Ocular surface changes may develop in patients with chronic renal failure (CRF) undergoing hemodialysis. In recent years, an association of CRF with dry eye syndrome has been emphasized. However, tear proteomics of CRF patients has not been analyzed. Here, we performed systematic profiling of the tear film proteins in CRF patients through use of isobaric tags for relative and absolute quantitative (iTRAQ) MS/MS, aiming to identify associations between dry eye symptoms and expression of tear proteomic changes in patients with CRF undergoing hemodialysis. Twenty CRF patients and ten healthy subjects underwent a series of ophthalmic examinations. Tear samples from the participants were analyzed by iTRAQ approach. A total of 1139 tear proteins were screened, and 212 differentially expressed proteins were identified. The pattern changes included 77 whose expression levels were upregulated (fold increase >1.2) whereas 135 others that were downregulated (fold decrease <1/1.2). Bioinformatics analysis showed that these proteins were significantly enriched in lipid metabolism, inflammatory, and immune response pathways. Furthermore, APOA1, APOA4, APOB, APOE, S100A8, S100A9, S100A4, HSP90B and other molecules were significantly changed. Our study elucidated the characteristics of tear dynamics and protein markers in CRF patients undergoing hemodialysis. Significance: Despite the association of chronic renal failure (CRF) with dry eye disease, there are no reports describing potentially important differentially expressed tear proteins in CRF patients undergoing hemodialysis. It is still a challenge to obtain a comprehensive description of the pathogenesis of dry eye in CRF patients which hinders establishing a patient specific therapeutic scheme. Our study is the first iTRAQ proteomics analysis of the tears of patients with CRF, which reveals the changes in the protein expression profile in CRF patients afflicted with dry eye disease. The identity was verified of some relevant differentially expressed proteins, and they may be candidate diagnostic markers of dry eye disease in patients with CRF. These tear film protein constituents found in hemodialysis patients can be of important clinical significance in treating this condition. SIGNIFICANCE: Despite the association of chronic renal failure (CRF) with dry eye disease, there are no reports describing potentially important differentially expressed tear proteins in CRF patients undergoing hemodialysis. It is still a challenge to obtain a comprehensive description of the pathogenesis of dry eye in CRF patients which hinders establishing a patient specific therapeutic scheme. Our study is the first iTRAQ proteomics analysis of the tears of patients with CRF, which reveals the changes in the protein expression profile in CRF patients afflicted with dry eye disease. The identity was verified of some relevant differentially expressed proteins, and they may be candidate diagnostic markers of dry eye disease in patients with CRF. These tear film protein constituents found in hemodialysis patients can be of important clinical significance in treating this condition.

Aberrantly sialylated cellular glycoconjugates were found to be involved in different processes during tumorigenesis. Such alteration was also noted in case of lung cancer, an important cause of cancer-related death throughout the world. Thus, study on lung cancer associated sialoglycoproteins is of paramount relevance to have a deeper insight into the mechanism of the disease pathogenesis. In the present study, sialic acid specific lectin (Maackia amurensis agglutinin and Sambcus nigra agglutinin)-based affinity chromatography followed by 2D-PAGE and MALDI-TOF/TOF mass spectrometric analysis were done to explore the disease-associated serum proteins of squamous cell carcinoma and adenocarcinoma [the major two subtypes of NSCLC (non-small cell lung carcinoma)] patients. Among seven identified proteins, α1-antitrypsin and haptoglobin-β were preferred for further studies. These two proteins were characterized as the disease associated serum-sialoglycoproteins of NSCLC-patients by western immunoblotting using each lectin specific inhibitor. The presence of these sialoglycoproteins was found on NSCLC cell lines (NCI-H520 & A549) by confocal microscopy. Both these proteins were also present in tissue samples of NSCLC origin and involved in proliferation, invasion and migration of NSCLC cells. Our findings suggest that α1-antitrypsin and haptoglobin-β may be the disease-associated sialoglycoproteins in NSCLC, which seem to be involved in disease progression. SIGNIFICANCE: Our contribution regarding the identification of the NSCLC associated sialoglycoproteins may provide a new vision towards the development of clinically useful newer strategies for the treatment of this disease.

Pecan (C. illinoinensis) pollen is an important cause of allergic respiratory disease. Pecan is distributed worldwide as shade, ornamental or cultivation tree. To date three well known pecan food allergens have been reported, however, pollen allergens have not been identified. Here, we describe the first identification of IgE recognized pecan pollen proteins, for which proteins were analyzed by 2-DE and immunoblotting using a pool of 8 sera from pecan sensitive patients as primary antibody. IgE recognized protein spots were analyzed by LC-MS/MS and identified using a database of translated protein sequences obtained by the assembly of C. illinoinensis public transcriptomic information. This study has identified 17 IgE binding proteins from pecan pollen including proteins widely recognized as allergens and panallergens. These findings will contribute to develop specific diagnosis and treatment of pecan pollen allergy. SIGNIFICANCE: Pecan is a tree highly valued for its fruits that have a great commercial value. To date three pecan seed storage proteins have been officially recognized by the WHO/IUIS allergen nomenclature subcommittee as food allergens (Car i 1, Car i 2 and Car i 4). Pecan tree pollen is highly allergenic and a clinically relevant cause of allergies in North America (USA and Mexico) and regions where the tree is extensively cultivated (Israel, South Africa, Australia, Egypt, Peru, Argentina, and Brazil). Here, we describe the first identification of IgE recognized pollen proteins using an immunoproteomics approach and a protein database created by the assembly of pecan public transcriptomic information. The findings described here will allow the development of new diagnostic and therapeutic modalities for pecan pollen allergy.

