Correlation of Venom Toxinome Composition of Indian Red Scorpion (Mesobuthus tamulus) with Clinical Manifestations of Scorpion Stings: Failure of Commercial Antivenom to Immune-Recognize the Abundance of Low Molecular Mass Toxins of This Venom.

The Indian red scorpion (Mesobuthus tamulus), with its life-threatening sting, is the world's most dangerous species of scorpion. The toxinome composition of M. tamulus venom was determined by tandem mass spectrometry (MS) analysis of venom protein bands separated by SDS-PAGE. A total of 110 venom toxins were identified from searching the MS data against the Buthidae family (taxid: 6855) of toxin entries in nonredundant protein databases. The Na+ and K+ ion channel toxins taken together are the most abundant toxins (76.7%) giving rise to the neurotoxic nature of this venom. The other minor toxin classes in the M. tamulus venom proteome are serine protease-like protein (2.9%), serine protease inhibitor (2.2%), antimicrobial peptide (2.3%), hyaluronidase (2.2%), makatoxin (2.1%), lipolysis potentiating peptides (1.2%), neurotoxin affecting Cl- channel (1%), parabutoporin (0.6%), Ca2+ channel toxins (0.8%), bradykinin potentiating peptides (0.2%), HMG CoA reductase inhibitor (0.1%), and other toxins with unknown pharmacological activity (7.7%). Several of these toxins have been shown to be promising drug candidates. M. tamulus venom does not show enzymatic activity (phospholipase A2, l-amino acid oxidase, adenosine tri-, di-, and monophosphatase, hyaluronidase, metalloproteinase, and fibrinogenolytic), in vitro hemolytic activity, interference with blood coagulation, or platelet modulation properties. The clinical manifestations post M. tamulus sting have been described in the literature and are well correlated with its venom proteome composition. An abundance of low molecular mass toxins (3-15 kDa) are responsible for exerting the major pharmacological effects of M. tamulus venom, though they are poorly immune-recognized by commercial scorpion antivenom. This is a major concern for the development of effective antivenom therapy against scorpion stings.

Quantitative proteomic analysis of venom from Southern India common krait (Bungarus caeruleus) and identification of poorly immunogenic toxins by immune-profiling against commercial antivenom.

OBJECTIVES: To study the venom proteome composition of Southern India (SI) Common Krait (Bungarus caeruleus) and immunological cross-reactivity between venom against commercial antivenom. METHODS: Proteomic analysis was done by nano LC-MS/MS and toxins were quantitated by label-free analysis. The immunological cross-reactivity of venom towards polyvalent antivenom (PAV) was assessed by ELISA, Immunoblotting, and immuno-chromatographic methods. RESULTS: A total of 57 enzymatic and non-enzymatic proteins belonging to 12 snake venom protein families were identified. The three finger toxins (3FTx) (48.3%) and phospholipase A2 (PLA2) (37.6%) represented the most abundant non-enzymatic and enzymatic proteins, respectively. ß-bungarotoxin (12.9%), a presynaptic neurotoxin, was also identified. The venom proteome composition is well correlated with its enzymatic activities, reported pharmacological properties, and clinical manifestations of krait envenomation. Immuno-cross-reactivity studies demonstrated better recognition of high molecular weight proteins (>45 kDa) of this venom by PAVs compared to low molecular weight (<15 kDa) toxins such as PLA2 and 3FTxs. CONCLUSION: The poor recognition of <15 kDa mass SI B. caeruleus venom proteins is of grave concern for the successful treatment of krait envenomation. Therefore, emphasis should be given to improve the immunization protocols and/or supplement of antibodies raised specifically against the <15 kDa toxins of this venom.

Molecular Networking Reveals Two Distinct Chemotypes in Pyrroloiminoquinone-Producing Tsitsikamma favus Sponges.

