Soybean seed basic 7S globulin (Bg7S)-like proteins are found in many plant species. Bg7S was originally thought to be a major seed storage protein but was later found to be multifunctional, with stress response, antibacterial activity, hormone receptor-like activity. Moreover, functional differences between Bg7S proteins from legumes and other plants have been revealed. In non-leguminous plants, Bg7S molecules inhibit the invasion of pathogenic microorganisms. However, although leguminous plants have a peptide called leg-insulin that can bind to Bg7S, non-leguminous plants do not have leginsulin. Bg7S in leguminous plants and other plants may have evolved in functionally different directions. Several homologs of Bg7S in plants are reported, but there is no homolog of this protein in peas, suggesting that the pea evolution might have followed a different route when compared to other leguminous plants. Although the functions of Bg7S are well documented in plants, recent studies suggest that this protein is also important in controlling blood glucose level, blood pressure and plasma cholesterol level, and cancer cell antiproliferative actions.

Intracranial saccular aneurysms (ISA) represent 90%-95% of all intracranial aneurysm cases, characterizing abnormal pockets at arterial branch points. Ruptures lead to subarachnoid hemorrhages (SAH) and poor prognoses. We applied mass spectrometry-based peptidomics to investigate the peptidome of twelve cerebrospinal fluid (CSF) samples collected from eleven patients diagnosed with ISA. For peptide profile analyses, participants were classified into: 1) ruptured intracranial saccular aneurysms (RIA), 2) unruptured intracranial saccular aneurysms (UIA), and late-ruptured intracranial saccular aneurysms (LRIA). Altogether, a total of 2199 peptides were detected by both Mascot and Peaks software, from which 484 (22.0%) were unique peptides. All unique peptides presented conserved chains, domains, regions of protein modulation and/or post-translational modification sites related to human diseases. Gene Ontology (GO) analyses of peptide precursor proteins showed that 42% are involved in binding, 56% in cellular anatomical entities, and 39% in intercellular signaling molecules. Unique peptides identified in patients diagnosed with RIA have a larger molecular weight and a distinctive developmental process compared to UIA and LRIA (P ≤ 0.05). Continued investigations will allow the characterization of the biological and clinical significance of the peptides identified in the present study, as well as identify prototypes for peptide-based pharmacological therapies to treat ISA. SIGNIFICANCE.

Psoriasis is a chronic skin disorder with undefined pathogenesis. Several biomarkers for this disease have been identified by proteomic analysis. We explored the whole-proteomic changes in 45 pairs of psoriatic and adjacent noninvolved skin tissues in a Chinese population. A total of 3686 proteins were identified, of which 3008 were quantified. A total of 102 and 124 proteins were upregulated and downregulated in lesional skin, respectively. SART1 (P = 3.55 × 10-5) and GLTP (P = 1.54 × 10-3) were the most significantly down- and upregulated proteins. Nearly 90% of these differentially regulated proteins exhibited the same expression trends as those in an online RNA sequencing dataset for psoriasis; 19 differentially regulated proteins exhibited a negative relationship with DNA methylation data for psoriatic lesions. The differentially expressed proteins were enriched in ribosomes, antigen processing and presentation, immune response, and IL-17 signalling pathways. This study identified multiple differentially regulated proteins in psoriatic lesions, which suggested that changes in the proteome play important regulatory roles in psoriasis-associated processes. SIGNIFICANCE: Proteomic analysis was performed in 45 pairs of psoriatic and adjacent noninvolved skin tissues in a Chinese population. More than 3000 proteins were quantified, of which 226 were differentially expressed in psoriatic skin tissues. These proteins were mainly enriched in the immune response, antigen processing and presentation and IL-17 signalling pathways, which have been reported to be associated with the pathogenesis of psoriasis.

Mitogen Activated Protein Kinase1 (MAPK1) of Leishmania donovani functions as key regulators of various cellular activities, which seem to be imperative for parasite survival, infectivity, drug resistance and post-translational modification of chaperones/co-chaperones. However, very less is known about LdMAPK1 target proteins. With recent advancements in proteomics, we aimed to identify phosphoproteins which were differentially expressed in LdMAPK1 overexpressing (Dd8++/++) and single replacement mutants (Dd8+/) as compared to wild type (Dd8+/+) parasites, utilizing LC-MS/MS approach. An in-depth label-free phospoproteomic analysis revealed that modulation of LdMAPK1 expression significantly modulates expression levels of miscellaneous phosphoproteins which may act as its targets/substrates. Out of 1974 quantified phosphoproteins in parasite, 140 were significantly differentially expressed in MAPK1 overexpressing and single replacement mutants. These differentially expressed phosphoproteins are majorly associated with metabolism, signal transduction, replication, transcription, translation, transporters and cytoskeleton/motor proteins, hence suggested that MAPK1 may act in concert to modulate global biological processes. The study further implicated possible role of LdMAPK1 in regulation and management of stress machinery in parasite through post translational modifications. Precisely, comparative phosphoproteomics study has elucidated significant role of LdMAPK1 in regulating various pathways contributing in parasite biology with relevance to future drug development. SIGNIFICANCE: MAPKinase1, the downstream kinase of MAPK signal transduction pathway, has drawn much attention as potential therapeutic drug target due to their indispensable role in survival and infectivity of Leishmania donovani. However, limited information is available about its downstream effector proteins/signaling networks. Utilizing label free LC-MS/MS analysis, phosphoproteome of LdMAPK1 over-expressing (Dd8++/++) and LdMAPK1 single replacement mutants (Dd8+/-) with wild type (Dd8+/+) parasites was compared and identified 140 LdMAPK1 modulated phosphoproteins, mainly involved in pathways like signal transduction, metabolism, transcriptional, translational, post-translational modification and regulation of heat shock proteins. Interestingly, LdMAPK1 interacts directly with only six phosphoproteins i.e. casein kinase, casein kinase II, HSP83/HSP90, LACK, protein kinase and serine/threonine protein kinase. Thus, the study elucidates significant role of LdMAPK1 in Leishmania biology which may drive drug-discovery efforts against visceral leishmaniasis.