A new method to probe the conformational changes of glycoproteins on a systems-wide scale, termed limited deglycosylation assay (LDA), is described. The method measures the differential rate of deglycosylation of N-glycans on natively folded proteins by the common peptide:N-glycosidase F (PNGase F) enzyme which in turn informs on their spatial presentation and solvent exposure on the protein surface hence ultimately the glycoprotein conformation. LDA involves 1) protein-level N-deglycosylation under native conditions, 2) trypsin digestion, 3) glycopeptide enrichment, 4) peptide-level N-deglycosylation and 5) quantitative MS-based analysis of formerly N-glycosylated peptides (FNGPs). LDA was initially developed and the experimental conditions optimized using bovine RNase B and fetuin. The method was then applied to glycoprotein extracts from LLC-MK2 epithelial cells upon treatment with dithiothreitol to induce endoplasmic reticulum stress and promote protein misfolding. Data from the LDA and 3D structure analysis showed that glycoproteins predominantly undergo structural changes in loops/turns upon ER stress as exemplified with detailed analysis of ephrin-A5, GALNT10, PVR and BCAM. These results show that LDA accurately reports on systems-wide conformational changes of glycoproteins induced under controlled treatment regimes. Thus, LDA opens avenues to study glycoprotein structural changes in a range of other physiological and pathophysiological conditions relevant to acute and chronic diseases. SIGNIFICANCE: We describe a novel method termed limited deglycosylation assay (LDA), to probe conformational changes of glycoproteins on a systems-wide scale. This method improves the current toolbox of structural proteomics by combining site and conformational-specific PNGase F enzymatic activity with large scale quantitative proteomics. X-ray crystallography, nuclear magnetic resonance spectroscopy and cryoEM techniques are the major techniques applied to elucidate macromolecule structures. However, the size and heterogeneity of the oligosaccharide chains poses several challenges to the applications of these techniques to glycoproteins. The LDA method presented here, can be applied to a range of pathophysiological conditions and expanded to investigate PTMs-mediated structural changes in complex proteomes.

Characterization of post-translational modifications is among the most challenging tasks in tandem mass spectrometry-based proteomics which has yet to find an efficient solution. The ultra-tolerant (open) database search attempts to meet this challenge. However, interpretation of the mass shifts observed in open search still requires an effective and automated solution. We have previously introduced the AA_stat tool for analysis of amino acid frequencies at different mass shifts and generation of hypotheses on unaccounted in vitro modifications. Here, we report on the new version of AA_stat, which now complements amino acid frequency statistics with a number of new features: (1) MS/MS-based localization of mass shifts and localization scoring, including shifts which are the sum of modifications; (2) inferring fixed modifications to increase method sensitivity; (3) inferring monoisotopic peak assignment errors and variable modifications based on abundant mass shift localizations to increase the yield of closed search; (4) new mass calibration algorithm to account for partial systematic shifts; (5) interactive integration of all results and a rated list of possible mass shift interpretations. With these options, we improve interpretation of open search results and demonstrate the utility of AA_stat for profiling of abundant and rare amino acid modifications. AA_stat is implemented in Python as an open-source tool available at https://github.com/SimpleNumber/aa_stat. SIGNIFICANCE: Mass spectrometry-based PTM characterization has a long history, yet most of the methods rely on a priori knowledge of modifications of interest and do not provide a whole proteome modification landscape in a blind manner. The open database search is an efficient attempt to address this challenge by identifying peptides with mass shifts corresponding to possible modifications. Then, interpreting these mass shifts is required. Therefore, development of bioinformatics software for post-processing of the open search results, which is capable of detection and accurate annotation of new or unexpected modifications, from characterization of sample preparation efficiency and quality control to discovery of rare post-translational modifications, is of high importance.

