resistant strategies. A. ilicifolius performed better under tidal flooding stress, which was reflected in the integrity of the morphological structure, more efficient use of carbon and energy, and a higher percentage of up-regulated proteins associated with carbon and energy metabolism. A. mollis could not survive in flooding conditions for a long time, as revealed by incomplete cell structures of the roots, less efficient use of carbon and energy, and a higher percentage of down-regulated proteins associated with carbon and energy metabolism. Energy provision and flux balance played a role in the flooding tolerance of A. ilicifolius and A. mollis.

Protein hydrolysates show great promise as bioactive food and feed ingredient and for valorization of side-streams from e.g. the fish processing industry. This study characterizes bulk emulsifying, foaming, and in vitro antioxidative properties of hydrolysates derived from cod frame by application of Alcalase and Neutrase, individually and sequentially as well as the influence of heat-treatment prior to hydrolysis. We present a novel approach that utilizes proteomics data for calculation of weighted mean peptide properties (length, molecular weight, and charge) and peptide-level abundance estimation. Using subsequent bioinformatic prediction of biofunctional properties to describe observed bulk properties, we are able to provide an in-depth hydrolysate characterization not previously seen. All hydrolysates displayed comparable or higher emulsifying activity and stability than sodium caseinate. Heat-treatment significantly increased stability but showed a negative effect on the activity and degree of hydrolysis. Combining peptide abundance with predicted emulsifying activity, we were able to identify several peptides that are likely linked to the observed differences in bulk emulsifying properties. In general, decreased hydrolysis resulted in significantly higher chelating activity, while the opposite was observed for radical scavenging activity. The study highlights the prospects of applying proteomics and bioinformatics for hydrolysate characterization and in food protein science.

Murine herpesvirus-68 (MHV-68) productively infects the mouse lungs, exhibiting a complex pathology characteristic of both acute viral infections and chronic respiratory diseases. We sought to discover proteins differentially expressed in bronchoalveolar lavage (BAL) from mice infected with MHV-68. Mice were infected intranasally with MHV-68. After 9 days, as the lytic phase of infection resolved, differential BAL proteins were identified by 2D electrophoresis and mass spectrometry. Of 23 unique proteins, acute phase proteins, vitamin A transport, and oxidative stress response factors Pdx6 and EC-SOD (Sod3) were enriched. Correspondingly, iNOS2 was induced in lung tissue by 7 days post infection. Oxidative stress was partly a direct result of MHV-68 infection, as reactive oxygen species (ROS) were induced in cultured murine NIH3T3 fibroblasts and human lung A549 cells infected with MHV-68. Finally, mice were infected with a recombinant MHV-68 co-expressing inflammatory cytokine murine interleukin 6 (IL6) showed exacerbated oxidative stress and soluble type I collagen characteristic of tissue recovery. Thus, oxidative stress appears to be a salient feature of MHV-68 pathogenesis, in part caused by lytic replication of virus and IL6. Proteins and small molecules in lung oxidative stress networks therefore may provide new therapeutic targets to ameliorate respiratory virus infections.

Human influenza A viruses (IAVs) cause global pandemics and epidemics. These viruses evolve rapidly, making current treatment options ineffective. To identify novel modulators of IAV-host interactions, we re-analyzed our recent transcriptomics, metabolomics, proteomics, phosphoproteomics, and genomics/virtual ligand screening data. We identified 713 potential modulators targeting 200 cellular and two viral proteins. Anti-influenza activity for 48 of them has been reported previously, whereas the antiviral efficacy of the remaining 665 is unknown. Studying anti-influenza efficacy, immuno-modulating properties and potential resistance of these compounds or their combinations may lead to the discovery of novel modulators of IAV-host interactions, which might be more effective than the currently available anti-influenza therapeutics.

