High-Accuracy Mass Spectrometry Based Screening Method for the Discovery of Cysteine Containing Peptides in Animal Venoms and Toxins.

Venom and toxin samples derived from animal origins are a rich source of bioactive peptides. A high proportion of bioactive peptides that have been identified in venom contain one or more disulfide bridges, which are thought to stabilize tertiary structure, and therefore influence the peptides' specificity and activity. In this chapter, we describe a label-free mass spectrometry-based screening workflow specifically to detect peptides that contain inter- and intramolecular disulfide bonds, followed by elucidation of their primary structure. This method is based on the determination of the normalized isotope shift (NIS) and the normalized mass defect (NMD) of peptides, two parameters which are heavily influenced by the presence of sulfur in a peptide, where cysteines are the main contributing residues. Using ant defensive secretions as an example, we describe the initial fractionation of the venom on strong cation exchange followed by nanoflow HPLC and mass spectrometry. High resolution zoom scan spectra of high-abundance peptides are acquired, allowing an accurate determination of both monoisotopic and average mass, which are essential for calculation of NMD and NIS. Candidate peptides exhibiting relative low NMD and high NIS values are selected for targeted de novo sequencing. By fine-tuning the collision energy for optimal fragmentation of each selected precursor ions, the full sequence of several novel inter- and intramolecular disulfide bond containing ant defensive peptides can be established.

The deep-subsurface sulfate reducer Desulfotomaculum kuznetsovii employs two methanol-degrading pathways.

Methanol is generally metabolized through a pathway initiated by a cobalamine-containing methanol methyltransferase by anaerobic methylotrophs (such as methanogens and acetogens), or through oxidation to formaldehyde using a methanol dehydrogenase by aerobes. Methanol is an important substrate in deep-subsurface environments, where thermophilic sulfate-reducing bacteria of the genus Desulfotomaculum have key roles. Here, we study the methanol metabolism of Desulfotomaculum kuznetsovii strain 17T, isolated from a 3000-m deep geothermal water reservoir. We use proteomics to analyze cells grown with methanol and sulfate in the presence and absence of cobalt and vitamin B12. The results indicate the presence of two methanol-degrading pathways in D. kuznetsovii, a cobalt-dependent methanol methyltransferase and a cobalt-independent methanol dehydrogenase, which is further confirmed by stable isotope fractionation. This is the first report of a microorganism utilizing two distinct methanol conversion pathways. We hypothesize that this gives D. kuznetsovii a competitive advantage in its natural environment.

High throughput techniques to reveal the molecular physiology and evolution of digestion in spiders.

BACKGROUND: Spiders are known for their predatory efficiency and for their high capacity of digesting relatively large prey. They do this by combining both extracorporeal and intracellular digestion. Whereas many high throughput ("-omics") techniques focus on biomolecules in spider venom, so far this approach has not yet been applied to investigate the protein composition of spider midgut diverticula (MD) and digestive fluid (DF). RESULTS: We here report on our investigations of both MD and DF of the spider Nephilingis (Nephilengys) cruentata through the use of next generation sequencing and shotgun proteomics. This shows that the DF is composed of a variety of hydrolases including peptidases, carbohydrases, lipases and nuclease, as well as of toxins and regulatory proteins. We detect 25 astacins in the DF. Phylogenetic analysis of the corresponding transcript(s) in Arachnida suggests that astacins have acquired an unprecedented role for extracorporeal digestion in Araneae, with different orthologs used by each family. The results of a comparative study of spiders in distinct physiological conditions allow us to propose some digestion mechanisms in this interesting animal taxon. CONCLUSION: All the high throughput data allowed the demonstration that DF is a secretion originating from the MD. We identified enzymes involved in the extracellular and intracellular phases of digestion. Besides that, data analyses show a large gene duplication event in Araneae digestive process evolution, mainly of astacin genes. We were also able to identify proteins expressed and translated in the digestive system, which until now had been exclusively associated to venom glands.

Cytochrome bd Displays Significant Quinol Peroxidase Activity.