This study describes the association between meat tenderness and abundance of soluble muscle proteins in Nellore bulls (Bos indicus) using a proteomic approach. We evaluated shear force (SF) of Longissimus thoracis muscle 24 h after slaughter and selected three experimental groups of animals with moderately tender (TE; SF = 3.9 ± 0.7 kg), moderately tough (TO; SF = 5.6 ± 0.7 kg) and very tough meat (TO+; SF = 7.9 ± 1.4 kg). Proteome was investigated by two-dimensional electrophoresis (2D-PAGE) in combination with electrospray ionization-tandem mass spectrometry (ESI-MS/MS). The metabolic proteins triosephosphate isomerase (TPI1) and phosphoglucomutase 1 (PGM1), the structural protein profilin 1 (PFN1), and cytosol aminopeptidase (LAP3) were up-regulated (P < 0.05) in the TE meat group when compared to the TO and TO+ groups. Actin structural proteins (ACTA1, ACTB, and ACTG1), the oxidative stress protein peroxiredoxin (PRDX6, PRDX2, PRDX1, and PARK7), heat shock protein isoforms, and co-chaperones (CDC37 and STIP1) were up-regulated (P < 0.05) in the TO and TO+ meat groups. In addition, we also identified proteins PFN1, LAP3, PRDX1, PRDX2, HSPD1, and ARHGDIA to be associated with beef tenderness. The results reported herein demonstrated that meat tenderness in Nellore cattle depends on the modulation and expression of a set of proteins involved in different biological pathways. SIGNIFICANCE: The manuscript entitled "Application of proteomic to investigate the different degrees of meat tenderness in Nellore breed" describes a classical proteomics work using two-dimensional gel electrophoresis (2D-PAGE), followed by mass spectrometry coupled to electrospray ionization ion trap (ESI-MS/MS) in order to understand the biochemical engineering involved in the process of meat tenderness. We evaluated shear force (SF) of Longissimus thoracis muscle samples of Nellore cattle (n = 90) and select three experimental groups of animals with moderately tender (TE; SF = 3.9 ± 0.7), moderately tough (TO; SF = 5.6 ± 0.7) and very tough meat (TO+; SF = 7.9 ± 1.4). The proteomic approach allowed observing that meat tenderness is influenced by structural proteins (ACTA1, ACTG1, ACTB, MYL1 and PFN1), co-chaperones (CDC37 and STIP1), heat shock proteins (HSP90AA1, HSP90AB1, HSPD1, HSPA1L, HSPA1A and HSPB1), regulatory protein (ARHGDIA), metabolic proteins (TPI1 and PGM1) and oxidative stress proteins (PRDX1, PRDX2, PRDX6, PARK7). Our results suggest that meat tenderness in Nellore depends on the modulation and expression of a set of proteins involved in different biological pathways.

Corynebacterium pseudotuberculosis (C.pseudotuberculosis) is a zoonotic pathogen that can cause cheese lymphadenitis in goats. In order to obtain detailed information about the pathogenesis and host immune response of goats infected with C.pseudotuberculosis, we used tandem mass tag (TMT)-labeling proteomic analysis to detect differentially expressed proteins (DEPs) in dairy goats infected with C.pseudotuberculosis, and confirmed the altered proteins with western blot. A total of 6611 trusted proteins were identified, and 126 proteins were differentially abundant. Gene ontology (GO) analysis showed that all DEPs were annotated as biological processes, cell composition, and molecular functions. Biological processes mainly involve acute inflammation and immune response; cell components mainly involve extracellular areas and high-density lipoprotein particles; molecular functions are mainly antigen binding, ferric iron binding, and iron ion binding. KEGG analysis showed that a total of 102 pathways were significantly enriched, mainly lysosomes, phagosomes, and mineral absorption pathways. Our findings provided the relevant knowledge of spleen protein levels in goats infected with C.pseudotuberculosis and revealed the complex molecular pathways and immune response mechanisms in the process of C.pseudotuberculosis infection. SIGNIFICANCE: C.pseudotuberculosis is the most fatal infectious disease in dairy goats, causing huge economic losses. However, the molecular pathways and immune response mechanisms of C.pseudotuberculosis infection in goats remain unclear. Therefore, we conducted a comparative quantitative proteomics study on dairy goats infected with C.pseudotuberculosis. The results provide a basis for better understanding the complexity of C.pseudotuberculosis infection, reveal the complex molecular pathways and immune response mechanisms in C.pseudotuberculosis infection, and provide some clues for identifying potential therapeutic targets. This is the first report to show that C.pseudotuberculosis infection in dairy goats can disrupt the immune response mechanism and lead to massive immune cell death. The study provided new findings on the interaction between C.pseudotuberculosis and the host.

Trypanosoma cruzi is a flagellate protozoa being the etiological agent of Chagas disease, a neglected tropical disease, which still poses a public health problem worldwide. The intricate molecular changes during T. cruzi-host interaction have been explored using different largescale omics techniques. However, protein stability is largely unknown. Thermal proteome profiling (TPP) methodology has the potential to characterize proteome-wide stability highlighting key proteins during T. cruzi infection and life stage transition from the invertebrate to the mammalian host. In the present work, T. cruzi epimastigotes and trypomastigotes cell lysates were subjected to TPP workflow and analyzed by quantitative large-scale mass spectrometry-based proteomics to fit a melting profile for each protein. A total of 2884 proteins were identified and associated to 1741 melting curves being 1370 in trypomastigotes (TmAVG 53.53 °C) and 1279 in epimastigotes (TmAVG 50.89 °C). A total of 453 proteins were identified with statistically different melting profiles between the two life stages. Proteins associated to pathogenesis and intracellular transport had regulated melting temperatures. Membrane and glycosylated proteins had a higher average Tm in trypomastigotes compared to epimastigotes. This study represents the first large-scale comparison of parasite protein stability between life stages. SIGNIFICANCE: Trypanosoma cruzi, a unicellular flagellate parasite, is the etiological agent of Chagas disease, endemic in South America and affecting more that 7 million people worldwide. There is an intense research to identify novel chemotherapeutic and diagnostic targets of Chagas disease. Proteomic approaches have helped in elucidating the quantitative proteome and PTMs changes of T. cruzi during life cycle transition and upon different biotic and abiotic stimuli. However, a comprehensive knowledge of the protein-protein interaction and protein conformation is still missing. In order to fill this gap, this manuscript elucidates the T. cruzi Y strain proteome-wide thermal stability map in the epimastigote and trypomastigote life stages. Comparison between life stages showed a higher average melting temperature stability for trypomastigotes than epimastigotes indicating a host temperature adaptation. Both presented a selective thermal stability shift for cellular compartments, molecular functions and biological processes based on the T. cruzi life stage. Membrane and glycosylated proteins presented a higher thermal stability in trypomastigotes when compared to the epimastigotes.

