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Spider venoms are composed of a complex mixture of bioactive molecules. The structural and functional characterization of these molecules in the venom of the Brazilian spider Acanthoscurria natalensis, has been little explored. The venom was fractionated using reversed-phase liquid chromatography. The fraction with hyaluronidase activity was named AnHyal. The partial sequencing of AnHyal revealed the presence of a CRISP-like protein, in addition to hyaluronidase, comprising 67% coverage for hyaluronidase from Brachypelma vagans and 82% for CRISP-like protein from Grammostola rosea. 1D BN-PAGE zymogram assays of AnHyal confirmed the presence of enzymatically active 53 kDa monomer and 124 and 178 kDa oligomers. The decomposition of the complexes by 2D BN/SDS-PAGE zymogram assays showed two subunits, 53 (AnHyalH) and 44 kDa (AnHyalC), with sequence similarity to hyaluronidase and CRISP proteins, respectively. The secondary structure of AnHyal is composed by 36% of α-helix. AnHyal presented maximum activity at pH between 4.0 and 6.0 and 30 and 60 °C, showed specificity to hyaluronic acid substrate and presented a KM of 617.9 μg/mL. Our results showed that hyaluronidase and CRISP proteins can form a complex and the CRISP protein may contribute to the enzymatic activity of AnHyalH.

An increased knowledge on the real impacts of ectomycorrhizal symbiosis in forest 32 species is needed to optimize forest sustainable productivity and thus to improve forests 33 services and their capacity to act as carbon sinks. In this study we investigated the 34 response of an oak species to ectomycorrhizae formation using a proteomics approach 35 complemented by biochemical analysis of carbohydrates levels. Comparative proteome 36 analysis between mycorrhizal and non-mycorrhizal cork oak plants revealed no 37 differences at the foliar level. However, the protein profile of 34 unique oak proteins 38 was altered in the roots. Consistent with the results of the biochemical analysis, the 39 proteome analysis of the mycorrhizal roots suggests a decreasing utilization of sucrose 40 for the metabolic activity of mycorrhizal roots which is consistent with an increased 41 allocation of carbohydrates from the plant to the fungus in order to sustain the 42 symbiosis. In addition, a promotion of protein unfolding mechanisms, attenuation of 43 defense reactions, increased nutrient mobilization from the plant-fungus interface (N 44 and P), as well as cytoskeleton rearrangements and induction of plant cell wall 45 loosening for fungal root accommodation in colonized roots, are also suggested by the 46 results. The suggested improvement in root capacity to take up nutrients accompanied 47 by an increase of root biomass without apparent changes in aboveground biomass 48 strongly re-enforce the potential of mycorrizal inoculation to improve cork oak forest 49 resistance capacity to cope with coming climate change.

The C2C12 myoblast is a model that has been used extensively to study the process of skeletal muscle differentiation. Proteomics has advanced our understanding of skeletal muscle biology and also the differentiation process of skeletal muscle cells. However, there is still no comprehensive analysis of C2C12 myoblast proteomes, which is important for the understanding of key drivers for the differentiation of skeletal muscle cells. Here, we conducted multidimensional proteome profiling to get a comprehensive analysis of proteomes and PTMomes of C2C12 myoblasts with a TiSH strategy. A total of 8313 protein groups were identified, including 7827 protein groups from nonmodified peptides, 3803 phosphoproteins, and 977 formerly sialylated N-linked glycoproteins. Integrated analysis of proteomic and PTMomic data showed that almost all of the kinases and transcription factors in the muscle cell differentiation pathway were phosphorylated. Further analysis indicated that sialylation might play a role in the differentiation of C2C12 myoblasts. Further functional analysis demonstrated that C2C12 myoblasts showed a decreased level of sialylation during skeletal muscle cell differentiation. Inhibition of sialylation with the sialyltransferase inhibitor 3Fax-Neu5Ac resulted in the lower expression of MHC and suppression of myoblast fusion. In all, these results indicate that sialylation has an effect on the differentiation of skeletal muscle cells.

Scorpion venoms are formed by toxins harmful to various organisms, including humans. Several techniques have been developed to understand the role of proteins in animal venoms, including proteomics approach. Rhopalurus agamemnon (Koch, 1839) is the largest scorpion in the Buthidae family in the Brazilian Cerrado, measuring up to 110 mm in total length. The accident with R. agamemnon is painful and causes some systemic reactions, but the specie's venom remains uninvestigated. We explore the venom protein composition using a proteomic and a biological-directed approach identifying 230 protein compounds including enzymes like Hyaluronidase, metalloproteinase, L-amino acid oxidase and amylase, the last two are first reported for scorpion venoms. Some of those new reports are important to demonstrate how distant we are from a total comprehension of the diversity about venoms in general, due to their diversity in composition and function. Biological significance In this study, we explored the composition of venom proteins from the scorpion Rhopalurus agamemnon. We identified 230 proteins from the venom including new enzyme reports. These data highlight the unique diversity of the venom proteins from the scorpion R. agamemnon, provide insights into new mechanisms of envenomation and enlarge the protein database of scorpion venoms. The discovery of new proteins provides a new scenario for the development of new drugs and suggests molecular targets to venom components.

Intestinal ischemia reperfusion injury (iIRI) is a severe clinical condition presenting high morbidity and mortality worldwide. Some of the systemic consequences of IRI can be prevented by applying ischemic preconditioning (IPC), a series of short ischemia/reperfusion events preceding the major ischemia. Although neutrophils are key players in the pathophysiology of ischemic injuries, neither the dysregulation presented by these cells in iIRI nor the protective effect of iIPC have their regulation mechanisms fully understood. Protein phosphorylation, as well as the regulation of the respective phosphatases and kinases are responsible for regulating a large number of cellular functions in the inflammatory response. Moreover, in previous work we found hydrolases and transferases to be modulated in iIR and iIPC, suggesting the possible involvement of phosphatases and kinases in the process. Therefore, in the present study, we analyzed the phosphoproteome of neutrophils from rats submitted to mesenteric ischemia and reperfusion, either submitted or not to IPC, compared to quiescent controls and sham laparotomy. Proteomic analysis was performed by multi-step enrichment of phosphopeptides, isobaric labeling, and LC-MS/MS analysis. Bioinformatics was used to determine phosphosite and phosphopeptide abundance and clustering, as well as kinases and phosphatases sites and domains. We found that most of the phosphorylation-regulated proteins are involved in apoptosis and migration, and most of the regulatory kinases belong to CAMK and CMGC families. An interesting finding revealed groups of proteins that are modulated by iIR, but such modulation can be prevented by iIPC. Among the regulated proteins related to the iIPC protective effect, Vamp8 and Inpp5d/Ship are discussed as possible candidates for control of the iIR damage.

