Search

Lectins are renowned hemagglutinins and multivalent proteins with a well known quality for sugar-binding specificity that participate significantly in invertebrate defense functions. Studies on biological activity of lectin from coleopteran insect are very scarce. In this study, lectin from the hemolymph in the grub of banana pest, Odoiporus longicollis was subjected to purification, biochemical and functional characterizations. The lectin was purified by PEG precipitation and ion-exchange chromatography using Q-Sepharose as a matrix. The purified lectin showed hemagglutination activity against rat erythrocytes, heat-labile, cation independent and insensitive to EDTA. Further, the carbohydrate affinity of this lectin was found with mannitol, adonitol, L-arabinose, L-rhamnose, D-galactose and sorbitol. The native form of purified lectin was calculated as 360 kDa by FPLC system. Denatured gel electrophoresis of the purified lectin consisted of five distinct polypeptides with molecular weights approximately 160, 60, 52, 40 and 38 kDa, respectively. The amino acid sequences obtained through peptide mass fingerprinting analysis exhibited homologies to the known conserved regions of galactose binding lectins. Further, the purified lectin exhibited bacterial inhibition with LPS from Serratia marcescens. In addition, isolated lectin also exerted bacterial agglutination, antibacterial and anti-proliferative activity against Mycobacterium smegmatis, Bacillus pumilus and Neuro 2a cell line, respectively.

Two IgM heavy (H) chain sub-isotypes (80 and 40 kDa) and two light (L) chain variants (25 and 30 kDa) were detected in the serum of giant grouper (Epinephelus lanceolatus), purified by ammonium sulphate precipitation followed by protein A affinity chromatography. This method yielded 5.6 mg/mL high purity IgM from grouper serum, with efficiency estimated at 39.5% recovery from crude serum. The H and L chains were identified by SDS-PAGE and mass spectrometry (MS). Nanopore long-read sequencing was used to generate a genomic contig (MW768935), containing Cμ, Cδ loci, VH regions, and a H chain Joining segment. cDNA sequencing of Cμ transcripts (MW768933 and MW768934) were used to polish the genomic contig and determine the exons and introns of the corresponding locus. MS peptide mapping revealed that the 80 kDa H chain consisted of CH1-4 domains while peptides from the 40 kDa H chain only mapped to CH1-2 domains. Our genomic contig showed the Cμ locus has a Cμ1-Cμ2-Cμ3-Cμ4 arrangement on the same strand as the other Ig loci identified in this genomic sequence. Our study corrects the NCBI annotations of the opposing Cμ loci (LOC117268697 and LOC117268550) in chromosome 16 (NC_047006). Further, we identified both κ and λ L chain isotypes in serum IgM. The molecular weight differences observed may result from different combinations of CL and VL genes. Putative IgM sub-isotypes have also been reported in Epinephelus itajara and Epinephelus coioides. The presence of IgM sub-isotypes may be a conserved trait among Epinephelus species.

Chinese black sleeper (Bostrychus sinensis) is a fish that lives both in seawater and freshwater, feeds on crustaceans, aquatic insects and occasionally shellfish. The existence of digestive enzyme in viscera to act on chitinous exoskeleton of the prey is of interest. In this study, a chitinase was purified to homogeneity using ammonium sulfate precipitation, DEAE-Sephacel ion exchange, Sephacryl S-200 HR and Superdex 200 gel filtration columns. The purified protein presents a molecular mass of 58 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and results in a single band on native PAGE. According to peptide mass fingerprinting, two peptides containing a total of 20 amino acid residues, were 95% identical to a chitinase from yellow perch (Perca flavescens) and 100% identical to the chitinase from greater amberjack (Seriola dumerili). The purified chitinase showed optimum activity at pH 6.0, and was stable at acidic conditions and temperature below 55°C. The enzymatic activity was quite stable in the presence of NaCl, even at 1 mol/L. The chitinase was capable of degrading chitosan into low molecular mass chitooligosaccharides (COS) with sizes in a range of 200–700 Da, and the circular dichroism profile of the COS greatly differed from native chitosan. Full-length cDNA encoding the present chitinase was cloned and the transcript levels of chitinase in various tissues were determined by quantitative real-time PCR. The results showed that the transcript level of chitinase was highest in esophagus and hepatopancreas.