The temperate marine sponge, Tsitsikamma favus, produces pyrroloiminoquinone alkaloids with potential as anticancer drug leads. We profiled the secondary metabolite reservoir of T. favus sponges using HR-ESI-LC-MS/MS-based molecular networking analysis followed by preparative purification efforts to map the diversity of new and known pyrroloiminoquinones and related compounds in extracts of seven specimens. Molecular taxonomic identification confirmed all sponges as T. favus and five specimens (chemotype I) were found to produce mainly discorhabdins and tsitsikammamines. Remarkably, however, two specimens (chemotype II) exhibited distinct morphological and chemical characteristics: the absence of discorhabdins, only trace levels of tsitsikammamines and, instead, an abundance of unbranched and halogenated makaluvamines. Targeted chromatographic isolation provided the new makaluvamine Q, the known makaluvamines A and I, tsitsikammamine B, 14-bromo-7,8-dehydro-3-dihydro-discorhabdin C, and the related pyrrolo-ortho-quinones makaluvamine O and makaluvone. Purified compounds displayed different activity profiles in assays for topoisomerase I inhibition, DNA intercalation and antimetabolic activity against human cell lines. This is the first report of makaluvamines from a Tsitsikamma sponge species, and the first description of distinct chemotypes within a species of the Latrunculiidae family. This study sheds new light on the putative pyrroloiminoquinone biosynthetic pathway of latrunculid sponges.

Proteomic Analysis and Immuno-Profiling of Eastern India Russell's Viper ( Daboia russelii) Venom: Correlation between RVV Composition and Clinical Manifestations Post RV Bite.

The proteomes of Russell's viper venom (RVV) from Burdwan (RVV B) and Nadia (RVV N), the two districts of West Bengal, eastern India (EI), were investigated by gel-filtration chromatography (GFC) followed by tandem mass spectrometry of tryptic fragments of the fractions. A total of 73 and 69 proteins belonging to 15 snake venom protein families were identified in RVV B and RVV N, respectively, by MS/MS search against Viperidae (taxid 8689) protein entries of the nonredundant NCBI database. The minor differences in venom composition of both the EI RV were established unequivocally by their biochemical and pharmacological properties and by SDS-PAGE, gel filtration chromatography, and LC-MS/MS analyses. The composition of EI RVVs was well correlated with published reports on the pathophysiology of RV-envenomed patients from this part of the country. Venom-antivenom cross-reactivity determined by ELISA, Western blotting, and antivenomics approaches demonstrated poor recognition of low molecular mass (<20 kDa) RVV proteins by commercial polyvalent antivenoms, which was substantiated by neutralization of RVV enzymes by antivenom.

Unraveling the Proteome Composition and Immuno-profiling of Western India Russell's Viper Venom for In-Depth Understanding of Its Pharmacological Properties, Clinical Manifestations, and Effective Antivenom Treatment.

The proteome composition of western India (WI) Russell's viper venom (RVV) was correlated with pharmacological properties and pathological manifestations of RV envenomation. Proteins in the 5-19 and 100-110 kDa mass ranges were the most predominate (∼35.1%) and least abundant (∼3.4%) components, respectively, of WI RVV. Non-reduced SDS-PAGE indicated the occurrence of multiple subunits, non-covalent oligomers, self-aggregation, and/or interactions among the RVV proteins. A total of 55 proteins belonging to 13 distinct snake venom families were unambiguously identified by ESI-LC-MS/MS analysis. Phospholipase A2 (32.5%) and Kunitz-type serine protease inhibitors (12.5%) represented the most abundant enzymatic and non-enzymatic proteins, respectively. However, ATPase, ADPase, and hyaluronidase, detected by enzyme assays, were not identified by proteomic analysis owing to limitations in protein database deposition. Several biochemical and pharmacological properties of WI RVV were also investigated. Neurological symptoms exhibited by some RV-bite patients in WI may be correlated to the presence of neurotoxic phospholipase A2 enzymes and Kunitz-type serine protease inhibitor complex in this venom. Monovalent antivenom was found to be better than polyvalent antivenom in immuno-recognition and neutralization of the tested pharmacological properties and enzyme activities of WI RVV; nevertheless, both antivenoms demonstrated poor cross-reactivity and neutralization of pharmacological activities shown by low-molecular-mass proteins (<18 kDa) of this venom.

Combination of virtual and experimental 2DE together with ESI LC-MS/MS gives a clearer view about proteomes of human cells and plasma.

Virtual and experimental 2DE coupled with ESI LC-MS/MS was introduced to obtain better representation of the information about human proteome. The proteins from HEPG2 cells and human blood plasma were run by 2DE. After staining and protein spot identification by MALDI-TOF MS, the protein maps were generated. The experimental physicochemical parameters (pI/Mw) of the proteoforms further detected by ESI LC-MS/MS in these spots were obtained. Next, the theoretical pI and Mw of identified proteins were calculated using program Compute pI/Mw (http://web.expasy.org/compute_pi/pi_tool-doc.html). Accordingly, the relationship between theoretical and experimental parameters was analyzed, and the correlation plots were built. Additionally, virtual/experimental information about different protein species/proteoforms from the same genes was extracted. As it was revealed from the plots, the major proteoforms detected in HepG2 cell line have pI/Mw parameters similar to theoretical values. In opposite, the minor protein species have mainly very different from theoretical pI and Mw parameters. A similar situation was observed in plasma in much higher degree. It means that minor protein species are heavily modified in cell and even more in plasma proteome.