In the past decade, relative proteomic quantification using isobaric labeling technology has developed into a key tool for comparing the expression of proteins in biological samples. Although its multiplexing capacity and flexibility make this a valuable technology for addressing various biological questions, its quantitative accuracy and precision still pose significant challenges to the reliability of its quantification results. Here, we give a detailed overview of the different kinds of isobaric mass tags and the advantages and disadvantages of the isobaric labeling method. We also discuss which precautions should be taken at each step of the isobaric labeling workflow, to obtain reliable quantification results in large-scale quantitative proteomics experiments. In the last section, we discuss the broad applications of the isobaric labeling technology in biological and clinical studies, with an emphasis on thermal proteome profiling and proteogenomics.

We have investigated the potential for analysis of the complex peptide mixtures secreted from frog skin using off-line LC-MALDI MS/MS. Since only limited information about the sequence of such peptides is available, de novo sequencing followed by Blast search was needed. An automated workflow has been applied based on automated collection of the chromatographic fractions on the MALDI target followed by automated recording of the MS/MS spectra of the 10 most intense peaks in each fraction. The MS/MS spectra were submitted to de novo sequencing by the Peaks software and searched for potential matches in an amphibian database extracted from UniProt. A number of known skin secreted peptides as well as precursor proteins for these were identified. However, most of the recorded MS/MS data and deduced sequences did not result in matches with the databases. These sequences might serve as a reference for future studies of peptides secreted from frogs.

Grapevine downy mildew is an important disease affecting crop production leading to severe yield losses. This study aims to identify the grapevine cultivar-specific adjustments of leaf proteome that allow the discrimination between resistance and susceptibility towards P. viticola (constitutive (0 h) and in after inoculation (6, 12 and 24 h). Leaf proteome analysis was performed using 2D difference gel electrophoresis followed by protein identification via mass spectrometry. In addition, we analysed ROS production, antioxidant capacity, lipid peroxidation and gene expression. Proteins related to photosynthesis and metabolism allowed the discrimination of resistant and susceptible grapevine cultivars prior to P. viticola inoculation. Following inoculation increase of hydrogen peroxide levels, cellular redox regulation, establishment of ROS signalling and plant cell death seem to be key points differentiating the resistant genotype. Lipid associated signalling events, particularly related to jasmonates appear also to play a major role in the establishment of resistance. The findings from this study contribute to a better understanding of genotype-specific differences that account for a successful establishment of a defence response to the downy mildew pathogen. Biological significance Here, we present for the first time grapevine cultivar-specific adjustments of leaf proteome that allow the discrimination between resistance and susceptibility towards P. viticola (constitutive (0 h) and in after inoculation (6, 12 and 24 h). We have highlighted that, following inoculation, the major factors differentiating the resistant from the susceptible grapevine cultivars are the establishment of effective ROS signalling together with lipid-associated signalling events, particularly related to jasmonates. It is believed that plants infected with biotrophic pathogens suppress JA-mediated responses, however recent evidences shown that jasmonic acid signalling pathway in grapevine resistance against Plasmopara viticola. Our results corroborate those evidences and highlight the importance of lipid- signalling for an effective resistance response against the downy mildew pathogen.

Chagas disease is caused by the protozoan Trypanosoma cruzi, affecting around 8 million people worldwide. After host cell invasion, the infective trypomastigote form remains 2–4 hours inside acidic phagolysosomes to differentiate into replicative amastigote form. In vitro acidic-pH-induced axenic amastigogenesis was used here to study this step of the parasite life cycle. After three hours of trypomastigote incubation in amastigogenesis promoting acidic medium (pH 5.0) or control physiological pH (7.4) medium samples were subjected to three rounds of centrifugation followed by ultrafiltration of the supernatants. The resulting exoproteome samples were trypsin digested and analysed by nano flow liquid chromatography coupled to tandem mass spectrometry. Computational protein identification searches yielded 271 and 483 protein groups in the exoproteome at pH 7.4 and pH 5.0, respectively, with 180 common proteins between both conditions. The total amount and diversity of proteins released by parasites almost doubled upon acidic incubation compared to control. Overall, 76.5% of proteins were predicted to be secreted by classical or non-classical pathways and 35.1% of these proteins have predicted transmembrane domains. Classical secretory pathway analysis showed an increased number of mucins and mucin-associated surface proteins after acidic incubation. However, the number of released trans-sialidases and surface GP63 peptidases was higher at pH 7.4. Trans-sialidases and mucins are anchored to the membrane and exhibit an enzyme-substrate relationship. In general, mucins are glycoproteins with immunomodulatory functions in Chagas disease, present mainly in the epimastigote and trypomastigote surfaces and could be enzymatically cleaved and released in the phagolysosome during amastigogenesis. Moreover, evidence for flagella discard during amastigogenesis are addressed. This study provides the first comparative analysis of the exoproteome during amastigogenesis, and the presented data evidence the dynamism of its profile in response to acidic pH-induced differentiation.

Amino acid analysis is among the most accurate methods for absolute quantification of proteins and peptides. Here, we combine acid hydrolysis with the addition of isotopically labeled standard amino acids and analysis by mass spectrometry for accurate and sensitive protein quantitation. Quantitation of less than 10 fmol of protein standards with errors below 10% has been demonstrated using this method.

Amino acid analysis is among the most accurate methods for absolute quantification of proteins and peptides. Here we combine acid hydrolysis with the addition of isotopically labeled standard amino acids and analysis by mass spectrometry for accurate and sensitive protein quantitation. Quantitation of less than 10 fmol of protein standards with errors below 10% has been demonstrated using this method.