In ageing tissues, long-lived extracellular matrix (ECM) proteins are susceptible to the accumulation of structural damage due to diverse mechanisms including glycation, oxidation and protease cleavage. Peptide location fingerprinting (PLF) is a new mass spectrometry (MS) analysis technique capable of identifying proteins exhibiting structural differences in complex proteomes. PLF applied to published young and aged intervertebral disc (IVD) MS datasets (posterior, lateral and anterior regions of the annulus fibrosus), identified 268 proteins with age-related structural differences. For several ECM assemblies (collagens I, II and V and aggrecan), these differences were markedly conserved between degeneration-prone (posterior and lateral) and resistant (anterior) regions. Significant differences in peptide yields, observed within collagen I, II and V α-chains (COL1A2, COL2A1, COL5A1), were located within their triple helical regions and/or cleaved C-terminal propeptides, indicating potential accumulation of damage and impaired maintenance in ageing. Several proteins (COL5A1, COL2A1 and aggrecan) also exhibited tissue region (lateral)-specific differences in structure between aged and young, suggesting that some ageing mechanisms may act locally within tissues. This study not only provides evidence of age-related changes in ECM protein structures which are tissue-region specific, but also highlights the ability of PLF to identify potential protein biomarkers of localised tissue remodelling.

Data independent acquisition - mass spectrometry (DIA-MS) is becoming widely utilised for robust and accurate quantification of samples in quantitative proteomics. Here, we describe the systematic evaluation of the effects of DIA precursor mass range on total protein identification and quantification. We show that a narrow mass range of precursors (~250 m/z) for DIA-MS enables a higher number of protein identifications. Subsequent application of DIA with narrow precursor range (from 400 to 650 m/z) on Arabidopsis sample with spike-in of known proteins identified 34.7% more proteins than in conventional DIA (cDIA) with a wide precursor range of 400-1200 m/z. When combining several DIA-MS analyses with narrow precursor ranges (i.e., 400-650, 650-900 and 900-1200 m/z), we were able to quantify 10,099 protein groups with a median coefficient of variation of <6%. These findings represent a 59.4% increase in the number of proteins quantified than with cDIA analysis. This is particularly important for low abundance proteins, as exemplified by the 6-protein mix spike-in. In cDIA only 5 out of the 6-protein mix were quantified while our approach allowed accurate quantitation of all six proteins.

Grapevine is one of the most relevant crops in the world being used for economically important products such as wine. Yet, relevant grapevine cultivars are heavily affected by diseases such as the downy mildew disease caused by Plasmopara viticola. Improvements on grapevine resistance are made mainly by breeding techniques where resistance traits are introgressed into cultivars with desired grape characteristics. However, there is still a lack of knowledge on how resistant or tolerant cultivars tackle the P. viticola pathogen. In this study, using a shotgun proteomics LC-MS/MS approach, we unravel the protein modulation of a highly tolerant grapevine cultivar, V. vinifera ‘Regent’, in the first hours post inoculation (hpi) with P. viticola. At 6 hpi, proteins related to defence and to response to stimuli are negatively modulated while at 12 hpi there is an accumulation of proteins belonging to both categories. The co-occurrence of effector-triggered susceptibility (ETS) and effector-triggered immunity (ETI) is detected at both time-points, showing that these defence processes present high plasticity. The results obtained in this study unravel the tolerant grapevine defence strategy towards P. viticola and may provide valuable insights on resistance associated candidates and mechanisms, which may play an important role in the definition of new strategies on breeding approaches.

This study aimed to assess the ecotoxicological effects of the interaction of fullerene (C₆₀) and benzo[a]pyrene (B[a]P) on the marine mussel, Mytilus galloprovincialis. The uptake of nC₆₀, B[a]P and mixtures of nC₆₀ and B[a]P into tissues was confirmed by GC-MS, LC-HRMS and ICP-MS. Biomarkers of DNA damage as well as proteomics analysis were applied to unravel the toxic effect of B[a]P and C₆₀. Antagonistic responses were observed at the genotoxic and proteomic level. Differentially expressed proteins (DEPs) were only identified in the B[a]P single exposure and the B[a]P mixture exposure groups containing 1 mg/L of C₆₀, the majority of which were down-regulated (~52%). No DEPs were identified at any of the concentrations of nC₆₀ (p < 0.05, 1% FDR). Using DEPs identified at a threshold of (p < 0.05; B[a]P and B[a]P mixture with nC₆₀), gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis indicated that these proteins were enriched with a broad spectrum of biological processes and pathways, including those broadly associated with protein processing, cellular processes and environmental information processing. Among those significantly enriched pathways, the ribosome was consistently the top enriched term irrespective of treatment or concentration and plays an important role as the site of biological protein synthesis and translation. Our results demonstrate the complex multi-modal response to environmental stressors in M. galloprovincialis.