Porcine rotavirus (PoRV), particularly group A, is one of the most important swine pathogens, causing substantial economic losses in the animal husbandry industry. To improve understanding of host responses to PoRV infection, we applied isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantitatively identify the differentially expressed proteins in PoRV-infected IPEC-J2 cells and confirmed the differentially accumulated proteins (DAPs) expression differences by performing RT-qPCR and Western blot analysis. Herein, in PoRV- and mock-infected IPEC-J2 cells, relative quantitative data were identified for 4724 proteins, 223 of which were DAPs (125 up-accumulated and 98 down-accumulated). Bioinformatics analyses further revealed that a majority of the DAPs are involved in numerous crucial biological processes and signaling pathways, such as metabolic process, immune system process, amino acid metabolism, energy metabolism, immune system, MHC class I peptide loading complex, Hippo signaling pathway, Th1 and Th2 cell differentiation, antigen processing and presentation, and tubule bicarbonate reclamation. The cellular localization prediction analysis indicated that these DAPs may be located in the Golgi apparatus, nucleus, peroxisomal, cytoplasm, mitochondria, extracellular, plasma membrane, and endoplasmic reticulum (ER). Expression levels of three up-accumulated (VAMP4, IKBKE, and TJP3) or two down-accumulated (SOD3 and DHX9) DAPs upon PoRV infection, were further validated by RT-qPCR and Western blot analysis. Collectively, this work is the first time to investigate the protein profile of PoRV-infected IPEC-J2 cells using quantitative proteomics; these findings provide valuable information to better understand the mechanisms underlying the host responses to PoRV infection in piglets. SIGNIFICANCE: The proteomics analysis of this study uncovered the target associated with PoRV-induced innate immune response or cellular damage, and provided relevant insights into the molecular functions, biological processes, and signaling pathway in these targets. Out of these 223 DAPs, the expression levels of three up-accumulated (VAMP4, IKBKE, and TJP3) and two down-accumulated (SOD3 and DHX9) DAPs upon PoRV infection, have been further validated using RT-qPCR and Western blot analysis. These outcomes could uncover how PoRV manipulated the cellular machinery, which could further our understanding of PoRV pathogenesis in piglets.

Stress-induced immunosuppression is one of the most widespread problems in the poultry industry. Understanding the molecular regulatory mechanism of immunosuppression induced by stress in the chicken spleen would provide a scientific foundation for the prevention of stress reactions and antistress molecular breeding in poultry. To assess the protein expression profile of spleen tissue in a stress-included immunosuppression model, we performed a TMT-based proteomic analysis of chicken spleen tissue in a Dex-induced immunosuppression model (group C) and a control group (group A). We identified 590 differentially abundant proteins (DAPs) in chicken spleen tissue. These DAPs were significantly enriched in the following functional categories: ECM-receptor interaction, DNA replication, p53 signaling pathway, PI3K-Akt signaling pathway and NF-kappa B signaling pathway. Integrative analysis of the proteome and our previous transcriptome data revealed 62 DAPs showing correlations with the expression of their encoding mRNAs. Complementary proteome- and transcriptome-level analyses revealed a complex molecular network of stress-included immunosuppression. DPP4 and ALDH1A3 were the most significantly upregulated DAPs. GBP and OASL were identified as important nodes in the network related to stress-induced immunosuppression. The candidate genes identified in this study may be useful for the marker-based breeding of new chicken varieties with reduced stress levels. SIGNIFICANCE: This study provides a large amount of new information about the spleen proteome of the Dex-induced immunosuppression in chicks, as well as the correlation of transcriptome and proteome. Analysis of this resource has enabled us to examine mechanism of protein and transcript diversification, which expands the understanding of the complexity of the mechanism of stress-induced immunosuppression.

Low temperature in winter was the most crucial abiotic stress that limits the mangrove afforestation northward. Previous study demonstrated that Sonneratia apetala initially transplanted to high latitude area exhibited a stronger plasticity of cold tolerance. To clarify the underlying mechanism, the physiological and proteomic responses to chilling stress were investigated in S. apetala leaves. Our results found that cold-acclimated seedlings had lower relative electrolyte leakage and MDA content than non-acclimated seedlings. On the contrary, higher chlorophyll content and photosynthetic capacity were observed in cold-acclimated seedlings. With proteomic analyses, the differentially accumulated proteins (DAPs) involved in ROS scavenging, photosynthesis and energy metabolism, carbohydrate metabolism, cofactor biosynthesis, and protein folding were suggested to play important roles in enhancing the cold tolerance of S. apetala. However, the down-regulation DAPs were suggested as a tradeoff between plant growth and chilling response. By the protein-protein interaction analyses, translation elongation factor G, chlorophyll A-B binding protein and ascorbate peroxidase 1 were suggested as the important regulators in cold-acclimated S. apetala seedlings under chilling stress. Based on the above results, a schematic diagram describing the mechanism of cold tolerance of exotic mangrove species S. apetala that was achieved by cold acclimation was presented in this study. SIGNIFICANCE: The major environmental factor limits the mangrove afforestation northward is the low temperature in winter. Previous study reported that Sonneratia apetala grew in high latitude exhibited a higher cold tolerance than that in low latitude, which was suggested as a result of cold acclimation. To further understand "how cold acclimation enhance the cold tolerance in S. apetala", the response of S. apetala subjected to chilling stress with or without cold acclimation was investigated in this study at the physiological and proteomic aspects. Our physiological results showed that S. apetala seedlings treated with cold acclimation exhibited a higher tolerance under chilling stress than that without cold acclimation. By using the comparative proteomic approaches and bioinformatic analyses, various biological processes were suggested to play an important role in enhancing the cold tolerance of S. apetala under chilling stress, such as ROS scavenging, photosynthesis and energy metabolism, carbohydrate metabolism, cofactor biosynthesis, and protein folding. Among these differentially accumulated proteins, translation elongation factor G (eEF-G), chlorophyll A-B binding protein (CAB) and ascorbate peroxidase 1 (APX1) were identified as the hub proteins function in coordinated regulating ROS scavenging, photosynthesis and protein biosynthesis in chloroplast and subsequently enhanced the cold tolerance of S. apetala under chilling stress. Our results provided a further understanding of cold acclimation in improving the cold tolerance in exotic mangrove species S. apetala.