Epidemiological and experimental studies have documented that long-term exposure to fine particulate matter (PM2.5) increases the risk of respiratory diseases. However, the details of the underlying mechanism remain unclear. In this study, male C57BL/6 mice were exposed to ambient PM2.5 (mean daily concentration ~64 µg/m3) for 12 weeks through a “real-world” airborne PM2.5 exposure system. We found that PM2.5 caused severe lung injury in mice as evidenced by histopathological examination. Then, tandem mass tag (TMT) labeling quantitative proteomic technology was performed to analyze protein expression profiling in the lungs from control and PM2.5-exposed mice. A total of 32 proteins were differentially expressed in PM2.5-exposed lungs versus the controls. Among these proteins, 24 and 8 proteins were up-and down-regulated, respectively. Gene ontology analysis indicated that PM2.5 exerts a toxic effect on lungs by affecting multiple biological processes, including oxidoreductase activity, receptor activity, and protein binding. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that extracellular matrix (ECM)–receptor interaction, phagosome, small cell lung cancer, and phosphatidylinositol 3-kinase(PI3K)-protein kinase B (Akt) signaling pathways contribute to PM2.5-induced pulmonary fibrosis. Taken together, these results provide a comprehensive proteomics analysis to further understanding of the molecular mechanisms underlying PM2.5-elicited pulmonary disease.

UNLABELLED: Redox homeostasis is essential for normal function of cells and redox imbalance has been recognised as a pathogenic factor of numerous human diseases. Oxidative modifications of cysteine thiols modulate function of many proteins, mediate signalling, and fine-tune transcriptional and metabolic processes. In this study we present the SNO/SOH TMT strategy, which enables simultaneous analysis of two different types of cysteine modification: S-nitrosylation (SNO) and S-sulfenylation (SOH). The method facilitates quantitation of modification changes corrected by changes in protein abundance levels and estimation of relative modification site occupancy in a single nLC-MSMS run. The approach was evaluated in vivo using an Escherichia coli based model of mild oxidative stress. Bacteria were grown anaerobically on fumarate or nitrate. Short-term treatment with sub-millimolar levels of hydrogen peroxide was used to induce SOH. We have identified and quantified 114 SNO and SOH modified peptides. In many instances SNO and SOH occupy the same site, suggesting an association between them. High site occupancy does not equate to a site of modification which responds to redox imbalance. The SNO/SOH TMT strategy is a viable alternative to existing methods for cysteine oxidation analysis and provides new features that will facilitate our understanding of the interplay between SNO and SOH.BIOLOGICAL SIGNIFICANCE: SNO/SOH TMT strategy outperforms other available strategies for cysteine oxidation analysis. It provides quantitative profiling of S-nitrosylation and S-sulfenylation changes simultaneously in two experimental conditions. It allows correction of modification levels by protein abundance changes and determination of relative modification site occupancy - all in a single nLC-MSMS experiment based on commercially available reagents. The method has proven precise and sensitive enough to detect and quantify endogenous levels of oxidative stress on proteome-wide scale.

Intestinal ischemia and reperfusion injury are widely used models, which result into tissue injury and multiple organ failure also observed after trauma and surgery. Ischemic preconditioning (IPC) preceding ischemia and reperfusion (IR) was shown to attenuate this injury and has a potential therapeutic application; however the exact underlying mechanism is not clear. Neutrophils play an important role in the mechanism of injuries caused by ischemia and reperfusion while IPC led to a decrease in neutrophil stimulation and activation. The effect of preconditioning on the neutrophil proteome is unclear. Proteomic analysis has been ratified as an appropriate tool for studying complex systems. In order to evaluate the effect of IPC preceding 45 min of ischemia on the proteome of neutrophils we used Wistar rats divided in four experimental groups: Control, sham laparotomy, intestinal ischemia reperfusion and ischemic preconditioning. After neutrophil separation, proteins were extracted, trypsin digested and the resulting peptides were iTRAQ labeled followed by HILIC fractionation and nLC-MS/MS analysis. After database searches, normalization and statistical analysis our proteomic analysis resulted in the identification of 2437 protein groups that were assigned to five different clusters based on the relative abundance profiles among the experimental groups. The clustering followed by statistical analysis led to the identification of significantly up and downregulated proteins in IR and IPC. Cluster based KEGG pathways analysis revealed up- regulation of actin cytoskeleton, metabolism, Fc gamma R mediated phagocytosis, chemokine signaling, focal adhesion and leukocyte transendothelial migration whereas downregulation in ribosome, spliceosome, RNA transport, protein processing in endoplasmic reticulum and proteasome, after intestinal ischemic preconditioning. Furthermore, enzyme prediction analysis revealed the regulation of some important antioxidant enzymes and having their role in reactive oxygen species production. To our knowledge, this work describes the most comprehensive and detailed quantitative proteomic study of the neutrophil showing the beneficial role of ischemic preconditioning and its effects on the neutrophil proteome. This data will be helpful to understand the effect of underlying protective mechanisms modulating the role of PMNs after IPC and provide a trustworthy basis for future studies. Biological significance Preconditioning is a relevant strategy to overcome clinical implications from ischemia and reperfusion. Such implications have the neutrophil as a major player. Although many publications describe specific biochemical and physiological roles of the neutrophil in such conditions, there is no report of a proteomic study providing a broader view of this scenario. Here we describe a group of proteins significantly regulated by ischemia and reperfusion being such regulation prevented by preconditioning. Such finding may provide relevant information for a deeper understanding of the mechanisms involved, as well as serve as basis for future biomarker or drug target assays.

In clinical conditions trauma is associated with high mortality and morbidity. Neutrophils play a key role in the development of multiple organ failure after trauma. To have a detailed understanding of the neutrophil activation at primary stages after trauma, neutrophils were isolated from control and surgical trauma rats in this study. Extracted proteins were analyzed using nano liquid chromatography coupled to tandem mass spectrometry. A total of 2924 rat neutrophil proteins were identified in our analysis, of which 393 were found differentially regulated between control and trauma groups. By using functional pathways analysis of the 190 proteins up-regulated in surgical trauma we found proteins related to transcription initiation and protein biosynthesis. On the other hand, among the 203 proteins down-regulated in surgical trauma we found enrichment for proteins of the immune response, proteasome degradation and actin cytoskeleton. Overall, enzyme prediction analysis revealed that regulated enzymes are directly involved in neutrophil apoptosis, directional migration and chemotaxis. Our observations were then confirmed by in silico protein-protein interaction analysis. Collectively, our results reveal that neutrophils drastically regulate their biochemical pathways after the early stages of surgical trauma, showing lower activity. This implies higher susceptibility of the trauma patients to infection and bystander tissues damage. This article is protected by copyright. All rights reserved.