Cytochrome bd is a prokaryotic terminal oxidase that catalyses the electrogenic reduction of oxygen to water using ubiquinol as electron donor. Cytochrome bd is a tri-haem integral membrane enzyme carrying a low-spin haem b558, and two high-spin haems: b595 and d. Here we show that besides its oxidase activity, cytochrome bd from Escherichia coli is a genuine quinol peroxidase (QPO) that reduces hydrogen peroxide to water. The highly active and pure enzyme preparation used in this study did not display the catalase activity recently reported for E. coli cytochrome bd. To our knowledge, cytochrome bd is the first membrane-bound quinol peroxidase detected in E. coli. The observation that cytochrome bd is a quinol peroxidase, can provide a biochemical basis for its role in detoxification of hydrogen peroxide and may explain the frequent findings reported in the literature that indicate increased sensitivity to hydrogen peroxide and decreased virulence in mutants that lack the enzyme.

Unravelling the one-carbon metabolism of the acetogen Sporomusa strain An4 by genome and proteome analysis.

The Sporomusa genus comprises anaerobic spore-forming acetogenic bacteria that stain Gram-negative. Sporomusa species typically grow with one-carbon substrates and N-methylated compounds. In the degradation of these compounds methyltransferases are involved. In addition, Sporomusa species can grow autotrophically with H2 and CO2 , and use a variety of sugars for acetogenic growth. Here we describe a genome analysis of Sporomusa strain An4 and a proteome analysis of cells grown under five different conditions. Comparison of the genomes of Sporomusa strain An4 and Sporomusa ovata strain H1 indicated that An4 is a S. ovata strain. Proteome analysis showed a high abundance of several methyltransferases, predominantly trimethylamine methyltransferases, during growth with betaine, whereas trimethylamine is one of the main end-products of betaine degradation. In methanol degradation methyltransferases are also involved. In methanol-utilizing methanogens, two methyltransferases catalyse methanol conversion, methyltransferase 1 composed of subunits MtaB and MtaC and methyltransferase 2, also called MtaA. The two methyltransferase 1 subunits MtaB and MtaC were highly abundant when strain An4 was grown with methanol. However, instead of MtaA a methyltetrahydrofolate methyltransferase was synthesized. We propose a novel methanol degradation pathway in Sporomusa strain An4 that uses a methyltetrahydrofolate methyltransferase instead of MtaA.

Screening Method for the Discovery of Potential Bioactive Cysteine-Containing Peptides Using 3D Mass Mapping.

Animal venoms and toxins are a valuable source of bioactive peptides with pharmacologic relevance as potential drug leads. A large subset of biologically active peptides discovered up till now contain disulfide bridges that enhance stability and activity. To discover new members of this class of peptides, we developed a workflow screening specifically for those peptides that contain inter- and intra-molecular disulfide bonds by means of three-dimensional (3D) mass mapping. Two intrinsic properties of the sulfur atom, (1) its relatively large negative mass defect, and (2) its isotopic composition, allow for differentiation between cysteine-containing peptides and peptides lacking sulfur. High sulfur content in a peptide decreases the normalized nominal mass defect (NMD) and increases the normalized isotopic shift (NIS). Hence in a 3D plot of mass, NIS, and NMD, peptides with sulfur appear in this plot with a distinct spatial localization compared with peptides that lack sulfur. In this study we investigated the skin secretion of two frog species; Odorrana schmackeri and Bombina variegata. Peptides from the crude skin secretions were separated by nanoflow LC, and of all eluting peptides high resolution zoom scans were acquired in order to accurately determine both monoisotopic mass and average mass. Both the NMD and the NIS were calculated from the experimental data using an in-house developed MATLAB script. Candidate peptides exhibiting a low NMD and high NIS values were selected for targeted de novo sequencing, and this resulted in the identification of several novel inter- and intra-molecular disulfide bond containing peptides. Graphical Abstract ᅟ.

Perchlorate and chlorate reduction by the Crenarchaeon Aeropyrum pernix and two thermophilic Firmicutes.