Bladder cancer (BC) is the fifth most common cancer with a high prevalence rate. It is classically classified in two groups, namely non-muscle invasive (NMIBC) and muscle invasive (MIBC). NMIBC accounts for 75% of cases and has a better prognosis than MIBC. However, 30-50% of the NMIBC patients will show recurrences throughout their lives, and about 10-20% of them will progress to MIBC, with frequent metastasis and a reduced survival rate. The diagnosis of bladder cancer is confirmed by direct visualization of the tumour and other mucosal abnormalities with endoscopic excision using cystoscopy and transurethral resection of the bladder (TURBT). An adequate TURBT requires complete resection of all visible tumour with appropriate sampling of the bladder to assess the depth of invasion. However, for many years, researchers have attempted to identify and utilise urinary markers for bladder cancer detection. Voided urine cytology has been the mainstay of urine-based diagnosis of bladder cancer since originally described by Papanicolau and Marshall. Nonetheless, urine cytology has several drawbacks, including a poor sensitivity for low-grade/stage tumours, a lack of interobserver consistency and a variable range of readings (e.g., atypical, atypical-suspicious, non-diagnostic). These shortcomings have inspired the search for more sensitive bladder cancer biomarkers. To bring precision medicine to genitourinary oncology, the analysis of the plasma/serum wide genome and proteome offers promising possibilities.

Metaproteomics is becoming widely used in microbiome research for gaining insights into the functional state of the microbial community. Current metaproteomics studies are generally based on high-throughput tandem mass spectrometry (MS/MS) coupled with liquid chromatography. In this paper, we proposed a deep-learning-based algorithm, named DeepFilter, for improving peptide identifications from a collection of tandem mass spectra. The key advantage of the DeepFilter is that it does not need ad hoc training or fine-tuning as in existing filtering tools. DeepFilter is freely available under the GNU GPL license at https://github.com/Biocomputing-Research-Group/DeepFilter. SIGNIFICANCE: The identification of peptides and proteins from MS data involves the computational procedure of searching MS/MS spectra against a predefined protein sequence database and assigning top-scored peptides to spectra. Existing computational tools are still far from being able to extract all the information out of MS/MS data sets acquired from metaproteome samples. Systematical experiment results demonstrate that the DeepFilter identified up to 12% and 9% more peptide-spectrum-matches and proteins, respectively, compared with existing filtering algorithms, including Percolator, Q-ranker, PeptideProphet, and iProphet, on marine and soil microbial metaproteome samples with false discovery rate at 1%. The taxonomic analysis shows that DeepFilter found up to 7%, 10%, and 14% more species from marine, soil, and human gut samples compared with existing filtering algorithms. Therefore, DeepFilter was believed to generalize properly to new, previously unseen peptide-spectrum-matches and can be readily applied in peptide identification from metaproteomics data.

Ammonia (NH3) is considered as the main pollutant in livestock houses and air environment, and its adverse effects on animal and human health have attracted widespread attention. However, trachea proteomics respond to NH3 is lacking, which is crucial to understanding how NH3 induces respiratory damage. In this study, we performed labeled quantitative proteomic (TMT-MS) analysis in the trachea of fatting pigs exposed to NH3 for 30 days. The proteomic results were then validated by Immunohistochemistry (IHC) and Parallel Reaction Monitoring (PRM). The results showed that a total of 126 differentially abundant proteins (DAPs) were identified (fold change <0.83 or > 1.2 and P < 0.05), including 70 differentially up-regulated proteins (DUPs) and 56 differentially down-regulated proteins (DDPs). These proteins were mainly located in intracellular regions and involved in immune response, metabolism and protein synthesis. The results of DAPs (EHHADH, RPL28, SLC25A6, TUBB6, CD14, CTSS, RPS11, RPL19, SLC25A5, RPS8, FABP3, RPL21, RPL34, RPL32, PDIA3, FBP1, HSPH1, SAR1A and SEC24C) verified by IHC and PRM were consistent with the proteomic results. The results of this study provided a basis and a novel insight for understanding the mechanism of NH3-induced tracheal injury. SIGNIFICANCE: Ammonia (NH3) is considered as the main pollutant in livestock houses and air environment, and its adverse effects on animal and human health have attracted widespread attention. However, trachea proteomics respond to NH3 is lacking, which is crucial to understanding how NH3 induces respiratory damage. Therefore, in this study, labeled quantitative proteomics (TMT-MS) was used to detect trachea tissue samples from finishing pigs in NH3 exposure group and control group, and PRM method was used to further verify the highly abundant proteins in NH3 exposure samples, so as to identify new diagnostic markers for NH3 poisoning. The results of this study provided a basis and a novel insight for understanding the molecular pathological mechanism of NH3-induced tracheal injury.