Studies of protein N-glycosylation are important for answering fundamental questions on the diverse functions of glycoproteins in plant growth and development. Here we generated and characterised a comprehensive collection of Lotus japonicusLORE1 insertion mutants, each lacking the activity of one of the 12 enzymes required for normal N-glycan maturation in the glycosylation machinery. The inactivation of the individual genes resulted in altered N-glycan patterns as documented using mass spectrometry and glycan-recognising antibodies, indicating successful identification of null mutations in the target glyco-genes. For example, both mass spectrometry and immunoblotting experiments suggest that proteins derived from the α1,3-fucosyltransferase (Lj3fuct) mutant completely lacked α1,3-core fucosylation. Mass spectrometry also suggested that the Lotus japonicus convicilin 2 was one of the main glycoproteins undergoing differential expression/N-glycosylation in the mutants. Demonstrating the functional importance of glycosylation, reduced growth and seed production phenotypes were observed for the mutant plants lacking functional mannosidase I, N-acetylglucosaminyltransferase I, and α1,3-fucosyltransferase, even though the relative protein composition and abundance appeared unaffected. The strength of our N-glycosylation mutant platform is the broad spectrum of resulting glycoprotein profiles and altered physiological phenotypes that can be produced from single, double, triple and quadruple mutants. This platform will serve as a valuable tool for elucidating the functional role of protein N-glycosylation in plants. Furthermore, this technology can be used to generate stable plant mutant lines for biopharmaceutical production of glycoproteins displaying relative homogeneous and mammalian-like N-glycosylation features.

In this study, we used a mass spectrometry-based quantification approach employing isotopic (ICPL) and isobaric (iTRAQ) labeling to investigate the pattern of protein deposition during castor oil seed (Ricinus communis L.) development, including that of proteins involved in fatty acid metabolism, seed-storage proteins (SSPs), toxins, and allergens. Additionally, we have used off-line hydrophilic interaction chromatography (HILIC) as a step of peptide fractionation preceding the reverse-phase nanoLC coupled to a LTQ Orbitrap. We were able to identify a total of 1875 proteins, and from these 1748 could be mapped to extant castor gene models, considerably expanding the number of proteins so far identified from developing castor seeds. Cluster validation and statistical analysis resulted in 975 protein trend patterns and the relative abundance of 618 proteins. The results presented in this work give important insights into certain aspects of the biology of castor oil seed development such as carbon flow, anabolism, and catabolism of fatty acid and the pattern of deposition of SSPs, toxins, and allergens such as ricin and 2S albumins. We also found, for the first time, some genes of SSP that are differentially expressed during seed development.

Besides being a public health problem, scorpion venoms have a potential biotechnological application since they contain peptides that may be used as drug leads and/or to reveal novel pharmacological targets. A comprehensive Tityus serrulatus venom proteome study with emphasis on the phosphoproteome and N-glycoproteome was performed to improve our knowledge on the molecular diversity of the proteinaceous toxins. We combined two peptide identification methodologies, i.e., database search and de novo sequencing, to achieve a more comprehensive overview of the molecular diversity of the venoms. A total of 147 proteins were identified, including neurotoxins, enzymes, bradykinin-potentiating peptides, and molecules with antimicrobial and diuretic activities. Among those, three proteins were found to be phosphorylated, and one N-glycosylated. Finally, cleavage of toxin polypeptide chains seems to be a common post-translational modification in the venom since 80% of the identified molecules were, in fact, products of toxins proteolysis.

Exposure of cells to the diarrhetic shellfish poison, okadaic acid, leads to a dramatic reorganization of cytoskeletal architecture and loss of cell-cell contact. When cells are exposed to high concentrations of okadaic acid (100–500 nM), the morphological rearrangement is followed by apoptotic cell death. Okadaic acid inhibits the broad acting Ser/Thr protein phosphatases 1 and 2A, which results in hyperphosphorylation of a large number of proteins. Some of these hyperphosphorylated proteins are most likely key players in the reorganization of the cell morphology induced by okadaic acid. We wanted to identify these phosphoproteins and searched for them in the cellular lipid rafts, which have been found to contain proteins that regulate cytoskeletal dynamics and cell adhesion. By using stable isotope labeling by amino acids in cell culture cells treated with okadaic acid (400 nM) could be combined with control cells before the isolation of lipid rafts. Protein phosphorylation events and translocations induced by okadaic acid were identified by mass spectrometry. Okadaic acid was shown to regulate the phosphorylation status and location of proteins associated with the actin cytoskeleton, microtubules and cell adhesion structures. A large number of these okadaic acid-regulated proteins have previously also been shown to be similarly regulated prior to cell proliferation and migration. Our results suggest that okadaic acid activates general cell signaling pathways that induce breakdown of the cortical actin cytoskeleton and cell detachment. publishedVersion

The protozoan parasite Trypanosoma cruzi causes Chagas disease, one of the major neglected infectious diseases. It has the potential to infect any nucleated mammalian cell. The secreted/excreted protein repertoire released by T. cruzi trypomastigotes is crucial in host-pathogen interactions. In this study, mammalian tissue culture-derived trypomastigotes (Y strain) were used to characterize the exoproteome of the infective bloodstream life form. Proteins released into the serum-free culture medium after 3 h of incubation were harvested and digested with trypsin. NanoLC-MS/MS analysis resulted in the identification of 540 proteins, the largest set of released proteins identified to date in Trypanosoma spp. Bioinformatic analysis predicted most identified proteins as secreted, predominantly by non-classical pathways, and involved in host-cell infection. Some proteins possess predicted GPI-anchor signals, these being mostly trans-sialidases, mucin associated surface proteins and surface glycoproteins. Moreover, we enriched phosphopeptides and glycopeptides from tryptic digests. The majority of identified glycoproteins are trans-sialidases and surface glycoproteins involved in host-parasite interaction. Conversely, most identified phosphoproteins have no Gene Ontology classification. The existence of various proteins related to similar functions in the exoproteome likely reflects this parasite's enhanced mechanisms for adhesion, invasion, and internalization of different host-cell types, and escape from immune defenses.