Membrane (M) proteins of coronaviruses are the most abundant component of the virus envelope and play crucial roles in virus assembly, virus budding and the regulation of host immunity. To understand more about these functions in the context of PEDV M protein, forty host cell proteins interacting with the M protein were identified in the present study by exploiting the proximity-labeling enzyme APEX2 (a mutant soybean ascorbate peroxidase). Bioinformatic analysis showed that the identified host cell proteins were related to fifty-four signal pathways and a wide diversity of biological processes. Interaction between M and five of the identified proteins (RIG-I, PPID, NHE-RF1, S100A11, CLDN4) was confirmed by co-immunoprecipitation (Co-IP). In addition, knockdown of PPID and S100A11 genes by siRNA significantly improved virus production, indicating that the proteins encoded by the two genes were interfering with or down-regulating virus replication in infected cells. Identification of the host cell proteins accomplished in this study provides new information about the mechanisms underlying PEDV replication and immune evasion. SIGNIFICANCE: PEDV M protein is an essential structural protein implicated in viral infection, replication and assembly although the precise mechanisms underlying these functions remain enigmatic. In this study, we have identified 40 host cell proteins that interact with PEDV M protein using the proximity-labeling enzyme APEX2. Co-immunoprecipitation subsequently confirmed interactions between PEDV M protein and five host cell proteins, two of which (S100A11 and PPID) were involved in down-regulating virus replication in infected cells. This study is significant in that it formulates a strategy to provide new information about the mechanisms relating to the novel functions of PEDV M protein.

Wound repair is a multistep process which involves coordination of multiple molecular players from different cell types and pathways. Though the cellular processes that are taking place in order to repair damage is already known, molecular players involved in crucial pathways are still scarce. In this regard, the present study intends to uncover crucial players that are involved in the central repair events through proteomics approach which included 2-D GE and LC-MS/MS using Caenorhabditis elegans wound model. Initial gel-based 2-D GE and following protein-protein interaction (PPI) network analyses revealed active role of calcium signaling, acetylcholine transport and serotonergic neurotransmitter pathways. Further, gel-free LC-MS/MS and following PPI network analyses revealed the incidence of actin nucleation at the initial hours immediately after injury. Further by visualizing the PPI network and the interacting players, pink-1, a mitochondrial Serine/threonine-protein kinase which is known to regulate mitochondrial dynamics, was found to be the central player in facilitating the mitochondrial fission and its role was further verified using qPCR analysis and pink-1 transgenic worms. Overall, the study delivers new insights from crucial regulatory pathways and central players involved in wound repair using high throughput proteomic approaches and the mass spectrometry Data (PXD024629/PXD024744) are available via ProteomeXchange. SIGNIFICANCE.

Chronic stressors represented risk factors for the etiology or exacerbation of several gastrointestinal diseases. The goal of the present study was to examine whether chronic restraint stress (CRS) could initiate and aggravate colonic inflammation, integrity damage and metabolic disturbance of rats. Firstly, increased inflammatory cytokines (interferon-γ (IFN-γ), tumor necrosis factor-α(TNF-α) and interleukin-10(IL-10)) and decreased tight junction (TJ) proteins (occludin and zonula occludins-1 (ZO-1)) in rat colon were observed. Secondly, untargeted metabolomics based on ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass (UPLC-Q-TOF/MS) revealed that TRP metabolism was the most prominently affected. Thirdly, quantification of TRP and its metabolites via prominence ultrafast liquid chromatography coupled with a QTRAP 5500 mass (UFLC-QTRAP-5500/MS) showed that TRP, kynurenine (KYN), kynurenic acid (KA) and 3-hydroxykynurenine (3-HK) were significantly increased. At the same time, 5-hydroxytryptamine (5-HT) was unchanged and 5-hydroxyindolacetic acid (5-HIAA) was significantly decreased in the colon of CRS rats. Besides, TRP metabolic enzyme changes were with the same trends as the corresponding metabolites. Thus, our data showed that CRS could initiate colonic inflammation, integrity damage and colonic metabolism disturbance, especially TRP-KYN metabolism pathway of rats, which may provide an experimental background for future research on stress-related gastrointestinal dysfunction. SIGNIFICANCE: Chronic exposure to psychological stress could induce metabolic imbalance of the body, and stressful life events were intimately correlated with frequent relapses in patients with intestinal disorders. The present study showed that chronic restraint stress (CRS) could initiate and aggravate colonic inflammation, integrity damage and metabolic disturbance, especially tryptophan-kynurenine metabolism of rats. Tryptophan-kynurenine pathway may be involved in the initiation and development of diseases induced by chronic stress. This research may shed light on future research on stress-related gastrointestinal dysfunction.