Distribution of Marine Lipophilic Toxins in Shellfish Products Collected from the Chinese Market.

To investigate the prevalence of lipophilic marine biotoxins in shellfish from the Chinese market, we used hydrophilic interaction liquid chromatography-tandem mass spectrometry (LC-MS/MS) to measure levels of okadaic acid (OA), azaspiracid (AZA1), pectenotoxin (PTX2), gymnodimine (GYM), and spirolide (SPX1). We collected and analyzed 291 shellfish samples from main production sites along a wide latitudinal transect along the Chinese coastline from December 2008 to December 2009. Results revealed a patchy distribution of the five toxins and highlighted the specific geographical distribution and seasonal and species variation of the putative toxigenic organisms. All five lipophilic marine biotoxins were found in shellfish samples. The highest concentrations of OA, AZA1, PTX2, GYM, and SPX1 were 37.3, 5.90, 16.4, 14.4, and 8.97 µg/kg, respectively. These values were much lower than the legislation limits for lipophilic shellfish toxins. However, the value might be significantly underestimated for the limited detection toxins. Also, these toxins were found in most coastal areas of China and were present in almost all seasons of the year. Thus, these five toxins represent a potential threat to human health. Consequently, studies should be conducted and measures should be taken to ensure the safety of the harvested product.

Evaluation of photostability of azelnidipine tablets and structure determination of its photoproducts.

Photo-exposure has a crucial effect on the natures of photosensitive pharmaceuticals in addition to their contents in medicines through the photodegradation. Generated photoproducts might be more bioactive and contribute to the expression of adverse side effects. This study aimed to clarify the photochemical behavior of medicines of azelnidipine, which is a member of dihydropyridine antihypertensive drugs, by the evaluation of its photostability and the determination of chemical structures of generated photoproducts. Calblock® tablets and its altered forms (powders and suspensions) were UV-irradiated by a black light. Residual amounts of active pharmaceutical ingredients (APIs) were monitored by high-performance liquid chromatography. The chemical structures of two photoproducts were determined by electrospray ionization tandem mass spectrometry. API of Calblock® tablets was photodegraded with the generation of several photoproducts. Its photodegradability was more significant when Calblock® tablets were crushed or suspended. Structural determination revealed that two photoproducts were benzophenone and a pyridine derivative. It was speculated that these photoproducts were generated by the elimination of diphenyl methylene radical and additional chemical reaction including oxidation and hydrolysis. Azelnidipine was photosensitive and its photodegradation in Calblock® tablets was promoted by the change of the dosage form. This difference might be derived from the light emission efficiency. This study suggests that API contents of Calblock® tablets might decrease when tablets or its altered forms are exposed to sunlight irradiation with the generation of benzophenone, which is a toxicological potent.

Structure Determination of Felodipine Photoproducts in UV-Irradiated Medicines Using ESI-LC/MS/MS.

Dihydropyridine drugs are well known as photodegradable pharmaceuticals. Herein, we evaluate the photostability of felodipine (FL) medicine (Splendil® (SPL) tablets) and its altered forms (powders and suspensions). FL is a type of dihydropyridine drug, but its photochemical behavior is unknown. FL contents after ultraviolet light (UV) irradiation for 24 h were monitored using high-performance liquid chromatography (HPLC). Values of the residual amounts of FL in UV-irradiated SPL powders and suspensions were 32.76 ± 4.88% and 0.79 ± 0.74%, respectively, with the generation of two photoproducts (FL photoproduct 1 and 2). To identify the chemical structures of these photoproducts, electrospray ionization liquid chromatography mass spectrometry (ESI-LC/MS/MS) analysis was performed. Based on their mass-to-charge ratio values and fragment patterns, it was proposed that FL photoproduct 1 was a pyridine derivative and FL photoproduct 2 was an FL dimer. Interestingly, generation rates of FL photoproduct 1 and 2 were dependent on the presence of the aqueous media. The photodimerization of FL was induced in UV-irradiated SPL suspensions. This is the first report evaluating the photostability of SPL tablets and its altered forms and estimating FL photoproducts induced by UV irradiation in the formulation of SPL.