Lactoferrin, a protein showing an array of biochemical properties, including immuno-modulation, iron-binding ability, as well as antioxidant, antibacterial and antiviral activities, but which may also represent a potential milk allergen, was isolated from donkey milk by ion exchange chromatography. The characterization of its primary structure, by means of enzymatic digestions, SPITC derivatization of tryptic digest, reversed-phase high performance liquid chromatography, electrospray and matrix-assisted laser desorption/ionization mass spectrometry, is reported. Our results allowed the almost complete characterization of donkey lactoferrin sequence, that, at least for the covered sequence, differs from the horse genomic deduced sequence (UniProtKB Acc. Nr. O77811) by five point substitutions located at positions 91 (Arg → His), 328 (Thr → Ile/Leu), 466 (Ala → Gly), 642 (Asn → Ser) and 668 (Ser → Ala). Analysis of the glycosylated protein showed that glycans in donkey lactoferrin are linked to the protein backbone via an amide bond to asparagine residues located at the positions 137, 281 and 476.

Snake venoms are composed of approximately 90% of proteins with several pharmacological activities having high potential in research as biological tools. One of the most abundant compounds is phospholipases A2 (PLA2), which are the most studied venom protein due to their wide pharmacological activity. Using a combination of chromatographic steps, a new PLA2 K49 was isolated and purified from the whole venom of the Bothriopsis taeniata and submitted to analyses mass spectrometry. An automatic “de novo” sequencing of this new PLA2 K49 denominated Btt-TX was performed using Peaks Studio 6 for analysis of the spectra. Additionally, a triplex approach CID/HCD/ETD has been performed, to generate higher coverage of the sequence of the protein. Structural studies correlating biological activities were made associating specific Btt-TX regions and myotoxic activity. Lysine acetylation was performed to better understand the mechanism of membrane interaction, identifying the extreme importance of the highly hydrophobic amino acids L, P and F for disruption of the membrane. Our myotoxical studies show a possible membrane disruption mechanism by Creatine Kinase release without a noticeable muscle damage, that probably occurred without phospholipid hydrolyses, but with a probable penetration of the hydrophobic amino acids present in the C-terminal region of the protein.

A comprehensive monosaccharide composition of the N-glycans of donkey milk lactoferrin, isolated by ion exchange chromatography from an individual milk sample, was obtained by means of chymotryptic digestion, TiO2 and HILIC enrichment, reversed-phase high-performance liquid chromatography, electrospray mass spectrometry, and high collision dissociation fragmentation. The results obtained allowed identifying 26 different glycan structures, including high mannose, complex and hybrid N-glycans, linked to the protein backbone via an amide bond to asparagine residues located at the positions 137, 281 and 476. Altogether, the N-glycan structures determined revealed that most of the N-glycans identified in donkey milk lactoferrin are neutral complex/hybrid. Indeed, 10 neutral non-fucosylated complex/hybrid N-glycans and 4 neutral fucosylated complex/hybrid N-glycans were found. In addition, two high mannose N-glycans, four sialylated fucosylated complex N-glycans and six sialylated non-fucosylated complex N-glycans, one of which containing N-glycolylneuraminic acid (Neu5Gc), were found. A comparison of the monosaccharide composition of the N-glycans of donkey milk lactoferrin with respect to that of human, bovine and goat milk lactoferrin is reported. Data are available via ProteomeXchange with identifier PXD004289.

DM43 is a circulating dimeric antitoxin isolated from Didelphis aurita, a South American marsupial naturally immune to snake envenomation. This endogenous inhibitor binds non-covalently to jararhagin, the main hemorrhagic metalloproteinase from Bothrops jararaca snake venom, and efficiently neutralizes its toxicity. The aim of this study was to apply mass spectrometry (MS) and surface plasmon resonance (SPR) to improve the molecular characterization of this heterocomplex. The stoichiometry of the interaction was confirmed by nanoelectrospray ionization-quadrupole-time-of-flight MS; from native solution conditions, the complex showed a molecular mass of ~94 kDa, indicating that one molecule of jararhagin (50 kDa) interacts with one monomer of DM43 (43 kDa). Although readily observed in solution, the dimeric structure of the inhibitor was barely preserved in the gas phase. This result suggests that, in contrast to the toxin-antitoxin complex, hydrophobic interactions are the primary driving force for the inhibitor dimerization. For the real-time interaction analysis, the toxin was captured on a sensor chip derivatized with the anti-jararhagin monoclonal antibody MAJar 2. The sensorgrams obtained after successive injections of DM43 in a concentration series were globally fitted to a simple bimolecular interaction, yielding the following kinetic rates for the DM43/jararhagin interaction: k(a) = 3.54 ± 0.03 × 10(4) M(-1) s(-1) and k(d) = 1.16 ± 0.07 × 10(-5) s(-1), resulting in an equilibrium dissociation constant (K(D) ) of 0.33 ± 0.06 nM. Taken together, MS and SPR results show that DM43 binds to its target toxin with high affinity and constitute the first accurate quantitative study on the extent of the interaction between a natural inhibitor and a metalloproteinase toxin, with unequivocal implications for the use of this kind of molecule as template for the rational development of novel antivenom therapies.