Noise Induced Hearing Loss (NIHL) is caused by excessive noise exposure due to occupational activities thus affects communication and quality of life. Prolonged occupational and environmental exposure to loud noise damages key molecules present in the micro-machinery of the ear which are required for the mechano-electrical transduction of sound waves in cochlea. Specific proteins are known to be associated with hearing loss and related structural and functional disabilities in the human inner, outer hair cells and cochlea. Rationale of this study was to identify the cochlear proteins associated with the pathophysiology of NIHL using proteomic approaches in mining based industrial workers. Total (n = 210) samples were collected from mining based industrial workers of central India. Subjects were categorized based on audiometric analysis. Proteome changes of the host serum were investigated using one and two-dimensional electrophoresis in combination with LC-MS/MS and MALDI-TOF-MS. Up-regulated 46 cochlear proteins among confirmed NIHL cases were identified by MASCOT. Shrinkage discriminant analysis provided top 25 discriminating feature proteins namely myosin, transthyretin, SERPIN, CCDC50, enkurin, transferin etc. The identified potential proteins may be used as biomarkers for early detection and to understand the pathogenic mechanism of NIHL. Evaluation of these biomarkers in follow-up cases may further aid in improving NIHL diagnosis. SIGNIFICANCE: Human proteome study in Noise Induced Hearing Loss (NIHL) cases has not been published till date. This study represents most comprehensive proteomic analysis in NIHL cases taken from Indian mine workers. The identified key twenty-five discriminating feature proteins which are upregulated when an individual develops (or is in stage of development of) NIHL, provides insights into the potential roles of these varied proteins in disease progression. The proteins thus identified by proteomic approach may be used as early diagnostic biomarker to predict the occurrence of disease at very early stage.

We used two fossil teeth from South American Pleistocene mammals to obtain subsuperficial acid etching samples. We employed samples from the species Notiomastodon platensis and Myocastor cf. coypus for the enamel etchings. The controls included an extant rodent (rat). After the first etching was discarded, a second 20-s etching (i.e., subsuperficial) was directly collected with a ZipTip and injected into an LTQ Orbitrap Velos for MS analysis. The peptides were identified with different software programs that used Peptide Spectrum Match (PSM) and de novo sequencing including similarity search strategies. Most of the peptides that were recovered from the enamel of the fossils belonged to enamel-specific proteins. To our knowledge, this is the first study that has described the recovery of enamel peptide molecules from extinct South American taxa, indicating that enamel peptide data from late Pleistocene fossils can be employed as an additional parameter for phylogenetic analysis, and that the sample can be obtained by a very conservative acid etching, with almost no damage to the fossils. SIGNIFICANCE: This study shows that it is possible to obtain information based on plenty of ancient peptides recovered from subsuperficial enamel of fossil teeth from South American Pleistocene. The quality of the data suggests that peptides are likely the best preserved biomolecules under certain harsh environmental conditions. The recovery procedure only lasted 20 s and was minimally destructive to the fossils. This opens a myriad of new possibilities for the study of the past.

Cobras are the most medically important elapid snakes in Africa. The African genera Naja and Hemachatus include snakes with neurotoxic and cytotoxic venoms, with shared biochemical, toxinological and antigenic characteristics. We have studied the antigenic cross-reactivity of four sub-Saharan Africa cobra venoms against an experimental monospecific Hemachatus haemachatus antivenom through comparative proteomics, preclinical assessment of neutralization, and third generation antivenomics. The venoms of H. haemachatus, N. annulifera, N. mossambica and N. nigricollis share an overall qualitative family toxin composition but depart in their proportions of three-finger toxin (3FTxs) classes, phospholipases A2 (PLA2s), snake venom metalloproteinases (SVMPs), and cysteine-rich secretory proteins (CRISPs). A monospecific anti-Hemachatus antivenom produced by Costa Rican Instituto Clodomiro Picado neutralized the lethal activity of the homologous and heterologous neuro/cytotoxic (H. haemachatus) and cyto/cardiotoxic (N. mossambica and N. nigricollis) venoms of the three spitting cobras sampled, while it was ineffective against the lethal and toxic activities of the neurotoxic venom of the non-spitting snouted cobra N. annulifera. The ability of the anti-Hemachatus-ICP antivenom to neutralize toxic (dermonecrotic and anticoagulant) and enzymatic (PLA2) activities of spitting cobra venoms suggested a closer kinship of H. haemachatus and Naja subgenus Afrocobra spitting cobras than to Naja subgenus Uraeus neurotoxic taxa. These results were confirmed by third generation antivenomics. BIOLOGICAL SIGNIFICANCE: African Naja species represent the most widespread medically important elapid snakes across Africa. To gain deeper insight into the spectrum of medically relevant toxins, we compared the proteome of three spitting cobras (Hemachatus haemachatus, Naja mossambica and N. nigricollis) and one non-spitting cobra (N. annulifera). Three finger toxins and phospholipases A2 are the two major protein families among the venoms analyzed. The development of antivenoms of broad species coverage is an urgent need in sub-Saharan Africa. An equine antivenom raised against H. haemachatus venom showed cross-reactivity with the venoms of H. haemachatus, N. mossambica and N. nigricollis, while having poor recognition of the venom of N. annulifera. This immunological information provides clues for the design of optimum venom mixtures for the preparation of broad spectrum antivenoms.