Phospholipase Dα1 (PLDα1) belongs to phospholipases, a large phospholipid hydrolyzing protein family. PLDα1 has a substrate preference for phosphatidylcholine leading to enzymatic production of phosphatidic acid, a lipid second messenger with multiple cellular functions. PLDα1 itself is implicated in biotic and abiotic stress responses. We present here a shot-gun differential proteomic analysis on roots of two pldα1 mutants compared to the Col-0 wild type. Our data suggest new roles of PLDα1 in endomembrane transport, mitochondrial protein import and protein quality control and glucosinolate biosynthesis. Thus, we identified proteins involved in endocytosis, endoplasmic reticulum-Golgi transport and attachment sites of endoplasmic reticulum and plasma membrane (V-type proton ATPases, protein transport protein SEC13 homolog A, vesicle-associated protein 1-2, vacuolar protein sorting-associated protein 29, syntaxin-32, all upregulated in the mutants), mitochondrial import and electron transport chain (mitochondrial import inner membrane translocase subunits TIM23-2 and TIM13, mitochondrial NADH dehydrogenases, ATP synthases, cytochrome c oxidase subunit 6b-1, ADP,ATP carrier protein 2, downregulated in the mutants) and glucosinolate biosynthesis (3-isopropylmalate dehydrogenases 1, 2 and 3, methylthioalkylmalate synthase 1, cytochrome P450 83B1, Glutathione S-transferase F9, indole glucosinolate O-methyltransferase 1, adenylyl-sulfate kinase 1, all upregulated in mutants). Our results suggest broader biological roles of PLDα1 as anticipated so far.

Protein phosphorylation and ADP-ribosylation (ADPr), as two types of post-translational modifications (PTM), are the process of adding phosphate group and ADP-ribose moieties to proteins, respectively. Although both PTM types can occur on many amino acid types, serine is the most common. Serine phosphorylation (pS), serine ADPr (SADPr), and their in situ crosstalks (pSADPr) play essential roles in biological processes. Although in silico classifiers have been developed for predicting pS and SADPr sites, the classifier for predicting pSADPr sites is unavailable. In this study, we developed classifiers to predict pSADPr sites. Specifically, we collected 3250 human pSADPr, 7520 SADPr, 151,227 pS and 80,096 unmodified serine sites. Based on them, we investigated the characteristics of pSADPr sites and constructed three classifiers to predict pSADPr sites from the pS dataset, the SADPr dataset and the protein sequences separately. We built and evaluated five deep-learning classifiers in ten-fold cross-validation and independent test datasets. Three of them (e.g. Convolutional Neural Network with the One-Hot encoding, dubbed CNNOH) performed better than the rest two. For instance, CNNOH had the AUC values of 0.700, 0.914 and 0.954 for recognizing pSADPr sites from the SADPr, pS and unmodified serine sites.Therefore, it is challenging to distinguish pSADPr sites from SADPr sites compared to the other two. It is consistent with our observation that pSADPr's characteristics are more similar to those of SADPr than the rest. Furthermore, we used the classifiers as base classifiers to develop a few stacking-based ensemble classifiers to improve performance. However, none of the ensemble classifiers showed better performances, suggesting that the base classifiers have good enough performances. Finally, we developed an online tool for extensively predicting human pSADPr sites based on the CNNOH classifier, dubbed EdeepSADPr. It is freely available through http://edeepsadpr.bioinfogo.org/.