Transformation of hempseed (Cannabis sativa L.) oil cake proteome, structure and functionality after extrusion.

The entire protein fractions from hempseed, its oil cake (30-40% protein) and the extruded protein isolate (>90% protein) were investigated. The first semi-quantitative mass spectrometry-based proteomics on hempseed was performed, leading to a sum of 1879 differentially abundant proteins being identified from individual pairwise comparisons of each extruded group compared to unextruded hempseed cake. The 'free-form' amino acid content and total amino acid content of hempseed oil cake were enhanced by up to 315% and 18%, respectively, after extrusion. Changes in the structure and thermal properties of hempseed protein were confirmed through circular dichroism, Fourier transform infrared spectroscopy, scanning electron microscopy and differential scanning calorimetry. The proteomic and structural transformation in the extruded hempseed protein fractions contributed to greater values in majority of tested functionality parameters, such as protein solubility, water and oil binding capacity, emulsification properties, and in vitro digestibility, as compared to their unextruded counterparts.

Cyanotoxin Screening in BACA Culture Collection: Identification of New Cylindrospermopsin Producing Cyanobacteria.

Microcystins (MCs), Saxitoxins (STXs), and Cylindrospermopsins (CYNs) are some of the more well-known cyanotoxins. Taking into consideration the impacts of cyanotoxins, many studies have focused on the identification of unknown cyanotoxin(s)-producing strains. This study aimed to screen strains from the Azorean Bank of Algae and Cyanobacteria (BACA) for MCs, STX, and CYN production. A total of 157 strains were searched for mcy, sxt, and cyr producing genes by PCR, toxin identification by ESI-LC-MS/MS, and cyanotoxin-producing strains morphological identification and confirmation by 16S rRNA phylogenetic analysis. Cyanotoxin-producing genes were amplified in 13 strains and four were confirmed as toxin producers by ESI-LC-MS/MS. As expected Aphanizomenon gracile BACA0041 was confirmed as an STX producer, with amplification of genes sxtA, sxtG, sxtH, and sxtI, and Microcystis aeruginosa BACA0148 as an MC-LR producer, with amplification of genes mcyC, mcyD, mcyE, and mcyG. Two nostocalean strains, BACA0025 and BACA0031, were positive for both cyrB and cyrC genes and ESI-LC-MS/MS confirmed CYN production. Although these strains morphologically resemble Sphaerospermopsis, the 16S rRNA phylogenetic analysis reveals that they probably belong to a new genus.

Isolation and whole protein characterization of species A and B bovine rotaviruses from Chinese calves.

Rotaviruses (RVs) account for severe diarrhea in children and young animals globally. In the current study, the fecal samples of diarrheic calves from a beef farm in Inner Mongolia were screened for RVA by ELISA and RT-PCR, followed by culture of three positive RVA samples in the MA-104 cell line. After 10 blind passages, cytopathic effects (CPE) appeared as detachment, granulation, and clustering of the inoculated cells. The virus isolates were identified by RT-PCR (VP6 gene RVA) and ESI-LC-MS/MS for whole protein sequencing. The protein sequences demonstrated the presence of two strains from species A rotavirus and one RVB strain; RVA/Cow-tc/CHN/35333/2019/G6P[5] was mixed with one RVB strain (RVB/Cow-tc/CHN/35334/2019/G5P[3]) in two samples, and RVA/Cow-tc/CHN/10927/2019/G8P[7] was found in one sample. They are of genotype constellations (G6-P[5]-I2-R2-C2-M2-A3-N2-T6-E2-H3), (G8-P[7]-I5-R1-C1- M2-A1-N1-T1-E1-H1), and (G5-P[3]-I3-R5-C5-A5-N4-H5), respectively. Besides, phylogenetic analysis of the obtained sequences demonstrated viral evolution.

Development of a Highly Sensitive Ultra-High-Performance Liquid Chromatography Coupled to Electrospray Ionization Tandem Mass Spectrometry Quantitation Method for Fecal Bile Acids and Application on Crohn's Disease Studies.