Although antibody mediated rejection (AMR) accounts for 20-30% of all acute renal allograft rejections, introducing biomarkers for a timely detection of allograft rejection has been remained challenging. This study investigated novel diagnostic biomarkers of AMR by examining of urine proteome in renal transplant patients. Thirty-six patients with kidney transplantation including 22 AMR patients and 14 patients with stable renal function (control group) were enrolled in this study. Urinary samples were collected and Label free quantification (LFQ) proteomics technique was applied on urine samples and data was subjected to Random Forest (RF) algorithm to predict main candidate proteins contributing in AMR. Finally, applicability of candidate diagnostic biomarkers was evaluated in new sets of AMR subjects, stable patients and healthy volunteers. A total of 1020 proteins were detected in urine proteome. RF algorithm predicted 20 differentially expressed proteins with the highest sensitivity and specificity and combination of EGF, COL6A, and NID-1 was identified as possible panel for early diagnosis of AMR. Applicability of EGF as diagnostic biomarker was validated in urine samples of independent set of AMR subjects. This is the first urinary proteomics study in AMR patients demonstrating that urinary EGF might be used as early diagnostic biomarker for AMR. SIGNIFICANCE: Renal antibody mediated rejection (AMR) accounts for 20-30% of all acute rejections of allografted kidneys. Although several possible biomarkers have been proposed to predict AMR, ineffectiveness of current urinary biomarkers in early diagnosing of AMR patients and in distinguishing AMR subjects from patients with stable kidney function casts doubts on their applicability in clinic. Here for the first time and based on the analysis of urinary proteome we showed that uEGF and uEGF/Cr might be candidate biomarkers to predict AMR with high diagnostic power.

Faecal proteomics studies have focussed on identification of microbial proteins, however; stool represents a valuable resource to interrogate the host interactions with the microbiota without the need for invasive intestinal biopsies. As the widely used enrichment method (differential centrifugation, DC) enriches for microbial proteins, we compared two other methods for enrichment of host proteins, termed 'host enriched' (HE) and ALL (all proteins). The HE and ALL protocols identified 1.8-fold more host proteins than DC while detecting a similar number of microbial proteins, but the methods had limited overlap in the specific microbial proteins detected. To maximize identification of both host and microbial proteins, samples were subjected to HE and the remaining material was used to perform DC. These two fractions displayed large differences in relative taxonomic abundance and cellular compartmentalization, with proteins from Bacteroidales and extracellular vesicles were enriched in the soluble HE component. The combination of data generated from these two fractions may allow identification of more distinct proteins than simply performing samples in duplicate or more complex fractionation techniques, or a single fraction could be chosen to suit the experimental hypothesis. SIGNIFICANCE: We compared how different stool protein preparation methods influenced the taxonomic and functional characteristics of microbial and host proteins identified. Surprisingly, a method designed to enrich for host proteins recovered a similar number of microbial protein groups to the method that specifically enriched intact bacterial cells. However, the taxonomic and subcellular origin of the microbial proteins differed considerably between the methods. By implementing a two-step method, we could maximize recovery of both host and microbial proteins derived from different cellular compartments and taxa.

Schistosoma mansoni causes schistosomiasis, which affects 240 million people, and 700 million people are living at risk of infection. Epigenetic mechanisms are important for transcriptional control and are well-known conserved transcriptional co-regulators in evolution, already described in mammal, yeast, protozoa and S. mansoni, responsible for heterochromatization and gene silence mechanisms through the formation of complexes of transcriptional repression in chromatin. Previous results from another group have shown that HP1 (SmCBX) proteins form chromatin complexes with SmMDB2/3 proteins and regulate stem cells and oviposition in parasite adult worms. In addition, results from other groups have shown that cercariae are transcriptionally silent and epigenetic mechanisms are involved in the regulation of gene expression in this stage. In this work, our aim was to give insights into SmHP1 and proteins involved in transcriptional regulation in the cercariae stage. Using monoclonal anti-HP1 antibody for Western blotting, immunoprecipitation, and mass spectrometry, we preliminarily determined nuclear proteins that putatively interact with HP1 to form complexes to regulate gene expression, heterochromatin formation, and translational complexes in the cercariae stage. So far, our data is to give some insights into nuclear interactors in S. mansoni cercariae. SIGNIFICANCE: The significance of this original paper is the evidence for Heterochromatin Protein (HP1), interaction with nuclear proteins in the cercariae stage. Schistosoma mansoni cercariae are the infective stage of the human beings in endemic areas of schistosomiasis, a neglected disease, most prevalent in Brazil and Africa. While cercariae are waiting for a host, it does not feed, gene expression is silent and protein synthesis is stopped. These biochemical mechanisms are recovered when cercariae find a human host, but all proteins and mechanisms are not still elucidated. Until now, literature shows that these phenomena are regulated by epigenetics mechanisms, dependent of histone posttranslational modifications. But we have few pieces of evidence about the other proteins that participates in these processes and which are the co-regulators of expression.

Kawasaki disease (KD) is a systemic vasculitis that can lead to severe cardiovascular complications, whereas the development and clinical usage of specific biomarkers might help diagnose KD and avoid certain complications. To this end, the molecular profiles of acute KD patients with coronary artery lesions (CAL) were first investigated through leukocyte proteomics and serum metabolomics assays. A total of 269 differentially abundant proteins and 35 differentially abundant metabolites with the top fold-changed levels were identified in acute KD patients compared to those in the healthy controls. Among them, several highly promising candidate marker proteins and metabolites indicative of KD progression were further analysed, such as the increased proteins ALPL, NAMPT, and S100P, as well as the decreased proteins C1QB and apolipoprotein family members. Moreover, metabolites, including succinic acid, dGMP, hyaluronic acid, L-tryptophan, propionylcarnitine, inosine, and phosphorylcholine, were found to be highly accurate at distinguishing between KD patients and healthy controls. Interestingly, the abnormal expression levels of a distinct set of proteins and metabolites in acute KD patients can be restored to normal levels upon intravenous immunoglobulin (IVIG) treatment. Overall, this work has revealed novel biomarkers and abnormal amino-acid metabolism as a prominent feature involved in KD patients with CAL. SIGNIFICANCE: KD is frequently concomitant with the development of life-threatening coronary vasculitis. Here, the profiles of leukocyte proteomics and serum metabolomics in acute KD patients with CALs were first investigated, and several hub molecules identified here could be used as supplemental biomarkers for KD diagnosis. Moreover, the metabolomic abnormalities especially the amino acids are particularly prominent in KD patients. Interestingly, the abnormal expression levels of a distinct set of proteins and metabolites in acute KD patients can be restored to normal levels upon IVIG treatment. Therefore, these findings might help understand the IVIG activities and also the underlying mechanisms of IVIG-resistant patients, thereby providing a new perspective for the exploration of mechanisms related to KD.