Background: In the present study, cisplatin, artemisinin and oleanolic acid were evaluated alone and in combination, on human ovarian A2780, A2780ZD0473R and A2780cisR cancer cell lines with aim of overcoming cisplatin resistance and side effects. Methods: Cytotoxicity was assessed by MTT reduction assay. CI values were used as a measure of combined drug effect. MALDI TOF/TOF MS/MS and 2-DE gel electrophoresis were used to identify protein biomarkers in ovarian cancer and to evaluate combination effects. Results: Synergism from combinations was dependent on concentration and sequence of administration. Generally, bolus was most synergistic. 49 proteins differently expressed by 2 ≥ fold were: CYPA, EIF5A1, Op18, p18, LDHB, P4HB, HSP7C, GRP94, ERp57, mortalin, IMMT, CLIC1, NM23, PSA3,1433Z, and HSP90B were down-regulated, whereas hnRNPA1, hnRNPA2/B1, EF2, GOT1, EF1A1, VIME, BIP, ATP5H, APG2, VINC, KPYM, RAN, PSA7, TPI, PGK1, ACTG and VDAC1 were up-regulated, while TCPA, TCPH, TCPB, PRDX6, EF1G, ATPA, ENOA, PRDX1, MCM7, GBLP, PSAT, Hop, EFTU, PGAM1, SERA and CAH2 were not-expressed in A2780cisR cells. The proteins were found to play critical roles in cell cycle regulation, metabolism and biosynthetic processes and drug resistance and detoxification. Conclusion: Results indicate that appropriately sequenced combinations of cisplatin with ART and OA may provide a means to reduce side effects and circumvent platinum resistance.

Plants attained cellular homeostasis of phosphate (Pi) through an integrated response pathway regulated by different families of transcription factors including MYB, WRKY, bHLH and ZFP. The systemic response to Pi limitation showed the critical role played by inositol pyrophosphate (PP-InsPs) as signaling molecule and SPX (SYG1/PHO81/XPR1) domain proteins as sensor of cellular Pi status. Binding of SPX to PP-InsPs regulates the transcriptional activity of the MYB-CC proteins, phosphate starvation response factors (PHR/PHL) as the central regulator of Pi-deficiency response in plants. Vacuolar phosphate transporter, VPT may sense the cellular Pi status by its SPX domain, and vacuolar sequestration is activated under Pi replete condition and the stored Pi is an important resource to be mobilized under Pi deficiency. Proteomic approaches led to new discoveries of proteins associated with Pi-deficient response pathways and post-translational events that may influence plants in achieving Pi homeostasis. This review provides current understanding on the molecular mechanisms at the transcriptional and translational levels for achieving Pi homeostasis in plants highlighting the importance of DNA-protein and protein-protein interactions. The potential strategies for employing the CRISPR technology to modify the gene sequences of key regulatory and response proteins for attaining plant Pi homeostasis are discussed.

Senescence-accelerated mouse prone 8 (SAMP8) mice exhibit cognitive defects and neuron loss with aging, and are used to study anti-aging effects of Dendrobium nobile alkaloids (DNLA). SAMP8 mice were orally given DNLA from ages 6 to 10 months. At 10 months of age, behavioral tests and neuron damage were evaluated. Protein was extracted and subjected to phosphorylated proteomic analysis. The cognitive deficits and neuron loss in hippocampus and cortex of aged SAMP8 mice were improved by DNLA. Hippocampal proteomic analysis showed differentially expressed protein/genes in SAMP8 compared to age-matched senescence-accelerated resistant mice, including altered tubulin binding, microtubule binding, etc. via Gene Oncology. KEGG revealed endocytosis, mRNA surveillance, tight junction, protein processing in endoplasmic reticulum, aldosterone synthesis and secretion, and glucagon signaling pathway changes. Upregulated protein/genes in the hippocampus of SAMP8 mice, such as Lmtk3, Usp10, Dzip1, Csnk2b, and Rtn1, were attenuated by DNLA; whereas downregulated protein/genes, such as Kctd16, Psd3, Bsn, Atxn2l, and Kif1a, were rescued by DNLA. The aberrant protein/gene expressions of SAMP8 mice were correlated with transcriptome changes of Alzheimer’s disease in the GEO database, and were attenuated by DNLA. Thus, DNLA improved cognitive dysfunction and ameliorated neuronal injury in aged SAMP8 mice, and attenuated aberrant protein/gene expressions.