In addition to their important role in fat digestion, bile acids are increasingly being used as markers for various diseases. The large diversity of bile acids results from the conversion of primary and conjugated bile acids into secondary bile acids by deconjugation and dehydroxylation reactions mediated by the intestinal microbiota. Here, we describe a fast and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for absolute quantitation of 45 bile acids in human or mouse feces in combination with a simple workup and extraction procedure. Method validation outlined excellent limits of detection and quantitation, linearity, selectivity, recovery, extraction loss, and precision. To investigate the connection between microbiome alterations and bile acid metabolism, the method was applied on a Crohn's disease study including patients with histologically documented active disease or remission as well as on a model using humanized mice. As the complex mechanism including genetic and environmental factors leading to the development of Crohn's disease is so far not completely understood, the study investigates the microbial metabolism of bile acids and the potential use of bile acid profiles to predict disease state.

Targeted LC-MS/MS Profiling of Bile Acids in Various Animal Tissues.

Bile acids are being increasingly investigated in humans and laboratory animals as markers for various diseases in addition to their important functions, such as promoting the emulsification in fat digestion and preventing gallstone formation. In humans and animals, primary bile acids are formed from cholesterol in the liver, converted in the intestine into various secondary bile acids by the intestinal microbiota and reabsorbed in the terminal ileum, and partially returned to the liver. A universal high-throughput workflow, including a simple workup, was applied as a tool for bile acid analysis in animal studies. The complex bile acid profiles in various tissues, organs, and body fluids from different animals were mapped using a newly developed comprehensive liquid chromatography-tandem mass spectrometry method. The method can also be used in screening food to obtain information about the nutritional content of bile acids. This could be relevant to investigations on various animal diseases and on the bioavailability of bile acids that pass through the gastric tract.

Cloning and expression of synthetic genes encoding native, hybrid- and bacteriocin-derived chimeras from mature class IIa bacteriocins, by Pichia pastoris (syn. Komagataella spp.).

In this work, synthetic genes designed from (a), the native amino acid sequence of the class IIa bacteriocins enterocin HF (EntHF) and enterocin CRL35 (EntCRL35), (b) from hybrid bacteriocins derived from fusion of enterocin A (EntA) to itself and to EntHF and EntCRL35 through a tri-glycine peptide linker, and (c) from bacteriocin-derived chimeras devised from fusion of the N-terminal region of EntA and enterocin P (EntP) to the C-terminal end of EntHF and EntCRL35, were cloned in plasmid pPICZαA for expression by P. pastoris X-33. Synthetic genes encoding EntHF and EntCRL35 were also cloned in plasmid pP-αhSUMO3 for expression of the hSUMO3-fused bacteriocins by P. pastoris. Only recombinant P. pastoris expressing the bacteriocin-derived chimeras displayed a direct antimicrobial activity whereas P. pastoris X-33, producer of EntP::EntHF, showed the highest antimicrobial activity in their supernatants and in the multi-step chromatographic purified fractions. The MRM-ESI-LC-MS/MS (QTRAP) analysis of purified fractions from P. pastoris producers of hybrid- and bacteriocin-derived chimeras, permitted detection in the samples of peptides with the expected molecular mass of the bacteriocins produced. The antimicrobial activity of the EntP::EntHF chimera compared to that of the synthetic EntP::EntHF peptide, suggest that the biologically-produced bacteriocin-derived chimera shows a higher specific antimicrobial activity than its synthetic counterpart against different Listeria strains, including L. monocytogenes. More important, the N-terminal region of EntA and EntP seems to drive the production, processing and secretion of hybrid- and bacteriocin-derived chimeras, by P. pastoris X-33.

Development of a CDC-certified total testosterone assay for adult and pediatric samples using LC-MS/MS.