Gluten proteins contribute to the rheological properties of dough. Mass spectrometric techniques help to understand the contribution of these proteins to the quality of the end product. This work aimed to apply modern proteomic techniques to characterize and provide a better understanding of gluten proteins in wheat flours of different technological qualities. Nine Brazilian wheat flours (Triticum aestivum) classified by rheological gluten force were used to extract the proteins. Extracts were pooled together by technological qualities in low (LW), medium (MD), and superior (SP). Peptides were analyzed by nanoUPLC and mass spectrometry multiplex method (MSE). Collectively, 3545 peptides and 1297 proteins were identified, and 116 proteins were found differentially abundant. Low molecular weight glutenin subunits (LMW-GS) were found up-regulated only in SP samples. Proteins related to wheat grain hardness, such as puroindoline-A, were found in significant concentration in LW samples. After domain prediction, LW presented a different pattern with a lower abundance of functional domains, and SP presented chaperones, known to be involved in adequate folding of the storage proteins. NanoUPLC-MSE was efficient in analyzing and distinguishing the proteomic pattern of wheat flours from different qualities, pointing out the differentially abundant gluten proteins and providing a better understanding of wheat flour quality. SIGNIFICANCE: Common wheat is one of the most important staple food sources in the world. The improvement and comprehension of wheat quality has been a major objective of plant breeders and cereal chemists. Our findings highlighted the application of a modern proteomic approach to obtain a better understanding of the impact of gluten proteins on the technological quality of different wheat flours. The obtained data revealed different abundances of wheat quality-related proteins in superior quality flours when compared with samples of low rheological properties. In addition, multivariate statistical analysis clearly distinguished the flours of different qualities. This work contributes to the consolidation of research in the field of wheat technological quality.

Pseudomonas aeruginosa is an important opportunistic human pathogen with high prevalence in nosocomial infections. This microorganism is a good model for understanding biological processes such as the quorum-sensing response, the metabolic integration of virulence, the mechanisms of global regulation of bacterial physiology, and the evolution of antibiotic resistance. Till now, P. aeruginosa proteomic data, although available in several on-line repositories, were dispersed and difficult to access. In the present work, proteomes of the PAO1 strain grown under different conditions and from diverse cellular compartments have been joined to build the Pseudomonas PeptideAtlas. This resource is a comprehensive mass spectrometry-derived peptide and inferred protein database with 71.3% coverage of the total predicted proteome of P. aeruginosa PAO1, the highest coverage among bacterial PeptideAtlas datasets. The proteins included cover 89% of metabolic proteins, 72% of proteins involved in genetic information processing, 83% of proteins responsible for environmental information processing, more than 88% of the ones related to quorum sensing and biofilm formation, and 89% of proteins responsible for antimicrobial resistance. It exemplifies a necessary tool for targeted proteomics studies, system-wide observations, and cross-species observational studies. The manuscript describes the building of the PeptideAtlas and the contribution of the different proteomic data used. SIGNIFICANCE: Pseudomonas aeruginosa is among the most versatile human bacterial pathogens. Studies of its proteome are very important as they can reveal virulence factors and mechanisms of antibiotic resistance. The construction of a proteomic resource such as the PeptideAtlas enables targeted proteomics studies, system-wide observations, and cross-species observational studies.

Non-obese, spontaneous, and genetically predisposed type 2 diabetic Chinese hamsters exhibit metabolic abnormalities similar to those observed in human T2DM. Here, tandem mass tag (TMT)-based quantitative proteomics technology was used to screen and identify differentially abundant proteins in the liver that are associated with diabetes in Chinese hamsters. GO and KEGG pathway enrichment analysis were conducted to validate the findings, as well as qRT-PCR and western blotting. In total, 103 proteins were identified in the livers of diabetic hamsters, of which 48 were up-regulated and 55 were down-regulated. KEGG pathway enrichment analysis further demonstrated that linoleic acid metabolism, arachidonic acid metabolism, bile secretion, and other pathways were affected. Moreover, AQP9 and EPHX1 were significantly down-regulated in the bile secretion pathway, whereas PTGES2, Cyp2c27, and Cyp2c70 were associated with the arachidonic acid metabolic pathway. Serum levels of bile acid (BA) and arachidonic acid (AA) in diabetic Chinese hamsters were significantly higher than those in control hamsters. Cumulatively, our findings indicate that the five candidate proteins may be associated with abnormal BA and AA metabolism, suggesting their involvement in pathological changes in the livers of Chinese hamsters with T2DM. SIGNIFICANCE: The liver proteomics of Chinese hamsters describes differentially abundant proteins associated with T2DM, while promoting this animal model as an appropriate and ideal platform for investigating underlying molecular mechanisms of T2DM. This study reveals abnormal bile acid and arachidonic acid metabolism in T2DM hamsters, which may provide insights for studying the relationship between candidate proteins and KEGG pathways to elucidate the underlying molecular mechanism associated with T2DM.