Angiotensin-(1-7) [Ang-(1-7)] is an endogenous ligand of the Mas receptor and induces vasodilation, positive regulation of insulin, and antiproliferative and antitumorigenic activities. However, little is known about the molecular mechanisms behind these biological properties. Aiming to identify proteins involved in the Ang-(1-7) signaling, we performed a mass spectrometry-based time-resolved quantitative phosphoproteome study of human aortic endothelial cells (HAEC) treated with Ang-(1-7). We identified 1288 unique phosphosites on 699 different proteins with 99% certainty of correct peptide identification and phosphorylation site localization. Of these, 121 sites on 79 proteins had their phosphorylation levels significantly changed by Ang-(1-7). Our data suggest that the antiproliferative activity of Ang-(1-7) is due to the activation or inactivation of several target phosphoproteins, such as forkhead box protein O1 (FOXO1), mitogen-activated protein kinase 1 (MAPK), proline-rich AKT1 substrate 1 (AKT1S1), among others. In addition, the antitumorigenic activity of Ang-(1-7) is at least partially due to FOXO1 activation, since we show that this transcriptional factor is activated and accumulated in the nucleus of A549 lung adenocarcinoma cells treated with Ang-(1-7). Moreover, Ang-(1-7) triggered changes in the phosphorylation status of several known downstream effectors of the insulin signaling, indicating an important role of Ang-(1-7) in glucose homeostasis. In summary, this study provides new concepts and new understanding of the Ang-(1-7) signal transduction, shedding light on the mechanisms underlying Mas activation.

We present here a new analytical strategy for identification and characterisation of fluorescent proteins from marine organisms. By applying basic proteomics tools it is possible to screen large sample collections for fluorescent proteins of desired characteristics prior to gene cloning. Our methodology which includes isolation, spectral characterisation, stability testing, gel-based separation and mass spectrometric identification was optimised on samples collected during the Danish Galathea 3 expedition. Four corals of the Fungia, Sarcophyton and Acropora species emitting green fluorescence were tested. Each of the fluorescent extracts behaves differently under denaturing conditions but complete fluorescence loss was not observed. Optimised electrophoretic conditions yielded effective separation of active fluorescent proteins in both 1DE and 2DE. Mass spectrometric analysis of the proteins in the fluorescent spots excised directly from unstained 2DE gels provides sequence information that might be sufficient to design degenerate primers for gene cloning. Identified fluorescent proteins are in agreement with the coral species determined by visual examination of the samples. The presented methodology is a viable alternative to direct gene cloning for the discovery of novel fluorescent proteins and will be further validated on other samples collected during the Galathea 3 expedition.

The triatomine bugs are obligatory haematophagous organisms that act as vectors of Chagas disease by transmitting the protozoan Trypanosoma cruzi. Their feeding success is strongly related to salivary proteins that allow these insects to access blood by counteracting host haemostatic mechanisms. Proteomic studies were performed on saliva from the Amazonian triatomine bugs: Rhodnius brethesi and R. robustus, species epidemiologically relevant in the transmission of T. cruzi. Initially, salivary proteins were separated by two-dimensional gel electrophoresis (2-DE). The average number of spots of the R. brethesi and R. robustus saliva samples were 129 and 135, respectively. The 2-DE profiles were very similar between the two species. Identification of spots by peptide mass fingerprinting afforded limited efficiency, since very few species-specific salivary protein sequences are available in public sequence databases. Therefore, peptide fragmentation and de novo sequencing using a MALDI-TOF/TOF mass spectrometer were applied for similarity-driven identifications which generated very positive results. The data revealed mainly lipocalin-like proteins which promote blood feeding of these insects. The redundancy of saliva sequence identification suggested multiple isoforms caused by gene duplication followed by gene modification and/or post-translational modifications. In the first experimental assay, these proteins were predominantly phosphorylated, suggesting functional phosphoregulation of the lipocalins.

The proangiogenic members of VEGF family and related receptors play a central role in the modulation of pathological angiogenesis. Recent insights indicate that, due to the strict biochemical and functional relationship between VEGFs and related receptors, the development of a new generation of agents able to target contemporarily more than one member of VEGFs might amplify the antiangiogenic response representing an advantage in term of therapeutic outcome. To identify molecules that are able to prevent the interaction of VEGFs with related receptors, we have screened small molecule collections consisting of >100 plant extracts. Here, we report the isolation and identification from an extract of the Malian plant Chrozophora senegalensis of the biflavonoid amentoflavone as an antiangiogenic bioactive molecule. Amentoflavone can to bind VEGFs preventing the interaction and phosphorylation of VEGF receptor 1 and 2 (VEGFR-1,VEGFR-2) and to inhibit endothelial cell migration and capillary-like tube formation induced by VEGF-A or placental growth factor 1 (PlGF-1) at low μm concentration. In vivo, amentoflavone is able to inhibit VEGF-A-induced chorioallantoic membrane neovascularization as well as tumor growth and associated neovascularization, as assessed in orthotropic melanoma and xenograft colon carcinoma models. In addition structural studies performed on the amentoflavone·PlGF-1 complex have provided evidence that this biflavonoid effectively interacts with the growth factor area crucial for VEGFR-1 receptor recognition. In conclusion, our results demonstrate that amentoflavone represents an interesting new antiangiogenic molecule that is able to prevent the activity of proangiogenic VEGF family members and that the biflavonoid structure is a new chemical scaffold to develop powerful new antiangiogenic molecules.

Protein nitration take place on tyrosine residues under oxidative stress conditions and may influence a number of processes including enzyme activity, protein-protein interactions and phospho-tyrosine signalling pathways. Nitrated proteins have been identified in a number of diseases, however, the study of these proteins has been compromised by the lack of good methods for identifying nitrated proteins, their nitration sites and the level of nitration. Here, we present a method for identification of nitrated peptides that allows the site specific assignment of nitration, is easy to use and reproducible, and opens up for the possibility to quantify the level of nitration of specific peptides as function of different oxidative conditions, namely combined fractional diagonal chromatography (COFRADIC) in combination with off-line nano-LC-MALDI. We identify six nitrated peptides from in vitro nitrated bovine serum albumin and propose that automated COFRADIC using nano-LC and off-line MALDI-MS might be a possibility for identification of tyrosine nitrated proteins and the nitration sites in complex samples.