BACKGROUND: Highly accurate and sensitive method to measure testosterone in hypogonadal male, female and children is vital for proper diagnosis of hormone-related conditions and their treatment. OBJECTIVE: To develop an accurate and robust total testosterone ESI-LC-MS/MS quantification method with a simple sample preparation workflow and sufficient sensitivity for serum or plasma samples of all gender and age groups, via ketone functional group derivatization (using Amplifex™ Keto Reagent). METHOD: A simple sample preparation method to accommodate both low and high numbers of samples was developed using simultaneous protein precipitation and derivatization with Amplifex™ Keto reagent, followed by centrifugation and direct injection of supernatant into an LC-MS/MS system (SCIEX Topaz™ IVD LC-MS/MS, in which MS is equivalent to a SCIEX 4500MD Mass Spectrometer). Total testosterone in human serum or plasma samples was quantified using an external calibration curve generated by calibrators spanning a broad concentration range of ∼1-2000 ng/dL (10-20,000 pg/mL), traceable to NIST 971 SRM. 13C3-enriched testosterone was used as an internal standard to correct for both analyte loss during sample preparation and matrix effect during analysis (Supplementary Information: SI Fig. 4C). Two methods, one using a 96-well filter plate and another using Eppendorf tubes, were developed. Both methods were certified by the Centers for Disease Control (CDC) hormone standardization (HoSt) program for total serum testosterone. The feasibility of implementing the method for plasma and serum samples was tested via a small-scale method comparison study between matched pediatric serum and plasma samples derived from the same donor. In addition, plasma samples originating from the same donor collected in two different anticoagulant tube types (Li-heparin and K2EDTA) were compared. RESULTS: Using in-house formulated NIST 971-traceable calibrators, the method was linear (r2 > 0.999) between 1 and 2000 ng/dL (10 and 20,000 pg/mL) with a limit of detection of approximately 1 ng/dL (10 pg/mL). The testosterone concentration bias against 40 reference samples from the HoSt certification program was absolute <3% with an average %CV of ∼3-4%. More than 78% of samples passed the CDC bias criterion of ±6.4%. Comparison between pediatric matched serum and plasma samples resulted in high correlation (r2 = 0.997) and bias of <5%. The calculated % difference between matched adult serum and plasma samples was ∼1%. CONCLUSIONS: Feasibility for an accurate and streamlined method suitable for measuring total testosterone in all human samples was demonstrated with a choice of sample preparation workflow to suit low or high number of samples. The method can potentially be used for plasma matrix from different blood collection tubes (Li-Heparin and K2EDTA).

Quantitative proteomic analysis and antivenom study revealing that neurotoxic phospholipase A2 enzymes, the major toxin class of Russell's viper venom from southern India, shows the least immuno-recognition and neutralization by commercial polyvalent antivenom.

The proteome composition of Russell's viper venom (RVV) from southern India (SI) was investigated by 1D-SDS-PAGE of venom followed by tandem mass spectrometry analysis of protein bands. A total of 66 proteins belonging to 14 snake venom protein families were identified by LC-MS/MS analysis against Viperidae (taxid 8689) protein entries from the non-redundant NCBI database. Phospholipase A2 (43.25%) and snaclec (14.57%) represented the most abundant enzymatic and non-enzymatic proteins, respectively. SI RVV was characterized as containing a higher quantity of PLA2 and a lower amount of Kunitz-type serine protease inhibitors, in comparison to RVV from other regions of the Indian subcontinent. The enzymatic activities, pharmacological properties, and clinical manifestations of RV envenomation in SI were well correlated with its proteome composition; however, ATPase, ADPase, and hyaluronidase enzymes were not identified by LC-MS/MS analysis, owing to paucity of the existing database. Neurological symptoms exhibited by RV-bite patients in SI were correlated to the presence of abundant neurotoxic phospholipase A2 enzymes (15.66%) in SI RVV. Neutralization studies, immunological cross-reactivity, and antivenomics studies unequivocally demonstrated the poor recognition and lowest neutralization of PLA2 enzymes by commercial polyvalent antivenom, which is a major concern for the treatment of RV-envenomed patients in SI.

Comparison of proteomic profiles of the venoms of two of the 'Big Four' snakes of India, the Indian cobra (Naja naja) and the common krait (Bungarus caeruleus), and analyses of their toxins.