Background: Reversibly glycosylated polypeptide (RGP), a kind of hydrosoluble and plasmodesmal-associated protein found in plants, plays a crucial role in the development of pollen. Objective: A novel RGP 2 was isolated and identified from rape (Brassica napus L.) bee pollen. Methods: RGP2 was isolated and purified by ion-exchange column and gel filtration chromatography, and characterized by MALDI-TOF-MS, LC-MS, immunological histological chemistry, and transmission electron microscope. Results: Our results indicated that the RGP2 is an acidic protein (pI=5.46) with the molecular weight 42388 Da. It contained 17 kinds of amino acids, among which aspartic acid had the highest amount (71.56 mg/g). Homologous alignment of amino acid sequence results showed that RGP2 was 80.33%, 85.02%, 86.06%, and 88.93% identical to Arabidopsis thaliana RGP2 (AtRGP2), Oryza sativa RGP (OsRGP), Triticum aestivum RGP (TaRGP), and Zea maize RGP (ZmRGP), respectively. The localization results showed that RGP2 in rape anther existed in exine and intine of anther cells of rape flower by immunological histological chemistry and the subcellular localization identified that RGP2 appeared around the Golgi apparatus in cytoplasm by transmission electron microscope. Conclusion: RGP2 has a highly conserved sequence of amino acid residues and potential glycosylation sites.

Medicinal plants are the basic source of medicinal compounds traditionally used for the treatment of human diseases. Calotropis gigantea is a medicinal plant belonging to the family of Apocynaceae in the plant kingdom and subfamily Asclepiadaceae usually bearing multiple medicinal properties to cure a variety of diseases. Background: The Peptide Mass Fingerprinting (PMF) identifies the proteins from a reference protein database by comparing the amino acid sequence that is previously stored in a database and identified. Method: The calculation of insilico peptide masses is done through the ExPASy PeptideMass and these masses are used to identify the peptides from MASCOT online server. Anticancer probability is calculated from the iACP server, docking of active peptides is done by CABS-dock the server. Objective: The purpose of the study is to identify the peptides having anti-cancerous properties by in silico peptide mass fingerprinting. Results : The anti-cancer peptides are identified with the MASCOT peptide mass fingerprinting server, the identified peptides are screened and only the anti-cancer are selected. De-novo peptide structure prediction is used for 3D structure prediction by PEP-FOLD 3 server. The docking results confirm strong bonding with the interacting amino acids of the receptor protein of breast cancer BRCA1 which shows the best peptide binding to the active chain, the human leukemia protein docking with peptides shows the accurate binding. Conclusion : These peptides are stable and functional and are the best way for the treatment of cancer and many other deadly diseases.

An efficient and simple method for the application of PEGylated affinity ligands in precipitative isolation of protein target molecules (TMs) from a biological fluid such as blood serum or small target molecules from an aqueous medium is presented for the first time. This approach is based on the high binding specificity of PEGylated recognition molecules (PEG-RMs) to their TMs and the unique physicochemical properties of PEG that result in their salt-assisted phase transformation. Addition of PEG-RM to blood serum results in the formation of an RM-specific macromolecular complex (PEG-RM + TM → PEG-RM.TM) that undergoes facile salt-assisted phase transformation to a separable semisolid with ammonium sulfate. PEG-RM.TM is then dissociated into its components by pH reduction or an increase of ionic strength (PEG-RM.TM → PEG-RM + TM). PEG-RM is salted out to afford pure TM in solution. The same phenomenon is observed when RM or TM are small molecules. The general applicability of the method was validated by PEGylation of two proteins (protein A, sheep antihuman IgG) and a small molecule (salicylic acid) used as model RMs for the isolation of Igs, IgG, and serum albumin from blood serum. The isolated protein TMs were shown to be pure and aggregate-free by gel electrophoresis and dynamic light scattering (DLS). IgG isolated by this method was further characterized by peptide mass fingerprinting. PEGylated protein A was used to demonstrate the recyclability and scale-up potential of PEG-RM. IgG isolated by this method from blood serum of a hepatitis C-vaccinated individual was tested for its binding to sheep antihuman IgG by UV spectroscopy, and its bioactivity was ascertained by comparison of its enzyme-linked immunosorbent assay (ELISA) result to that of a blood sample from the same individual. Reciprocity of RM and TM was ascertained using PEGylated salicylic acid to obtain pure serum albumin, and PEGylated serum albumin was utilized for near-exclusive isolation of one drug from an aqueous equimolar mixture of three drugs (salicylic acid, 91%; capecitabine, 6%; and deferiprone, 3%). Advantages of this approach, including target specificity and general applicability and celerity, over other affinity methods for the isolation of proteins are discussed at a molecular level.