Withaferin A (WA) is a steroidal lactone extracted from Withania somnifera, commonly known as Ashwagandha. WA has several therapeutic benefits. The current study aims to identify proteins that are potentially regulated by WA in prostate cancer (PCA) cells. We used a SILAC-based proteomic approach to analyze the expression of proteins in response to WA treatment at 4 h and 24 h time points in three PCA cell lines: 22Rv1, DU-145, and LNCaP. Ontology analysis suggested that prolonged treatment with WA upregulated the expression of proteins involved in stress-response pathways. Treatment with WA increased oxidative stress, reduced global mRNA translation, and elevated the expression of cytoprotective stress granule (SG) protein G3BP1. WA treatment also enhanced the formation of SGs. The elevated expression of G3BP1 and the formation of SGs might constitute a mechanism of cytoprotection in PCA cells. Knockdown of G3BP1 blocked SG formation and enhanced the efficacy of WA to reduce PCA cell survival. SIGNIFICANCE: Withaferin A, a steroidal lactone, extracted from Withania somnifera is a promising anti-cancer drug. Using a SILAC-based quantitative proteomic approach, we identified proteins changed by WA-treatment at 4 h and 24 h in three prostate cancer (PCA) cell lines. WA-treatment induced the expression of proteins involved in apoptosis and reduced the expression of proteins involved in cell growth at 4 h. WA-treatment for 24 h enhanced the expression of proteins involved in stress response pathways. WA-treated cells exhibited increased oxidative stress, reduced mRNA translation and enhanced SG formation. PCA is characterized by higher metabolic rate and increased oxidative stress. PCA with a higher stress tolerance can effectively adapt to anti-cancer treatment stress, leading to drug resistance and cellular protection. Enhancing the level of oxidative stress along with inhibition of corresponding cytoprotective stress response pathways is a feasible option to prevent PCA from getting adapted to treatment stress. WA-treatment induced oxidative stress, in combination with blocking SGs by G3BP1 targeting, offers a therapeutic strategy to reduce PCA cell survival.

BRG1, one of core subunits of the SWI/SNF chromatin remodeling complex, is frequently mutated in cancers. Previously, we reported significant downregulation of the phosphorylation level of BRG1 on Ser1452 (<10%) in cell lines derived from ovarian clear cell carcinoma with frequent recurrence and acquired drug resistance. In this study, we tried to elucidate the roles of BRG1 phosphorylation, using cell lines expressing wild-type, phosphorylation-mimic (brg1-S1452D), or non-phosphorylatable (brg1-S1452A) BRG1. Quantitative proteomic analyses revealed upregulation of proteins and phosphoproteins related to linker histone H1s, histone methylation, and protein ubiquitylation in brg1-S1452D cells, which may coordinately promote the chromatin inactivation and ubiquitin-dependent degradation of target proteins. Consistent with these results, brg1-S1452D cells exhibited an increase in condensed chromatin and polyubiquitylated proteins. In brg1-S1452D cells, we also detected downregulation of various cancer-related proteins (e.g., EGFR and MET) as well as decreased migration, proliferation, and sensitivity to taxanes and oxaliplatin. Together, our results reveal that BRG1 phosphorylation drives tumor malignancy by inhibiting the functions of SWI/SNF complex in chromatin activation, thereby promoting expression of various cancer-related proteins. SIGNIFICANCE: For the first time we demonstrated that the mutation on Ser1452 phosphorylation site of BRG1, a component of SWI/SNF chromatin remodeling complex, changed protein and phosphoprotein levels of linker histone H1s, binding competitor of histone H1s, and histone methylase/demethylase involved in the heterochromatic histone modifications to promote the chromatin inactivation. In phosphorylation-mimic mutant, significant decrease of various cancer-related proteins as well as migration, proliferation, and sensitivity to specific antitumor agents were detected. Our results reveal that BRG1 phosphorylation drives tumor malignancy by inhibiting the functions of SWI/SNF complex in chromatin activation, thereby promoting expression of various cancer-related proteins.