Milk is newborn’s food and an emulsion full of all necessary components for neonatal growth. Its consumption is worldwide and is the base for all dairy products. Because of the latter, many new technologies are growing, among them proteomics; in order to give new insights in milk’s compounds and to maximize the beneficial potential for consumers’ health. In this review, we aimed to gather data of proteomics studies for the majority of dairy animals and elucidate the role of milk bioactive compounds. Furthermore, special reference was made to milk fat globule membrane (MFGM) peptides and the result of thermal treatment in milk proteins. Finally, the proteomic approach regarding adulterations was included in the review.

Matrix metalloproteinases (MMPs) have been demonstrated to have both detrimental and protective functions in inflammatory diseases. Several MMP inhibitors, with the exception of Periostat®, have failed in Phase III clinical trials. As an alternative strategy, recent efforts have been focussed on the development of more selective inhibitors or targeting other domains than their active sites (e.g., exosites, ectosites) through specific small molecule inhibitors or monoclonal antibodies. Here, we present some examples that aim to better understand the mechanisms of conformational changes/allosteric control of MMPs functions. In addition to MMP inhibitors, we discuss unbiased global approaches such as proteomics and N-terminomics to identify new MMP substrates and achieve a better understanding of the roles of these proteases in diseases.

Today massive amounts of sequenced metagenomic and -transcriptomic data from different ecological niches and environmental locations are available. Scientific progress depends critically on methods that allow extracting useful information from the various types of sequence data. Here, we will first discuss types of information contained in the various flavours of biological sequence data, and how this information can be interpreted to increase our scientific knowledge and understanding. We argue that a mechanistic understanding is required to consistently interpret experimental observations, and that this understanding is greatly facilitated by the generation and analysis of dynamic mathematical models. We conclude that, in order to construct mathematical models and to test mechanistic hypotheses, time-series data is of critical importance. We review diverse techniques to analyse time-series data and discuss various approaches by which time-series of biological sequence data was successfully used to derive and test mechanistic hypotheses. Analysing the bottlenecks of current strategies in the extraction of knowledge and understanding from data, we conclude that combined experimental and theoretical efforts should be implemented as early as possible during the planning phase of individual experiments and scientific research projects.

About 50% of colorectal cancer patients develop liver metastases. Patients with metastatic colorectal cancer have 5-year survival rates below 20% despite new therapeutic regimens. Tumor heterogeneity has been linked with poor treatment response and clinical outcome, but was so far mainly studied via bulk genomic analyses. In this study we performed spatial proteomics via MALDI mass spectrometry imaging on six patient-matched CRC primary tumor and liver metastases to characterize interpatient, intertumor and intratumor hetereogeneity. We found several peptide features that were enriched in vital tumor areas of primary tumors and liver metastasis and tentatively derived from tumor cell specific proteins such as annexin A4 and prelamin A/C. Liver metastases of colorectal cancer showed higher heterogeneity between patients than primary tumors while within patients both entities show similar intratumor heterogeneity sometimes organized in zonal pattern. Together our findings give new insights into the spatial proteomic heterogeneity of primary CRC and patient-matched liver metastases.

The dynamic interplay of signaling networks in most major cellular processes is characterized by the orchestration of reversible protein phosphorylation. Consequently, analytic methods like quantitative phospho-peptidomics has been pushed forward from a highly specialized edge-technique to a powerful and versatile platform for comprehensively analyzing the phosphorylation profile of living organisms. Despite enormous progress in instrumentation and bioinformatics, a major problem remains a high number of missing values caused by the experimental procedure due to either a random phospho-peptide enrichment selectivity or borderline signal intensities, which both cause the exclusion for fragmentation using the commonly applied data dependent acquisition (DDA) mode. Consequently, an incomplete dataset reduces confidence in the subsequent statistical bioinformatic processing. Here, we successfully applied data independent acquisition (DIA) by using the filamentous fungus Magnaporthe oryzae as model organism and could prove that while maintaining data quality (such as phosphosite and peptide sequence confidence), the data completeness increases dramatically. Since the method presented here reduces the LC-MS/MS analysis from 3 h to 1 h and increases the number of phoshosites identified up to 10-fold in contrast to published studies in fungi, we pushed the phospho-proteomic technique beyond its current limits and could provide a sophisticated resource for investigation of signaling processes in filamentous fungi.