Folates are essential for nucleotide biosynthesis, amino acid metabolism and cellular proliferation. Following carrier-mediated uptake, folates are polyglutamylated by folylpoly-ɣ-glutamate synthetase (FPGS), resulting in their intracellular retention. FPGS appears as a long isoform, directed to mitochondria via a leader sequence, and a short isoform reported as a soluble cytosolic protein (cFPGS). However, since folates are labile and folate metabolism is compartmentalized, we herein hypothesized that cFPGS is associated with the cytoskeleton, to couple folate uptake and polyglutamylation and channel folate polyglutamates to metabolon compartments. We show that cFPGS is a cytoskeleton-microtubule associated protein: Western blot analysis revealed that endogenous cFPGS is associated with the insoluble cellular fraction, i.e., cytoskeleton and membranes, but not with the cytosol. Mass spectrometry analysis identified the putative cFPGS interactome primarily consisting of microtubule subunits and cytoskeletal motor proteins. Consistently, immunofluorescence microscopy with cytosol-depleted cells demonstrated the association of cFPGS with the cytoskeleton and unconventional myosin-1c. Furthermore, since anti-microtubule, anti-actin cytoskeleton, and coatomer dissociation-inducing agents yielded perinuclear pausing of cFPGS, we propose an actin- and microtubule-dependent transport of cFPGS between the ER-Golgi and the plasma membrane. These novel findings support the coupling of folate transport with polyglutamylation and folate channeling to intracellular metabolon compartments. SIGNIFICANCE: FPGS, an essential enzyme catalyzing intracellular folate polyglutamylation and efficient retention, was described as a soluble cytosolic enzyme in the past 40 years. However, based on the lability of folates and the compartmentalization of folate metabolism and nucleotide biosynthesis, we herein hypothesized that cytoplasmic FPGS is associated with the cytoskeleton, to couple folate transport and polyglutamylation as well as channel folate polyglutamates to biosynthetic metabolon compartments. Indeed, using complementary techniques including Mass-spectrometry proteomics and fluorescence microscopy, we show that cytoplasmic FPGS is associated with the cytoskeleton and unconventional myosin-1c. This novel cytoskeletal localization of cytoplasmic FPGS supports the dynamic channeling of polyglutamylated folates to metabolon compartments to avoid oxidation and intracellular dilution of folates, while enhancing folate-dependent de novo biosynthesis of nucleotides and DNA/protein methylation.

Comparative proteomes of Actinoplanes utahensis ZJB-03852 grown on various saccharides (glucose, maltotriose, maltose, glucose + maltose) were analyzed using 2D-DIGE and MALDI-TOF/TOF-MS. Acarbose was detected in all groups except in the glucose only culture. The abundance of acarbose synthesis proteins AcbV, AcbK, AcbL and AcbN was highest in the medium containing mixed glucose and maltose. The accumulation of Zwf and Xpk1 in acarbose-producing media indicated that the cyclitol moiety of acarbose was derived from pentose phosphate pathway. The elevation of GlnA supported that glutamine was a good nitrogen source of the nitrogen-atom in acarbose synthesis. SIGNIFICANCE: Non-insulin-dependent diabetes mellitus, also known as Type II diabetes, constitutes >90% of the diabetes mellitus worldwide. Acarbose is clinically utilized to treat Type II diabetes, but the fermentation process of acarbose-producing Actinoplanes is usually accompanied with structural analogues of acarbose. In this study, we compared the proteomics of Actinoplanes utahensis ZJB-03852 grown on various saccharides by 2D-DIGE and MALDI-TOF/TOF-MS. Our findings highlighted the importance of key proteins in the formation of acarbose and its analogues when A. utahensis was cultivated in various saccharides. These results revealed fundamental data to elucidate the complexity of formation of acarbose analogues.

Spider mites are a group of arachnids belonging to Acari (mites and ticks), family Tetranychidae, known to produce nanoscale silk fibers characterized by a high Young's modulus. The silk fibroin gene of spider mites has been computationally predicted through genomic analysis of Tetranychus urticae Koch, but it has yet to be confirmed by proteomic evidence. In this work, we sequenced and assembled the transcriptome from two genera of spider mites, Tetranychus kanzawai Kishida and Panonychus citri (McGregor), and combined it with silk proteomics of T. urticae and P. citri to characterize the fibroin genes through comparative genomics and multiomics analysis. As a result, two fibroins were identified, which were different genes than those previously predicted by computational methods. The amino acid composition and secondary structure suggest similarity to aciniform or cylindrical spidroins of spider silk, which partly mirrors their mechanical properties, exhibiting a high Young's modulus. The availability of full-length fibroin sequences of spider mites facilitates the study of the evolution of silk genes that sometimes emerge in multiple lineages in a convergent manner and in the industrial application of artificial protein fibers through the study of the amino acid sequence and the resulting mechanical properties of these silks. SIGNIFICANCE: Here we sequenced and assembled the transcriptome from two genera of spider mites, T. kanzawai and P. citri, and combined it with silk proteomics of T. urticae and P. citri to characterize the fibroin genes through comparative genomics and multiomics analysis. Spider mite silk is especially characterized by its extremely fine nano-scale diameter and high Young's modulus, even exceeding those of spider silks. The availability of full-length fibroin sequences of spider mites facilitates the study of the evolution of silk genes, which independently evolved in mites, insects, and spiders but yet show sequence convergence, and in the industrial application of artificial protein fibers through the study of the amino acid sequence and the resulting mechanical properties of these silks.