Udgivelsesdato: 2010 Breast cancer is by far the most common diagnosed form of cancer and the leading cause of cancer death in women today. Clinically useful biomarkers for early detection of breast cancer could lead to a significant reduction in mortality. Here we describe a detailed analysis using gel-based proteomics in combination with mass spectrometry and immunohistochemistry (IHC) of the tumour interstitial fluids (TIF) and normal interstitial fluids (NIF) collected from 69 prospective breast cancer patients. The goal of this study was to identify abundant cancer up-regulated proteins that are externalised by cells in the tumour microenvironment of most if not all these lesions. To this end, we applied a phased biomarker discovery research strategy to the analysis of these samples rather than comparing all samples among each other, with inherent inter and intra-sample variability problems. To this end, we chose to use samples derived from a single tumour/benign tissue pair (patient 46, triple negative tumour), for which we had well-matched samples in terms of epithelial cell numbers, to generate the initial dataset. In this first phase we found 110 proteins that were up-regulated by a factor of 2 or more in the TIF, some of which were confirmed by IHC. In the second phase, we carried out a systematic computer assisted analysis of the 2D gels of the remaining 68 TIF samples in order to identify TIF 46 up-regulated proteins that were deregulated in 90% or more of all the available TIFs, thus representing common breast cancer markers. This second phase singled out a set of 26 breast cancer markers, most of which were also identified by a complementary analysis using LC-MS/MS. The expression of calreticulin, cellular retinoic acid-binding protein II, chloride intracellular channel protein 1, EF-1-beta, galectin 1, peroxiredoxin-2, platelet-derived endothelial cell growth factor, protein disulfide isomerase and ubiquitin carboxyl-terminal hydrolase 5 were further validated using a tissue microarray containing 70 malignant breast carcinomas of various grades of atypia. A significant number of these proteins have already been detected in the blood/plasma/secretome by others. The next steps, which include biomarker prioritization based on the hierarchal evaluation of these markers, antibody and antigen development, assay development, analytical validation, and preliminary testing in the blood of healthy and breast cancer patients, are discussed.

The photoneuroendocrine circadian system of the brain consists of (a) specialized photoreceptors in the retina, (b) a circadian generator located in the forebrain that contains "clock genes," (c) specialized nuclei in the forebrain involved in neuroendocrine secretion, and (d) the pineal gland. The circadian generator is a nucleus, called the suprachiasmatic nucleus (SCN). The neurons of this nucleus contain "clock genes," the transcription of which exhibits a circadian rhythm. Most circadian rhythms are generated by the neurons of this nucleus and, via neuronal and humoral connections, the SCN controls circadian activity of the brain and peripheral tissues. The endogenous oscillator of the SCN is each day entrained to the length of the daily photoperiod by light that reach the retina, and specialized photoreceptors transmit impulses to the SCN via the optic nerves. Mass screening for day/night variations in gene expression in the circadian system as well as in the whole brain and peripheral tissues have, during the last decade, been performed. However, studies of circadian changes in the proteome have been less investigated. In this survey, the anatomy and function of the circadian-generating system in mammals is described, and recent proteomic studies that investigate day/night changes in the retina, SCN, and pineal gland are reviewed. Further circadian changes controlled by the SCN in gene and protein expression in the liver are discussed.

The small heat shock proteins (sHsps), which counteract heat and oxidative stress in an unknown way, belong to a protein family of sHsps and alpha-crystallins whose members form large oligomeric complexes. The chloroplast-localized sHsp, Hsp21, contains a conserved methionine- rich sequence, predicted to form an amphipatic helix with the methionines situated along one of its sides. Here, we report how methionine sulfoxidation was detected by mass spectrometry in proteolytically cleaved peptides that were produced from recombinant Arabidopsis thaliana Hsp21, which had been treated with varying concentrations of hydrogen peroxide. Sulfoxidation of the methionine residues in the conserved amphipatic helix coincided with a significant conformational change in the Hsp21 protein oligomer. (Less)

In this work, partial characterization of the primary structure of phycocyanin from the cyanobacterium Aphanizomenon flos-aquae (AFA) was achieved by mass spectrometry de novo sequencing with the aid of chemical derivatization. Combining N-terminal sulfonation of tryptic peptides by 4-sulfophenyl isothiocyanate (SPITC) and MALDI-TOF/TOF analyses, facilitated the acquisition of sequence information for AFA phycocyanin subunits. In fact, SPITC-derivatized peptides underwent facile fragmentation, predominantly resulting in y-series ions in the MS/MS spectra and often exhibiting uninterrupted sequences of 20 or more amino acid residues. This strategy allowed us to carry out peptide fragment fingerprinting and de novo sequencing of several peptides belonging to both α- and β-phycocyanin polypeptides, obtaining a sequence coverage of 67% and 75%, respectively. The presence of different isoforms of phycocyanin subunits was also revealed; subsequently Intact Mass Measurements (IMMs) by both MALDI- and ESI-MS supported the detection of these protein isoforms. Finally, we discuss the evolutionary importance of phycocyanin isoforms in cyanobacteria, suggesting the possible use of the phycocyanin operon for a correct taxonomic identity of this species. Copyright © 2008 John Wiley & Sons, Ltd.

Udgivelsesdato: 2008-Jun-11 We have previously shown that peptide amide hydrogens undergo extensive intramolecular migration (i.e., complete hydrogen scrambling) upon collisional activation of protonated peptides (Jørgensen et al. J. Am. Chem. Soc. 2005, 127, 2785-2793). The occurrence of hydrogen scrambling enforces severe limitations on the application of gas-phase fragmentation as a convenient method to obtain information about the site-specific deuterium uptake for proteins and peptides in solution. To investigate whether deprotonated peptides exhibit a lower level of scrambling relative to their protonated counterparts, we have now measured the level of hydrogen scrambling in a deprotonated, selectively labeled peptide using MALDI tandem time-of-flight mass spectrometry. Our results conclusively show that hydrogen scrambling is prevalent in the deprotonated peptide upon collisional activation. The amide hydrogens ((1)H/(2)H) have migrated extensively in the anionic peptide, thereby erasing the original regioselective deuteration pattern obtained in solution.