The molecular study of fat cell development in the human body is essential for our understanding of obesity and related diseases. Mesenchymal stem/stromal cells (MSC) are the ideal source to study fat formation as they are the progenitors of adipocytes. In this work, we used human MSCs, received from surgery waste, and differentiate them into fat adipocytes. The combination of several layers of information coming from lipidomics, metabolomics and proteomics enabled comprehensive analysis of the biochemical pathways in adipogenesis. Simultaneous analysis of metabolites, lipids and proteins in cell culture is challenging due to the compound’s chemical difference so that most studies involve separate analysis with unimolecular strategies. In this study, we employed a multimolecular approach using a two–phase extraction to monitor the crosstalk between lipid metabolism and protein-based signaling in a single sample (~105 cells). We developed an innovative analytical workflow including standardization with in-house produced 13C-isotopically labeled compounds, hyphenated high-end mass spectrometry (high-resolution Orbitrap MS) and chromatography (HILIC, RP) for simultaneous untargeted screening and targeted quantification. Metabolite and lipid concentrations ranged over 3-4 orders of magnitude and were detected down to the low fmol (absolute on column) level. Biological validation and data interpretation of the multiomics workflow was performed based on proteomics network reconstruction, metabolic modelling (MetaboAnalyst 4.0) and pathway analysis (OmicsNet). Comparing MSCs and adipocytes, we observed significant regulation of different metabolites and lipids such as triglycerides, gangliosides and carnitine with 113 fully reprogrammed pathways. The observed changes are in accordance with literature findings dealing with adipogenic differentiation of MSC. These results are a proof of principle for the power of multimolecular extraction combined with orthogonal LC-MS assays and network construction. Considering the analytical and biological validation performed in this study, we conclude that the proposed multiomics workflow is ideally suited for comprehensive follow-up studies on adipogenesis and is fit for purpose for different applications.

The alteration of the delicate balance that regulates the secretion and distribution of the tear film determines the dry eye (DE) syndrome, because the tear film represents the interface between the eye and the environment. Despite having a multifactorial origin, the main risk factors for the emergence of the ocular disease are female gender and advanced age. Likewise, morphological changes in several glands and in chemical composition of their secretions such as proteins, mucins, lipidics, aqueous tears, and salinity, are highly relevant factors to maintain a condition of good health of the ocular anterior segment. Another key factor of recurrence and onset of the disease is the presence of local and/or systemic infiammation that reflex on the ocular surface. However, it is one of the most commonly encountered disease in clinical practice and many other causes related to daily life and to lengthen the average life will contribute to the beginning. This review will consider how and what disorders of the ocular surface are responsible for a widespread pathology so. In the end, the most appropriate and new therapies will be briefly exposed according to the specific pathology.

Abstract Tetraspanin CD63 is a cluster of cell surface proteins with four transmembrane domains which associates with tetraspanin-enriched microdomains and typically localizes to late endosomes and lysosomes. CD63 plays an important role in cellular trafficking of different proteins, EV cargo sorting and vesicles formation. We have preciously shown that CD63 is important in LMP1 trafficking to EVs and this also affects LMP1 mediated intracellular signaling including MAPK/ERK, NF-κB and mTOR activation. Using the BioID combined with mass spectrometry, we sought to define the broad CD63 interactome and how LMP1 modulates this network of interacting proteins. We identified a total of 1600 total proteins as proximal interacting newtwork of proteins to CD63. Biological process enrichment analysis revealed significant involvement in signal transduction, cell communication, protein metabolism and transportation. The CD63 only interactome was enriched in Rab GTPases, SNARE proteins and sorting nexins while adding LMP1 into the interactome increased presence of signaling and ribosomal proteins. Our results showed that LMP1 alters the CD63 interactome, shifting the network of proteins enrichment from protein localization and vesicle mediated transportation to metabolic processes and translation. We also show that LMP1 interacts with mTor, Nedd4L and PP2A indicating formation of a multiprotein complex with CD63 thereby potentially regulating LMP1 dependent mTor signaling. Collectively, the comprehensive analysis of CD63 proximal interacting proteins provides insights into network of partners required for endocytic trafficking, extracellular vesicle cargo sorting, formation and secretion.