Snake venoms are mixtures of biologically-active proteins and peptides, and several studies have described the characteristics of some of these toxins. However, complete proteomic profiling of the venoms of many snake species has not yet been done. The Indian cobra (Naja naja) and common krait (Bungarus caeruleus) are elapid snake species that are among the 'Big Four' responsible for the majority of human snake envenomation cases in India. As understanding the composition and complexity of venoms is necessary for successful treatment of envenomation in humans, we utilized three different proteomic profiling approaches to characterize these venoms: i) one-dimensional SDS-PAGE coupled with in-gel tryptic digestion and electrospray tandem mass spectrometry (ESI-LC-MS/MS) of individual protein bands; ii) in-solution tryptic digestion of crude venoms coupled with ESI-LC-MS/MS; and iii) separation by gel-filtration chromatography coupled with tryptic digestion and ESI-LC-MS/MS of separated fractions. From the generated data, 81 and 46 different proteins were identified from N. naja and B. caeruleus venoms, respectively, belonging to fifteen different protein families. Venoms from both species were found to contain a variety of phospholipases A2 and three-finger toxins, whereas relatively higher numbers of snake venom metalloproteinases were found in N. naja compared to B. caeruleus venom. The analyses also identified less represented venom proteins including L-amino acid oxidases, cysteine-rich secretory proteins, 5'-nucleotidases and venom nerve growth factors. Further, Kunitz-type serine protease inhibitors, cobra venom factors, phosphodiesterases, vespryns and aminopeptidases were identified in the N. naja venom, while acetylcholinesterases and hyaluronidases were found in the B. caeruleus venom. We further analyzed protein coverage (Lys/Arg rich and poor regions as well as potential glycosylation sites) using in-house software. These studies expand our understanding of the proteomes of the venoms of these two medically-important species.

A comparative intracellular proteomic profiling of Pseudomonas aeruginosa strain ASP-53 grown on pyrene or glucose as sole source of carbon and identification of some key enzymes of pyrene biodegradation pathway.

Pseudomonas aeruginosa strain ASP-53, isolated from a petroleum oil-contaminated soil sample, was found to be an efficient degrader of pyrene. PCR amplification of selected hydrocarbon catabolic genes (alkB gene, which encodes for monooxygenase, and the C12O, C23O, and PAH-RHDα genes encoding for the dioxygenase enzyme) from the genomic DNA of P. aeruginosa strain ASP-53 suggested its hydrocarbon degradation potential. The GC-MS analysis demonstrated 30.1% pyrene degradation by P. aeruginosa strain ASP-53 after 144h of incubation at pH6.5, 37°C. Expressions of 115 and 196 intracellular proteins were unambiguously identified and quantitated by shotgun proteomics analysis when the isolate was grown in medium containing pyrene and glucose, respectively. The pyrene-induced uniquely expressed and up-regulated proteins in P. aeruginosa strain ASP-53 in addition to substrate (pyrene) metabolism are also likely to be associated with different cellular functions for example-related to protein folding (molecular chaperone), stress response, metabolism of carbohydrate, proteins and amino acids, and fatty acids; transport of metabolites, energy generation such as ATP synthesis, electron transport and nitrate assimilation, and other oxidation-reduction reactions. Proteomic analyses identified some important enzymes involved in pyrene degradation by P. aeruginosa ASP-53 which shows that this bacterium follows the salicylate pathway of pyrene degradation. SIGNIFICANCE: This study is the first report on proteomic analysis of pyrene biodegradation pathway by Pseudomonas aeruginosa, isolated from a petroleum-oil contaminated soil sample. The pathway displays partial similarity with deduced pyrene degradation mechanisms of Mycobacterium vanbaalenii PYR-1. The GC-MS analysis as well as PCR amplification of hydrocarbon catabolic genes substantiated the potency of the bacterium under study to effectively degrade high molecular weight, toxic PAH such as pyrene for its filed scale bioremediation experiments. The proteomics approach (LC-MS/MS analysis) identified the differentially regulated intracellular proteins of the isolate P. aeruginosa ASP-53 when grown in pyrene medium. This study identified some important pyrene biodegradation enzymes in Pseudomonas aeruginosa ASP-53 and highlights that the bacterium follows salicylate pathway for pyrene degradation.

Variety and Dynamics of Proteoforms in the Human Proteome: Aspects of Markers for Hepatocellular Carcinoma.

We have previously developed an approach, where two-dimensional gel electrophoresis (2DE) was used, followed by sectional analysis of the whole gel using high-resolution nano-liquid chromatography-mass spectrometry (ESI LC-MS/MS). In this study, we applied this approach on the panoramic analysis of proteins and their proteoforms from normal (liver) and cancer (HepG2) cells. This allowed us to detect, in a single proteome, about 20,000 proteoforms coded by more than 4000 genes. A set of 3D-graphs showing distribution of these proteoforms in 2DE maps (profiles) was generated. A comparative analysis of these profiles between normal and cancer cells showed high variability and dynamics of many proteins. Among these proteins, there are some well-known features like alpha-fetoprotein (FETA) or glypican-3 (GPC3) and potential hepatocellular carcinoma (HCC) markers. More detailed information about their proteoforms could be used for generation of panels of more specific biomarkers.