This study reports the ability of one hyperthermophilic and two thermophilic microorganisms to grow anaerobically by the reduction of chlorate and perchlorate. Physiological, genomic and proteome analyses suggest that the Crenarchaeon Aeropyrum pernix reduces perchlorate with a periplasmic enzyme related to nitrate reductases, but that it lacks a functional chlorite-disproportionating enzyme (Cld) to complete the pathway. Aeropyrum pernix, previously described as a strictly aerobic microorganism, seems to rely on the chemical reactivity of reduced sulfur compounds with chlorite, a mechanism previously reported for perchlorate-reducing Archaeoglobus fulgidus. The chemical oxidation of thiosulfate (in excessive amounts present in the medium) and the reduction of chlorite result in the release of sulfate and chloride, which are the products of a biotic-abiotic perchlorate reduction pathway in Ae. pernix. The apparent absence of Cld in two other perchlorate-reducing microorganisms, Carboxydothermus hydrogenoformans and Moorella glycerini strain NMP, and their dependence on sulfide for perchlorate reduction is consistent with the observations made on Ar. fulgidus. Our findings suggest that microbial perchlorate reduction at high temperature differs notably from the physiology of perchlorate- and chlorate-reducing mesophiles and that it is characterized by the lack of a chlorite dismutase and is enabled by a combination of biotic and abiotic reactions.

Biochemical, transcriptomic and proteomic analyses of digestion in the scorpion Tityus serrulatus: insights into function and evolution of digestion in an ancient arthropod.

Scorpions are among the oldest terrestrial arthropods and they have passed through small morphological changes during their evolutionary history on land. They are efficient predators capable of capturing and consuming large preys and due to envenomation these animals can become a human health challenge. Understanding the physiology of scorpions can not only lead to evolutionary insights but also is a crucial step in the development of control strategies. However, the digestive process in scorpions has been scarcely studied. In this work, we describe the combinatory use of next generation sequencing, proteomic analysis and biochemical assays in order to investigate the digestive process in the yellow scorpion Tityus serrulatus, mainly focusing in the initial protein digestion. The transcriptome generated database allowed the quantitative identification by mass spectrometry of different enzymes and proteins involved in digestion. All the results suggested that cysteine cathepsins play an important role in protein digestion. Two digestive cysteine cathepsins were isolated and characterized presenting acidic characteristics (pH optima and stability), zymogen conversion to the mature form after acidic activation and a cross-class inhibition by pepstatin. A more elucidative picture of the molecular mechanism of digestion in a scorpion was proposed based on our results from Tityus serrulatus. The midgut and midgut glands (MMG) are composed by secretory and digestive cells. In fasting animals, the secretory granules are ready for the next predation event, containing enzymes needed for alkaline extra-oral digestion which will compose the digestive fluid, such as trypsins, astacins and chitinase. The digestive vacuoles are filled with an acidic proteolytic cocktail to the intracellular digestion composed by cathepsins L, B, F, D and legumain. Other proteins as lipases, carbohydrases, ctenitoxins and a chitolectin with a perithrophin domain were also detected. Evolutionarily, a large gene duplication of cathepsin L occurred in Arachnida with the sequences from ticks being completely divergent from other arachnids probably due to the particular selective pressures over this group.

Cysteine cathepsins as digestive enzymes in the spider Nephilengys cruentata.

Cysteine cathepsins are widely spread on living organisms associated to protein degradation in lysosomes, but some groups of Arthropoda (Heteroptera, Coleoptera, Crustacea and Acari) present these enzymes related to digestion of the meal proteins. Although spiders combine a mechanism of extra-oral with intracellular digestion, the sporadic studies on this subject were mainly concerned with the digestive fluid (DF) analysis. Thus, a more complete scenario of the digestive process in spiders is still lacking in the literature. In this paper we describe the identification and characterization of cysteine cathepsins in the midgut diverticula (MD) and DF of the spider Nephilengys cruentata by using enzymological assays. Furthermore, qualitative and quantitative data from transcriptomic followed by proteomic experiments were used together with biochemical assays for results interpretation. Five cathepsins L, one cathepsin F and one cathepsin B were identified by mass spectrometry, with cathepsins L1 (NcCTSL1) and 2 (NcCTSL2) as the most abundant enzymes. The native cysteine cathepsins presented acidic characteristics such as pH optima of 5.5, pH stability in acidic range and zymogen conversion to the mature form after in vitro acidification. NcCTSL1 seems to be a lysosomal enzyme with its recombinant form displaying acidic characteristics as the native ones and being inhibited by pepstatin. Evolutionarily, arachnid cathepsin L may have acquired different roles but its use for digestion is a common feature to studied taxa. Now a more elucidative picture of the digestive process in spiders can be depicted, with trypsins and astacins acting extra-orally under alkaline conditions whereas cysteine cathepsins will act in an acidic environment, likely in the digestive vacuoles or lysosome-like vesicles.