Even in stallions with sperm quality within normal reference ranges at ejaculation, subtle differences in sperm quality exist that in many cases lead to reduced time frames for conservation of the ejaculate and/or reduced fertility. The spermatozoon is a cell highly suitable for proteomics studies, and the use of this technique is allowing rapid advances in the understanding of sperm biology. The aim of the present study was to investigate differences among stallions of variable sperm quality (based on motility and sperm velocities), although all horses had sperm characteristics within normal ranges. The proteome was studied using UHPLC/MS/MS and posterior bioinformatic and enrichment analysis; data are available via ProteomeXchange with identifier PXD025807. Sperm motility, linear motility and circular, straight line and average velocities (VCL, VSL, VAP) were measured using computer assisted sperm analysis (CASA). In stallions showing better percentages of motility, circular and average velocity predominated mitochondrial proteins with roles in the Citric acid cycle, pyruvate metabolism and oxidative phosphorylation. Interestingly, in stallions with better percentages of total motility, sperm proteins were also enriched in proteins within the gene ontology (G0) terms, single fertilization (G0: 0007338), fertilization (G0: 0009566), and zona pellucida receptor complex (GO:0002199). The enrichment of this proteins in samples with better percentages of total motility may offer a molecular explanation for the link between this parameter and fertility. SIGNIFICANCE: Proteomic analysis identified a high degree of specificity of stallion sperm proteins with discriminant power for motility, linear motility, and sperm velocities (VCL, VAP and VSL). These findings may represent an interesting outcome in relation to the molecular biology regulating the movement of the spermatozoa, and the biological meaning of the measurements that computer assisted sperm analysis (CASA) provide. Of a total of 903 proteins identified in stallion spermatozoa, 24 were related to the percentage of total motility in the sample; interestingly, gene ontology (G0) analysis revealed that these proteins were enriched in terms like single fertilization and fertilization, providing a molecular link between motility and fertility. Field studies indicate that the percentage of total motility is the CASA derived parameter with the best correlation with fertility in stallions.

The aim of this work was to gain insight into the molecular mechanisms underlying the effect of fulvic acid on drought-exposed tea plants. We performed proteomic analysis of fulvic acid-treated tea leaves from the target plants using tandem mass tag quantitative labeling technology and compared the results with those of a previous transcriptomic analysis. We identified 48 and 611 differentially abundant proteins in the leaves of tea plants treated with fulvic acid compared with the control under mild and severe drought, respectively. Comparative analysis showed that, under severe drought, 55 genes had similar expression patterns at the transcriptome and proteome levels, such as PAL, GBE, GBSS and bAS. Bioinformatic analysis revealed that those genes were mainly related to the starch and sucrose metabolism, phenylpropanoid biosynthesis and triterpenoid biosynthesis. SIGNIFICANCE: This study broadens the understanding of the molecular mechanisms underlying the improved drought resistance seen in tea plants in the presence of fulvic acid and provides a basis for further research on the genomics of drought tolerance in these plants. In addition, these findings could be used to develop new guidance strategies for improved drought management systems in tea plantation.

Cutaneous squamous cell carcinoma (CSCC) is a widespread malignancy but has a very low long-term survival rate for patients at the metastatic stage. Therefore, it is urgent to identify prognostic biomarkers for CSCC and improve our understanding of disease progression. Here we took advantage of a data-independent acquisition (DIA)-based nano liquid chromatography equipped with an orbitrap mass spectrometry (nLC-MS/MS) and ultraperformance LC coupled to a time-of-flight tandem MS (UPLC-TOF-MS/MS) technique to analyze cancer and corresponding noncancerous tissues from 20 CSCC patients for integrated proteomic and metabolomic analyses. Overall, 6241 tissue proteins were detected, while 136 proteins were significantly expressed in CSCC tissues. Further functional analysis revealed that various biological processes were highly enriched and participated in the pathogenesis of CSCC, especially DNA damage responses. Moreover, 641 named metabolites in total were identified, among which 181 were significantly changed in CSCC tissues. A total of 101 pathways were significantly enriched including apoptosis, autophagy, PI3K-Akt and mTOR signaling pathways. Interestingly, two pathways, protein digestion & absorption and platelet activation were both enriched in proteomic and metabolomic studies involving 5 proteins and 11 metabolites. Accordingly, a four-metabolite panel consisting of arachidonate, glutamine, glutamic acid, and proline (all area under the curve (AUC) values more than 0.9) was developed with a high accuracy (0.971) to distinguish the 20 detected cancer tissues from their noncancerous tissues. Collectively, our work highlighted the key elements and regulatory pathways involved in the pathogenesis of CSCC. More importantly, the present study not only provided potential biomarkers for the early diagnosis of CSCC, but also expanded our knowledge of the physiopathology of the disease. SIGNIFICANCE: CSCC is the second most common human cancer but has few treatment options and few sensitive biomarkers for diagnosis. Here we comprehensively revealed its molecular characteristics by performing integrated tissue proteomic and metabolomic analyses. Significantly distinct profiles and certain enriched pathways including DNA damage responses were identified as associated with CSCC. Moreover, protein digestion & absorption and platelet activation were both enriched in the proteome and metabolome. These identified molecular changes probably play significant roles in CSCC development. Finally, we developed a four-metabolite panel to distinguish CSCC with high accuracy. Overall, our data not only provided potential diagnostic biomarkers, but also extended knowledge on the pathogenesis of CSCC.