Cyclic disulfide-rich peptides have attracted significant interest in drug development and biotechnology. Here, we describe a protocol for producing cyclic peptide precursors in Pichia pastoris that undergo in vitro enzymatic maturation into cyclic peptides using recombinant asparaginyl endopeptidases (AEPs). Peptide precursors are expressed with a C-terminal His tag and secreted into the media, enabling facile purification by immobilized metal affinity chromatography. After AEP-mediated cyclization, cyclic peptides are purified by reverse-phase high-performance liquid chromatography and characterized by mass spectrometry, peptide mass fingerprinting, NMR spectroscopy, and activity assays. We demonstrate the broad applicability of this protocol by generating cyclic peptides from three distinct classes that are either naturally occurring or synthetically backbone cyclized, and range in size from 14 amino acids with one disulfide bond, to 34 amino acids with a cystine knot comprising three disulfide bonds. The protocol requires 14 d to identify and optimize a high-expressing Pichia clone in small-scale cultures (24 well plates or 50 mL tubes), after which large-scale production in a bioreactor and peptide purification can be completed in 10 d. We use the cyclotide Momordica cochinchinensis trypsin inhibitor II as an example. We also include a protocol for recombinant AEP production in Escherichia coli as AEPs are emerging tools for orthogonal peptide and protein ligation. We focus on two AEPs that preferentially cyclize different peptide precursors, namely an engineered AEP with improved catalytic efficiency [C247A]OaAEP1b and the plant-derived MCoAEP2. Rudimentary proficiency and equipment in molecular biology, protein biochemistry and analytical chemistry are needed. The authors present a protocol for producing cyclic peptide precursors in Pichia pastoris that undergo in vitro enzymatic maturation into cyclic peptides using recombinant asparaginyl endopeptidases. Assays for characterizing purified cyclic peptides are also described.

Vinculin is a well-known cytoskeletal protein and is a component of the integrin-mediated cell-matrix adhesion system. Recently, vinculin is also being reported from the nuclei from a number of organisms. However, there is no report yet on purification of nuclear vinculin from the native source of any organism. In the present study, by using western blotting, we show nuclear localization of vinculin in chicken liver. The chicken liver nuclear vinculin was purified to homogeneity and subsequently, the identity of vinculin was confirmed by peptide mass fingerprinting. Further, actin was co-immunoprecipitated with vinculin from chicken liver nuclear extract. Interestingly, the above immunoprecipitate (IP) demonstrated histone specific protease activity. Thus, the present study suggests plausible interaction of vinculin with actin and histone specific proteases in the chicken liver nuclei.

Our previous studies showed, bacterium Aquamicrobium sp. SK-2 could degrade biphenyl and polychlorinatedbiphenyls (PCBs). In the present study, proteins involved in the biphenyl degradation was evaluatedusing various molecular biology methods. The gene bphC present in the strain SK-2 was identifiedusing the polymerase chain reaction method. Further the key enzyme in biphenyl degradation, 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC) was purified through anion exchange and gel filtration chromatography,subsequently the enzyme activity was measured. The N-terminal amino acid sequence ofthe purified enzyme showed 92% homology with BphC enzyme of Gram-negative bacteria(Pseudomonas sp. KKS102, Comamonas testosterone, Burkholderiaceae bacterium, Delftia acidovorans, andAchromobacter denitrificans). Fractions collected during protein purification were applied on SDS-PAGEgel. Significant bands were selected in SDS-PAGE gel, and the gel pieces were cut out to analyze the proteinsusing peptide mass fingerprinting (PMF) method. PMF method provided useful information aboutthe proteins involved in biphenyl degradation. Apart from BphC, two other enzymes, benzoate dioxygenaseand catechol 2,3-dioxygenase which were involved in biphenyl degradation process were identified.The results indicate that catechol can be degraded to 2-hydroxymuconic-semialdehyde and this result isin accordance with the results from our previous study. Based on all these results we can conclude thatthe strain SK-2 is a potential candidate for the bioremediation of biphenyl contaminated places._ 2021 The Authors. Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd.This is an open access article under the CC BY-NC-ND license