Protein aggregation is indicative of failing protein quality control systems. These systems are responsible for the refolding or degradation of aberrant and misfolded proteins. Heat stress can cause proteins to misfold, triggering cellular responses including a marked increase in the ubiquitination of proteins. This response has been characterized in yeast, however more studies are needed within mammalian cells. Herein, we examine proteins that become ubiquitinated during heat shock in human tissue culture cells using diGly enrichment coupled with mass spectrometry. A majority of these proteins are localized in the nucleus or cytosol. Proteins which are conjugated under stress display longer sequence lengths, more interaction partners, and more hydrophobic patches than controls but do not show lower melting temperatures. Furthermore, heat-induced conjugation sites occur less frequently in disordered regions and are closer to hydrophobic patches than other ubiquitination sites; perhaps providing novel insight into the molecular mechanism mediating this response. Nuclear and cytosolic pools of modified proteins appear to have different protein features. Using a pulse-SILAC approach, we found that both long-lived and newly-synthesized proteins are conjugated under stress. Modified long-lived proteins are predominately nuclear and were distinct from newly-synthesized proteins, indicating that different pathways may mediate the heat-induced increase of polyubiquitination. SIGNIFICANCE: The maintenance of protein homeostasis requires a balance of protein synthesis, folding, and degradation. Under stress conditions, the cell must rapidly adapt by increasing its folding capacity to eliminate aberrant proteins. A major pathway for proteolysis is mediated by the ubiquitin proteasome system. While increased ubiquitination after heat stress was observed over 30 years ago, it remains unclear which proteins are conjugated during heat shock in mammalian cells and by what means this conjugation occurs. In this study, we combined SILAC-based mass spectrometry with computational analyses to reveal features associated to proteins ubiquitinated while under heat shock. Interestingly, we found that conjugation sites induced by the stress are less often located within disordered regions and more often located near hydrophobic patches. Our study showcases how proteomics can reveal distinct feature associated to a cohort of proteins that are modified post translationally and how the ubiquitin conjugation sites are preferably selected in these conditions. Our work opens a new path for delineating the molecular mechanisms leading to the heat stress response and the regulation of protein homeostasis.

Rifamycin B is produced by Amycolatopsis mediterranei S699 as a secondary metabolite. Its semi-synthetic derivatives have been used for curing tuberculosis caused by Mycobacterium tuberculosis. But the emergence of rifampicin-resistant strains required analogs of rifamycin B to be developed by rifamycin biosynthetic gene cluster manipulation. In 2014 genetic engineering of the rifamycin polyketide synthase gene cluster in S699 led to a mutant, A. mediterranei DCO#34, that produced 24-desmethylrifamycin B. Unfortunately, the productivity was strongly reduced to 20 mgL-1 as compared to 50 mgL-1 of rifamycin B. To understand the mechanisms leading to reduced productivity and rifamycin biosynthesis by A. mediterranei S699 during the early and late growth phase we performed a proteome study for wild type strain S699, mutant DCO#34, and the non-producer strain SCO2-2. Proteins identification and relative label-free quantification were performed by nLC-MS/MS. Data are available via ProteomeXchange with identifier PXD016416. Also, in-silico protein-protein interaction approach was used to determine the relationship between different structural and regulatory proteins involved in rifamycin biosynthesis. Our studies revealed RifA, RifK, RifL, Rif-Orf19 as the major regulatory hubs. Relative abundance expression values revealed that genes encoding RifC-RifI and the transporter RifP, down-regulated in DCO#34 and genes encoding RifR, RifZ, other regulatory proteins up-regulated. SIGNIFICANCE: The study is designed mainly to understand the underlying mechanisms of rifamycin biosynthesis in Amycolatopsis mediterranei. This resulted in the identification of regulatory hubs which play a crucial role in regulating secondary metabolism. It elucidates the complex mechanism of secondary metabolite biosynthesis and their conversion and extracellular transportation in temporal correlation with the different growth phases. The study also elucidated the mechanisms leading to reduced production of analog, 24-desmethylrifamycin B by the genetically modified strain DCO#34, derivatives of which have been found effective against rifampicin-resistant strains of Mycobacterium tuberculosis. These results can be useful while carrying out genetic manipulations to improve the strains of Amycolatopsis to produce better analogs/drugs and promote the eradication of TB. Thus, this study is contributing significantly to the growing knowledge in the field of the crucial drug, rifamycin B biosynthesis by an economically important bacterium Amycolatopsis mediterranei.

Soybean is one of the most commonly allergenic foods in the U.S. However, the variety of commercial soy ingredients used in the food industry makes soybean a challenging allergen to detect and quantify. The processing methods used to produce soy-derived ingredients result in protein modifications that often substantially impact detection and quantification with commonly used antibody-based methods. This study aimed to develop a mass spectrometry (MS)-based method capable of quantifying commercially processed soy ingredients in food matrices. A quantification strategy using external standards with internal calibrants was developed and evaluated, resulting in the ultimate use of a matrix-independent standard curve of non-roasted soy flour with milk proteins as carrier proteins. The method performance was evaluated by quantifying six soy-derived ingredients in incurred food matrices using three quantification strategies. Out of the twelve ingredient-matrix combinations with 10 ppm incurred total soy protein, eight had maximum recoveries between 60 and 120% using the full standard curve strategy. Other quantification strategies may be useful for internal quality control and interlaboratory calibrations. Compared with three commercial ELISA kits, the MS method showed a substantial advantage in quantifying the highly processed soy proteins in food matrices. SIGNIFICANCE: The ability to quantify undeclared soy protein in food products regardless of the soy ingredient source is essential for food allergen management, risk assessment, and regulatory enforcement. The MS-based method described here is able to reliably quantify six different soy-derived ingredients incurred in a model processed food. When compared with existing commercial ELISA methods, the MS method is much less affected by matrices and ingredient types, indicating its wider applicability to a range of soy-derived ingredients and processed products.