Proteomics in respiratory allergic diseases has such a battery of techniques and programs that one would almost think that there is nothing impossible to find, invent or mold. All these resources that we document here, are involved in solving problems in allergic diseases, both diagnostic, prognostic, treatment and immunotherapy development. The main perspectives, according to this version, are in three strands and / or lockout immunological system: 1) In blocking the diapedesis of cells involved, 2) modifications and blocking of paratopes and epitopes being understood by modifications to antibodies, antagonism or blocking them and 3) blocking FcεR1 receptors to prevent specific IgEs from sticking to mast cells and basophils. These tools and targets in the allergic landscape are, in our view, the prospects in the field.

Ferroptosis is a unique variety of non-apoptotic cell death, driven by massive lipid oxidation in an iron-dependent manner. Since Ferroptosis was introduced as a concept in 2012, it was shown it's essential role in the pathogenesis in neurodegenerative diseases and an important role in therapy-resistant cancer cells. Thus, a detailed molecular understanding of both canonical and alternative ferroptosis pathways are required. There is a set of widely used chemical agents to modulate ferroptosis using different pathway targets: Erastin blocks cystine-glutamate antiporter, system xc-; ML210 directly inactivate GPX4; L-buthionine sulfoximine (BSO) inhibits γ-glutamylcysteine synthetase, an essential enzyme for glutathione synthesis de novo. Most studies were focused on lipidomic profiling of model systems undergoing death in a ferroptotic modality.

The aim of the study was to gain deeper insights in the potential for polyclonal stimulation of PBMC from cows and pigs with banana lectin (BanLec) from Musa paradisiaca. BanLec induced a marked proliferative response in cow and pig PBMC. The response in pigs was even higher than to Concanavalin A. Molecular processes associated with respective responses were examined with differential proteome analyses. Discovery proteomic experiments was applied to BanLec stimulated PBMC and cellular and secretome responses were analyzed with label free LC-MS/MS. In PBMC, 3955 proteins were identified. After polyclonal stimulation with BanLec, 459 proteins showed significantly changed abundance in PBMC. In respective PBMC secretomes, 2867 proteins were identified with 231 differentially expressed candidates as reaction to BanLec stimulation. The transcription factor `E74 like ETS transcription factor 1 (ELF1)` was solely enriched in BanLec stimulated PBMC. BanLec induced secretion of several immune regulators, amongst them positive regulators of activated T cell proliferation and Jak-STAT signaling pathway. Top changed immune proteins were CD226, CD27, IFNG, IL18, IL2, CXCL10, LAT, ICOS, IL2RA, LAG3 and CD300C. BanLec stimulates PBMC of cows and pigs polyclonally and induces IL2 pathway and further proinflammatory cytokines. Proteomics data are available via ProteomeXchange with identifier PXD027505.

Reef-dwelling calcifiers face numerous environmental stresses associated with anthropogenic carbon dioxide emissions, including ocean acidification and warming. Photosymbiont-bearing calcifiers, such as large benthic foraminifera, are particularly sensitive. To gain insight into their resistance and adaptive mechanisms to climate change, Amphistegina lobifera from the Gulf of Aqaba were cultured under elevated pCO2 (492, 963, and 3182 ppm) fully-crossed with elevated temperature (28°C and 31°C) for two months. Differential protein abundances in host and photosymbionts amongst treatments were investigated alongside physiological responses and microenvironmental pH variations. Over 1000 proteins were identified, of which one-third varied significantly between treatments. Thermal stress induced protein depletions, along with reduced holobiont growth. Elevated pCO2 caused only minor proteomic alterations and color changes. However, combined stressors reduced pore sizes and increased microenvironmental pH, indicating adaptive modifications to gas exchange. Notably, substantial proteomic variations at moderate-pCO2 and 31°C indicate cellular stress, while stable physiological performance at high-pCO2 and 31°C is scrutinized by putative decreases in test stability. Our experiment shows that the effects of climate change can be missed when stressors are assessed in isolation, and that physiological responses should be assessed across organismal levels to make more realistic predictions for the fate of reef calcifiers.