Analytical characterization of complex, biotechnological feedstocks by pH gradient ion exchange chromatography for purification process development.

The accelerating growth of the market for proteins and the growing interest in new, more complex molecules are bringing new challenges to the downstream process development of these proteins. This results in a demand for faster, more cost efficient, and highly understood downstream processes. Screening procedures based on high-throughput methods are widely applied nowadays to develop purification processes for proteins. However, screening highly complex biotechnological feedstocks, such as complete cell lysates containing target proteins often expressed with a low titre, is still very challenging. In this work we demonstrate a multidimensional, analytical screening approach based on pH gradient ion exchange chromatography (IEC), gel electrophoresis and protein identification via mass spectrometry to rationally characterize a biotechnological feedstock for the purpose of purification process development. With this very simple characterization strategy a two-step purification based on consecutive IEC operations was rapidly laid out for the purification of a diagnostic protein from a cell lysate reaching a purity of ∼80%. The target protein was recombinantly produced using an insect cell expression system.

A high-throughput sample preparation method for cellular proteomics using 96-well filter plates.

A high-throughput sample preparation protocol based on the use of 96-well molecular weight cutoff (MWCO) filter plates was developed for shotgun proteomics of cell lysates. All sample preparation steps, including cell lysis, buffer exchange, protein denaturation, reduction, alkylation and proteolytic digestion are performed in a 96-well plate format, making the platform extremely well suited for processing large numbers of samples and directly compatible with functional assays for cellular proteomics. In addition, the usage of a single plate for all sample preparation steps following cell lysis reduces potential samples losses and allows for automation. The MWCO filter also enables sample concentration, thereby increasing the overall sensitivity, and implementation of washing steps involving organic solvents, for example, to remove cell membranes constituents. The optimized protocol allowed for higher throughput with improved sensitivity in terms of the number of identified cellular proteins when compared to an established protocol employing gel-filtration columns.

Archaeal (per)chlorate reduction at high temperature: an interplay of biotic and abiotic reactions.

Perchlorate and chlorate anions [(per)chlorate] exist in the environment from natural and anthropogenic sources, where they can serve as electron acceptors for bacteria. We performed growth experiments combined with genomic and proteomic analyses of the hyperthermophile Archaeoglobus fulgidus that show (per)chlorate reduction also extends into the archaeal domain of life. The (per)chlorate reduction pathway in A. fulgidus relies on molybdo-enzymes that have similarity with bacterial enzymes; however, chlorite is not enzymatically split into chloride and oxygen. Evidence suggests that it is eliminated by an interplay of abiotic and biotic redox reactions involving sulfur compounds. Biological (per)chlorate reduction by ancient archaea at high temperature may have prevented accumulation of perchlorate in early terrestrial environments and consequently given rise to oxidizing conditions on Earth before the rise of oxygenic photosynthesis.

Molecular characterization of Saccharomyces cerevisiae α-pheromone by mass spectrometry-based peptidomics.

Using modern peptide analytical MS technology ('Peptidomics'), it is possible to analyze yeast α-pheromone both qualitatively and semi-quantitatively directly from conditioned cell culture media. MS/MS analysis shows both forms of α-pheromone (MFα and MFα') detectable and identifiable straight from WT supernatants. In addition to the mature intact α-pheromones, also post-translationally modified α-pheromone peptides and fragments thereof are found to be present in the culture medium. This molecular analytical technique is complementary to the recently described quantitation method by Rogers et al. (2012, FEMS Yeast Res. 12:668) based on ELISA.

Identification of a quinone dehydrogenase from a Bacillus sp. involved in the decolourization of the lignin-model dye, Azure B.