BACKGROUND/OBJECTIVES: Cereal products like flour and bread are known to trigger diseases such as wheat allergy, celiac disease and non-celiac wheat sensitivity (NCWS). Some of these diseases are caused by allergenic proteins, the expression of which might vary depending on the grain type and manufacturing processes. Therefore, we examined the protein composition and abundance of potentially allergenic proteins in flours from bread wheat, spelt and rye, and corresponding breads. MATERIALS AND METHODS: Using Nano-LC-ESI-MS/MS and label free quantification (LFQ) we analyzed the proteome of six different bread flours (wholegrain and superfine flours from rye, spelt and bread wheat) and 14 bread types (yeast and sourdough fermented breads from all flours and wheat breads plus/minus bread improver). Potentially allergenic proteins in flours and breads were functionally categorized using the Pfam database and relatively quantified by LFQ. RESULTS: We could show that almost equal numbers of proteins can be identified in rye- and spelt samples compared to wheat samples using the Uniprot bread wheat protein database, indicating high sequence conservation between cereals. In total, 4424 proteins were identified in the 20 flour and bread samples. The average number of identified proteins in flour (2719 ± 243) was slightly higher than in bread (2283 ± 232; P < 0.001). In wheat- and spelt wholegrain flour higher protein numbers (wheat: 2891 ± 90; spelt: 2743 ± 140) were identified on average than in superfine flour (wheat: 2562 ± 79; P = 0.009; spelt: 2431 ± 140; P = 0.004). Neither the absolute number nor the abundance distribution of potentially allergenic proteins were dependent on the flour type or the fermentation process, but known allergenic proteins like gliadins showed higher relative abundance in spelt- and wheat samples, compared to rye samples. CONCLUSION: We provide comprehensive proteome data for six flour types and related breads showing that the grain species have greater influence on proteome composition than milling and fermentation processes. Our data indicate that allergenic proteins are not selectively degraded during bread production and are more abundant in bread wheat and spelt compared to rye. SIGNIFICANCE: Our proteomics study revealed that bread contains a number of potentially and proven allergenic proteins. Most likely allergenicity is not dependent on milling or conventional fermentation processes, but on the grain type. Relative abundance of allergenic proteins was higher in spelt- and wheat samples than in rye samples. Considering rye bread as better suited to atopic individuals predisposed to react to cereal allergens, clinical trials are warranted to verify this assumption.

Di-n-butyl phthalate (DBP), a common compound of phthalates, can pose a risk to humans as a contaminant in the food industry. At present, the molecular mechanism of gene and protein toxicity caused by DBP in human cells is unclear. This in vitro study investigated the potential of inactivated Lactobacillus acidophilus NCFM in alleviating the damage caused by DBP in Caco-2 cells. According to the results from transcriptome and proteome analyses, the Caco-2 cells treated by DBP was resulted finally endoplasmic reticulum stress and mitochondrial oxidative damage. The most important differentially expressed genes and proteins involved in Caco-2 cells treated with NCFM to relieve DBP's cytotoxicity were TNF, NF-κB, CREB, P21, GADD45, FOS and CASP3. The molecular mechanism of DBP toxicity alleviated by strain NCFM was involved the MAPK pathway, via DBP bind to strain NCFM and avoid the activation of TNF receptor by DBP, so down-regulated the NF-κB, CREB, P21, GADD45, and CASP3, relieving the apoptosis of Caco-2 cells. Overall, our data provide new insights into detoxification of phthalate by using Lactobacillus. SIGNIFICANCE: Here we sequenced and assembled the transcriptome from Caco-2 cells which were treated with 4 groups: Control, DBP, strain NCFM, and strain NCFM+DBP groups, and combined it with proteome to characterize DBP detoxification genes/proteins through multiomics analysis. The cell viability in DBP treated groups were significantly increased by NCFM strain, indicating NCFM strain has the ability to alleviate the cytotoxicity of DBP via their binding ability with toxins. Furthermore, the results of transcriptome and proteome analysis showed that the signaling pathway of strain NCFM can alleviate DBP toxicity through MAPK pathway, and the potential biomarkers were identified too. This research may provided new information for developing new detoxification strategies for DBP.

Liver fluke, Fascioloides magna, is an important non-native parasite introduced to Europe, posing a threat to survival of local wildlife populations. The aim of this study was to assess the serum and liver protein profile of control and F. magna infected wild boars, by means of shotgun tandem mass tag - based quantitative high resolution proteomics approach. In serum, 4 differentially abundant proteins were found out of total 1073 identified, while in liver from 3520 identified proteins, 116 were differentially abundant between healthy and F. magna infected wild boars. Pathway analysis revealed that most of the proteins differing in abundance are involved in metabolism, biological oxidations, cellular responses to stimuli, fatty acid metabolism, and others. Validation of proteomic results was performed for paraoxonase-1, ceruloplasmin, glutathione S-transferase and liver enzymes by ELISA and automated assays. Complementary analysis of liver and serum in F. magna infection enabled insight into changes of proteome profile of the host at local and sistemic level. Our findings showed that chronic infection with F. magna is associated with immune response in host, oxidative stress and metabolomic changes in liver. SIGNIFICANCE: Liver fluke infections are recognised as worldwide neglected diseases with considerable veterinary and public health importance. Pathological changes, clinical signs and outcome of F. magna infection are strongly related to the type of final hosts and their different tolerance to infection. In order to gain insight into host-parasite interactions in wild boars, dead-end host for F. magna, we assessed proteomics profile of serum and liver of control animals and those infected with F. magna. Proteomics analysis of serum and liver in parallel showed as advantageous and beneficial, demonstrating protein alterations mainly at local level. Bioinformatics analysis enabled elucidation of molecular pathways associated with F. magna infection. Identification and validation of proteins associated with infection may have added value to current tools for efficient liver fluke control.