Honey adulteration is a common practice that deceives consumers and devalues the unique curative and food properties of honey. For marketing, each honey must satisfy an internationally valid Codex standard. One of the quality parameters is diastase/amylase activity, which, if lowered, may be compensated for by the addition of foreign amylases. However, the estimation of enzyme activity does not enable identification of artificially added amylases. 45 honey samples were analyzed using label-free nanoLC-MS/MS proteomics. Four honeys were found to contain the foreign amylases from Aspergillus niger, Bacillus amyloliquefaciens and/or Bacillus licheniformis. This result was confirmed via proof of specificity at multiple levels. Furthermore, we identified a series of plant-related protein groups. Despite plant-related proteins constituting a significant portion of honey proteins, they were minor components compared to the major honey bee-derived proteins. Bioinformatic analysis also provided evidence for aphid and catalase proteins in honey, but the limited specificity of the MS/MS identified peptides must be considered. Overall, we demonstrate a proteomics approach employing LC-MS/MS that is useful for proving adulteration and assessing honey quality. As an resource useful for reference, we provide curated sequence databases. In addition, we provide many markers that are naturally found in honey for future studies. SIGNIFICANCE: Honey is unique natural product used since ancient times as a food and natural medicine. Humans strive to understand honey components because they can characterize different types of honey and be used for authentication and origin assessment. One of the important honey components are proteins. The proteins present in honey can naturally occur in honey, but some of them can be used to mask deficiencies in some honey quality properties. Diastases/amylases are such proteins, and their activity, a measure of honey freshness, can decrease in time or due to processing. To our knowledge, we for the first time specifically identify foreign amylases in honey. However, this study provided new information on other non-honey bee proteins in honey. Thus, this study is also of importance due to its identification of plant and aphid proteins and catalase-related proteins. This study provides a clue explaining the controversial presence of catalase in honey, since catalases can be identified and their origin determined via proteomics.

Persistent plant viruses multiply and circulate inside insect vectors following the route of midgut-hemolymph-salivary gland. Currently, how viruses interact with insect vectors after they are released into hemolymph is not entirely clear. In this study, we found that the hemolymph and fat body (HF) contained the highest Rice stripe virus (RSV) levels. Proteomic analysis on RSV-free and RSV-infected HF identified 156 differentially expressed proteins (DEPs), with the majority of them participating in metabolism, transportation, and detoxification. The RNA binding protein esf2 was the most downregulated protein. Knocking down the expression of esf2 did not influence the RSV burden, but caused the lethal effect to L. striatellus. In contrast, the mRNA decay protein ZFP36L1 was 69% more abundant upon RSV infection, and suppression of ZFP36L1 significantly increased the RSV burden. Our results reveal the potential role of ZFP36L1 in restricting the viral proliferation, and provide valuable clues for unravelling the interaction between RSV and L. striatellus in HF. SIGNIFICANCE: More than 76% of plant viruses are transmitted by insect vectors. For persistent propagative transmission, plant viruses multiply and circulate inside insects following the route of midgut-hemolymph-salivary gland. However, how viruses interact with vector insects after they are released into hemolymph is not entirely clear. Our study investigated the influence of rice stripe virus (RSV) on insect hemolymph and fat body by iTRAQ labeling method. Among the 156 differentially expressed proteins (DEPs) identified, two proteins associated with mRNA metabolism were selected for function analysis. We found that the mRNA decay activator protein ZFP36L1 influenced the RSV proliferation, and RNA binding protein esf2 caused the lethal effect to L. striatellus. Our results provide valuable clues for unveiling the interaction between RSV and L. striatellus, and might be useful in pest management.

Copper is an essential metal for life, but is toxic at high concentrations. In mammalian cells, two copper transporters are known, CTR1 and CTR2. In order to gain insights on the possible influence of the import pathway on cellular responses to copper, two copper challenges were compared: one with copper ion, which is likely to use preferentially CTR1, and one with a copper-polyacrylate complex, which will be internalized via the endosomal pathway and is likely to use preferentially CTR2. A model system consisting in the J774A1 mouse macrophage system, with a strong endosomal/lysosomal pathway, was used. In order to gain wide insights into the cellular responses to copper, a proteomic approach was used. The proteomic results were validated by targeted experiments, and showed differential effects of the import mode on cellular physiology parameters. While the mitochondrial transmembrane potential was kept constant, a depletion in the free glutahione content was observed with copper (ion and polylacrylate complex). Both copper-polyacrylate and polyacrylate induced perturbations in the cytoskeleton and in phagocytosis. Inflammatory responses were also differently altered by copper ion and copper-polyacrylate. Copper-polyacrylate also perturbed several metabolic enzymes. Lastly, enzymes were used as a test set to assess the predictive value of proteomics. SIGNIFICANCE: Proteomic profiling provides an in depth analysis of the alterations induced on cells by copper under two different exposure modes to this metal, namely as the free ion or as a complex with polyacrylate. The cellular responses were substantially different between the two exposure modes, although some cellular effects are shared, such as the depletion in free glutathione. Targeted experiments were used to confirm the proteomic results. Some metabolic enzymes showed altered activities after exposure to the copper-polyacrylate complex. The basal inflammatory responses were different for copper ion and for the copper-polyacrylate complex, while the two forms of copper inhibited lipopolysaccharide-induced inflammatory responses.