In this study we have investigated the molecular background of the previously reported dye decolourization potential of Bacillus sp. LD003. Strain LD003 was previously isolated on Kraft lignin and was able to decolourize various lignin model dyes. Specifically Azure B (AB) was decolourized efficiently. Proteins possibly involved in AB decolourization were partially purified, fractionated by gel electrophoresis and identified via mass spectrometry. Five candidate enzymes were selected and expressed in Escherichia coli. Of these, only a quinone dehydrogenase was shown to decolourize AB. Thus, this quinone dehydrogenase was identified as an AB decolourizing enzyme of Bacillus sp. LD003.

(89)Zr-labeled paramagnetic octreotide-liposomes for PET-MR imaging of cancer.

PURPOSE: Dual-modality PET/MR platforms add a new dimension to patient diagnosis with high resolution, functional, and anatomical imaging. The full potential of this emerging hybrid modality could be realized by using a corresponding dual-modality probe. Here, we report pegylated liposome (LP) formulations, housing a MR T(1) contrast agent (Gd) and the positron-emitting (89)Zr (half-life: 3.27 days), for simultaneous PET and MR tumor imaging capabilities. METHODS: (89)Zr oxophilicity was unexpectedly found advantageous for direct radiolabeling of preformed paramagnetic LPs. LPs were conjugated with octreotide to selectively target neuroendocrine tumors via human somatostatin receptor subtype 2 (SSTr2). (89)Zr-Gd-LPs and octreotide-conjugated homolog were physically, chemically and biologically characterized. RESULTS: (89)Zr-LPs showed reasonable stability over serum proteins and chelator challenges for proof-of-concept in vitro and in vivo investigations. Nuclear and paramagnetic tracking quantified superior SSTr2-recognition of octreotide-LP compared to controls. CONCLUSIONS: This study demonstrated SSTr2-targeting specificity along with direct chelator-free (89)Zr-labeling of LPs and dual PET/MR imaging properties.

The chains of the heterodimeric amphibian skin antimicrobial peptide, distinctin, are encoded by separate messenger RNAs.

Using a primer to a conserved nucleotide sequence of previously cloned skin peptides of Phyllomedusa species, two distinct cDNAs were "shotgun" cloned from a skin secretion-derived cDNA library of the frog, Phyllomedusa burmeisteri. The two ORFs separately encode chains A and B of an analog of the previously reported heterodimeric peptide, distinctin. LC-MS/MS analysis of native versus dithiotreitol reduced crude venom, confirmed the predicted primary sequences as well as the cystine link between the two monomers. Distinctin predominantly exists in the venom as a heterodimer (A-B), neither of the constituent peptides were detected as monomer, whereas of the two possible homodimers (A-A or B-B), only B-B was detected in comparatively low quantity. In vitro dimerization of synthetic replicates of the monomers demonstrated that besides heterodimer, both homodimers are also formed in considerable amounts. Distinctin is the first example of an amphibian skin dimeric peptide that is formed by covalent linkage of two chains that are the products of different mRNAs. How this phenomenon occurs in vivo, to exclude significant homodimer formation, is unclear at present but a "favored steric state" type of interaction between chains is most likely.

In vivo phosphorylation of Ser21 and Ser83 during nutrient-induced activation of the yeast protein kinase A (PKA) target trehalase.

The readdition of an essential nutrient to starved, fermenting cells of the yeast Saccharomyces cerevisiae triggers rapid activation of the protein kinase A (PKA) pathway. Trehalase is activated 5-10-fold within minutes and has been used as a convenient reporter for rapid activation of PKA in vivo. Although trehalase can be phosphorylated and activated by PKA in vitro, demonstration of phosphorylation during nutrient activation in vivo has been lacking. We now show, using phosphospecific antibodies, that glucose and nitrogen activation of trehalase in vivo is associated with phosphorylation of Ser(21) and Ser(83). Unexpectedly, mutants with reduced PKA activity show constitutive phosphorylation despite reduced trehalase activation. The same phenotype was observed upon deletion of the catalytic subunits of yeast protein phosphatase 2A, suggesting that lower PKA activity causes reduced trehalase dephosphorylation. Hence, phosphorylation of trehalase in vivo is not sufficient for activation. Deletion of the inhibitor Dcs1 causes constitutive trehalase activation and phosphorylation. It also enhances binding of trehalase to the 14-3-3 proteins Bmh1 and Bmh2, suggesting that Dcs1 inhibits by preventing 14-3-3 binding. Deletion of Bmh1 and Bmh2 eliminates both trehalase activation and phosphorylation. Our results reveal that trehalase activation in vivo is associated with phosphorylation of typical PKA sites and thus establish the enzyme as a reliable read-out for nutrient activation of PKA in vivo.