Udgivelsesdato: 2008-Aug-15 Nanodiscs are self-assembled soluble discoidal phospholipids bilayers encirculated by an amphipathic protein that together provide a functional stabilized membrane disk for the incorporation of membrane-bound and membrane-associated molecules. The scope of the present work is to investigate how nanodiscs and their incorporated membrane receptors can be attached to surface plasmon resonance sensorchips and used to measure the kinetics of the interaction between soluble molecules and membrane receptors inserted in the bilayer of nanodiscs. Cholera toxin and its glycolipid receptor G(M1) constitute a system that can be considered a paradigm for interactions of soluble proteins with membrane receptors. In this work, we have investigated different technologies for capturing nanodiscs containing the glycolipid receptor G(M1) in lipid bilayers, enabling measurements of binding of its soluble interaction partner cholera toxin B subunit to the receptor with the sensorchip-based surface plasmon resonance (SPR) technology. The measured stoichiometric and kinetic values of the interaction are in agreement with those reported by previous studies, thus providing proof-of-principle that nanodiscs can be employed for kinetic SPR studies.

Udgivelsesdato: 2008-Jul-21 Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) followed by mass spectrometric identification of the proteins in the protein spots has become a central tool in proteomics. CMT167(H), CMT64(M) and CMT170(L) cell lines, selected from a spontaneous mouse lung adenocarcinoma, with high-, middle- or low-metastatic potential have been characterized in vivo. In this study, the comprehensive protein expression profiles of the CMT cell lines were analyzed at passages 5, 15 and 35 in order to assess the cell line stability. During the passages 5 to 15, the expression profiles of CMT cells remained reasonably stable as evidenced by only 0.7%, 3.9% and 1.1% proteins changed in CMT167(H), CMT64(M) and CMT170(L) respectively. However, the number of differentially expressed proteins were considerably increased at passage 35 in CMT64(M) and CMT170(L) while CMT167(H) remained stable. Based on our selection criteria, 22, 109 and 84 spots in CMT167(H), CMT64(M) and CMT170(L) were selected for protein identification by MS and 99 unique proteins were identified. Bioinformatics analysis indicated that most of these proteins participate in cellular metabolism. In conclusion, proteomics was found to be a useful tool for assessing differences in cell line stability. This approach provided a tool to select the best cell line and optimal subculture period for studies of cancer related phenomena and for testing the effect of potential anticancer drugs.

Udgivelsesdato: 2008-May In the literature, a variety of ways have been used to obtain anoxia, and most often results are compared between studies without taking into consideration how anoxia has been obtained. Here, we provide a comprehensive study of two types of anoxia, using a proteomics approach to compare changes in protein expression. The two investigated situations were 30 min of chemical anoxia (10 mM NaN(3)) followed by reoxygenation overnight (CR) and 2 h of N(2)-induced anoxia (achieved by flushing with N(2)) followed by reoxygenation overnight (NR), after which samples were resolved by 2-DE. Forty-five protein spots changed their abundance in response to CR and 35 protein spots changed their abundance in response to NR, but only six proteins changed their abundance in response to both stimuli. By the means of MS/MS, 40 protein spots were identified including proteins involved in processes like cell protection and protein synthesis. It was also revealed that the level of a number of keratins was down-regulated. This study therefore provides a valuable comparison of two different anoxia models and shows that great care should be taken when comparing the effects of anoxia in studies that have used different types and durations of anoxia.

Thirteen peroxidase spots on two-dimensional gels were identified by comprehensive proteome analysis of the barley seed. Mass spectrometry tracked multiple forms of three different peroxidase isozymes: barley seed peroxidase 1, barley seed-specific peroxidase BP1 and a not previously identified putative barley peroxidase. The presence of multiple spots for each of the isozymes reflected variations in post-translational glycosylation and protein truncation. Complete sequence coverage was achieved by using a series of proteases and chromatographic resins for sample preparation prior to mass spectrometric analysis. Distinct peroxidase spot patterns divided the 16 cultivars tested into two groups. The distribution of the three isozymes in different seed tissues (endosperm, embryo, and aleurone layer) suggested the peroxidases to play individual albeit partially overlapping roles during germination. In summary, a subset of three peroxidase isozymes was found to occur in the seed, whereas products of four other barley peroxidase genes were not detected. The present analysis documents the selective expression profiles and post-translational modifications of isozymes from a large plant gene family.

Udgivelsesdato: 2007-Jul An improved method for sample preparation for MALDI-MS and MS/MS using AnchorChip targets is presented. The method, termed the SMW method (sample, matrix wash), results in better sensitivity for peptide mass fingerprinting as well as for sequencing by MS/MS than previously published methods. The method allows up-concentration and desalting directly on the mass spectrometric target and should be amenable for automation. A draw back caused by extensive oxidation of methionine and tryptophan in the SMW method can be alleviated by the addition of n-octyl glucopyranoside and DTT to the sample solution. The method was validated for protein identification from a 2-DE based liver proteome study. The SMW method resulted in identification of many more proteins and in most cases with a better score than the previously published methods.

Ten genes from two multigene families encode barley α-amylases. To gain insight into the occurrence and fate of individual isoforms during seed germination, the α-amylase repertoire was mapped by using a proteomics approach consisting of 2D gel electrophoresis, western blotting, and mass spectrometry. Mass spectrometric analysis confirmed that the 29 α-amylase positive 2D gel spots contained products of one (GenBank accession gi|113765) and two (gi|4699831 and gi|166985) genes encoding α-amylase 1 and 2, respectively, but lacked products from seven other genes. Eleven spots were identified only by immunostaining. Mass spectrometry identified 12 full-length forms and 12 fragments from the cultivar Barke. Products of both α-amylase 2 entries co-migrated in five full-length and one fragment spot. The α-amylase abundance and the number of fragments increased during germination. Assessing the fragment minimum chain length by peptide mass fingerprinting suggested that α-amylase 2 (gi|4699831) initially was cleaved just prior to domain B that protrudes from the (βα)8-barrel between β-strand 3 and α-helix 3, followed by cleavage on the C-terminal side of domain B and near the C-terminus. Only two shorter fragments were identified of the other α-amylase 2 (gi|166985). The 2D gels of dissected tissues showed α-amylase degradation to be confined to endosperm. In contrast, the aleurone layer contained essentially only full-length α-amylase forms. While only products of the above three genes appeared by germination also of 15 other barley cultivars, the cultivars had distinct repertoires of charge and molecular mass variant forms. These patterns appeared not to be correlated with malt quality.