Miniaturized mass-spectrometry-based analysis system for fully automated examination of conditioned cell culture media.

We present a fully automated setup for performing in-line mass spectrometry (MS) analysis of conditioned media in cell cultures, in particular focusing on the peptides therein. The goal is to assess peptides secreted by cells in different culture conditions. The developed system is compatible with MS as analytical technique, as this is one of the most powerful analysis methods for peptide detection and identification. Proof of concept was achieved using the well-known mating-factor signaling in baker's yeast, Saccharomyces cerevisiae. Our concept system holds 1 mL of cell culture medium and allows maintaining a yeast culture for, at least, 40 hours with continuous supernatant extraction (and medium replenishing). The device's small dimensions result in reduced costs for reagents and open perspectives towards full integration on-chip. Experimental data that can be obtained are time-resolved peptide profiles in a yeast culture, including information about the appearance of mating-factor-related peptides. We emphasize that the system operates without any manual intervention or pipetting steps, which allows for an improved overall sensitivity compared to non-automated alternatives. MS data confirmed previously reported aspects of the physiology of the yeast-mating process. Moreover, matingfactor breakdown products (as well as evidence for a potentially responsible protease) were found.

PTM-driven differential peptide display: survey of peptides containing inter/intra-molecular disulfide bridges in frog venoms.

Amphibian defensive skin secretions are complex species-specific mixtures of biologically active molecules, including many uncharacterized peptides. Many of these peptides are post-translationally modified and amongst the modifications discovered so far on amphibian defense peptides, disulfide bonds are quite frequently encountered. The presence of this PTM often complicates the MS-based sequencing. Here we demonstrate a method to target peptides containing inter/intra-molecular S-S bonds applying a PTM-driven differential display. Upon reduction of the disulfide bond both molecular mass and retention time of a peptide are altered. Assembling the LC-MS data by plotting the m/z data against retention time generates a peptide display and overlaying peptide displays of untreated and DTT-reduced material yields a differential display. From such an overlay, peptides originally carrying a disulfide bond are recognized due to the shift in both retention time and m/z values, whereas non cystine containing peptides remain unaltered in the differential display. The success of this approach is demonstrated by the visualization of the cystines-containing peptides in the skin secretion of Odorrana schmackeri, Phyllomedusa burmeisteri, Phyllomedusa rohdei, Kassina senegalensis, and Bombina variegata. The venoms from these different species yield complicated differential displays, showing interesting peptides, allowing one to target them for more detailed structural characterization.

Multi-dimensional fractionation and characterization of crude protein mixtures: toward establishment of a database of protein purification process development parameters.

A multi-dimensional fractionation and characterization scheme was developed for fast acquisition of the relevant molecular properties for protein separation from crude biological feedstocks by ion-exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), and size-exclusion chromatography. In this approach, the linear IEX isotherm parameters were estimated from multiple linear salt-gradient IEX data, while the nonlinear IEX parameters as well as the HIC isotherm parameters were obtained by the inverse method under column overloading conditions. Collected chromatographic fractions were analyzed by gel electrophoresis for estimation of molecular mass, followed by mass spectrometry for protein identification. The usefulness of the generated molecular properties data for rational decision-making during downstream process development was equally demonstrated. Monoclonal antibody purification from crude hybridoma cell culture supernatant was used as case study. The obtained chromatographic parameters only apply to the employed stationary phases and operating conditions, hence prior high throughput screening of different chromatographic resins and mobile phase conditions is still a prerequisite. Nevertheless, it provides a quick, knowledge-based approach for rationally synthesizing purification cascades prior to more detailed process optimization and evaluation.