An rare pandemic of viral pneumonia occurs in December 2019 in Wuhan, China, which is now recognized internationally as Corona Virus Disease 2019 (COVID-19), the etiological agent classified as Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2). According to the World Health Organization (WHO), it has so far expanded to more than 213 countries/territories worldwide. Our study aims to find the viral peptides of SARS-COV-2 by peptide mass fingerprinting (PMF) in order to predict its novel structure and find an inhibitor for each viral peptide. For this reason, we calculated the mass of amino acid sequences translated from the SARS-CoV2 whole genome and identify the peptides that may be a target for inhibition. Molecular peptide docking with Moringa oleifera, phytochemicals (aqueous and ethanolic) leaf extracts of flavonoids (3.56 ± 0.03), (3.83 ± 0.02), anthraquinone (11.68 ± 0.04), (10.86 ± 0.06) and hydroxychloroquine present therapy of COVID-19 in Pakistan for comparative study. Results indicate that 15 peptides of SARS-CoV2 have been identified from PMF, which is then used as a selective inhibitor. The maximum energy obtained from AutoDock Vina for hydroxychloroquine is –5.1 kcal/mol, kaempferol (flavonoid) is –6.2 kcal/mol, and for anthraquinone –6 kcal/mol. Visualization of docking complex, important effects are observed regarding the binding of peptides to drug compounds. In conclusion, it is proposed that these compounds are effective antiviral agents against COVID-19 and can be used in clinical trials. Communicated by Ramaswamy H. Sarma

The effects of detergent sodium dodecyl sulfate (SDS) on protein structure and dynamics are fundamental to the most common laboratory technique used to separate proteins and determine their molecular weights: polyacrylamide gel electrophoresis. However, the mechanism by which SDS induces protein unfolding and the microstructure of protein–SDS complexes remain largely unknown. Here, we report a detailed account of SDS-induced unfolding of two proteins—I27 domain of titin and β-amylase—obtained through all-atom molecular dynamics simulations. Both proteins were found to spontaneously unfold in the presence of SDS at boiling water temperature on the time scale of several microseconds. The protein unfolding was found to occur via two distinct mechanisms in which specific interactions of individual SDS molecules disrupt the protein’s secondary structure. In the final state of the unfolding process, the proteins are found to wrap around SDS micelles in a fluid necklace-and-beads configuration, where the number and location of bound micelles changes dynamically. The global conformation of the protein was found to correlate with the number of SDS micelles bound to it, whereas the number of SDS molecules directly bound to the protein was found to define the relaxation time scale of the unfolded protein. Our microscopic characterization of SDS–protein interactions sets the stage for future refinement of SDS-enabled protein characterization methods, including protein fingerprinting and sequencing using a solid-state nanopore.

Adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) (AK) is the enzyme that catalyzes the reversible conversion of MgATP + AMP to MgADP + ADP, a principal step in adenine nucleotide metabolism and cellular energy homeostasis. To enrich the information about fish AKs, we isolated this enzyme from the lateral muscle of bastard halibut Paralichthys olivaceus and biochemically characterized it. The halibut enzyme (PoAK) extracted from the lateral muscle was purified by column chromatographies on TOYOPEARL SP-650 M and Superdex 75 10/300. The purified PoAK showed a single protein band of ~ 22 kDa on SDS–polyacrylamide gel electrophoresis, and optimal temperature and pH at around 40 °C and 7, respectively. PoAK was appreciably heat stable, e.g., the temperature that caused 50% inactivation during 30-min incubation was 54 °C. The molar ratio for ATP:ADP:AMP in the equilibrium state of the reaction was ~ 1:1:1. Peptide mass fingerprinting indicated that PoAK is the product of adenylate kinase isoform 1 gene (GenBank, XP_019937160.1) encoded in the halibut genome. The deduced amino-acid sequence of the halibut AK comprised 194 residues and showed 92, 91 and 81% amino-acid identities to those of a putative rainbow trout AK, a carp AK and a chicken AK, respectively.

Novel tick-borne phleboviruses in the Phenuiviridae family, which are highly pathogenic in humans and all closely related to Uukuniemi virus (UUKV), have recently emerged on different continents. How phleboviruses assemble, bud, and exit cells remains largely elusive. Here, we performed high-resolution, label-free mass spectrometry analysis of UUKV immunoprecipitated from cell lysates and identified 39 cellular partners interacting with the viral envelope glycoproteins. The importance of these host factors for UUKV infection was validated by silencing each host factor by RNA interference. This revealed Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1), a guanine nucleotide exchange factor resident in the Golgi, as a critical host factor required for the UUKV life cycle. An inhibitor of GBF1, Golgicide A, confirmed the role of the cellular factor in UUKV infection. We could pinpoint the GBF1 requirement to UUKV replication and particle assembly. When the investigation was extended to viruses from various positive and negative RNA viral families, we found that not only phleboviruses rely on GBF1 for infection, but also Flavi-, Corona-, Rhabdo-, and Togaviridae. In contrast, silencing or blocking GBF1 did not abrogate infection by the human adenovirus serotype 5 and immunodeficiency retrovirus type 1, the replication of both requires nuclear steps. Together our results indicate that UUKV relies on GBF1 for viral replication, assembly and egress. This study also highlights the proviral activity of GBF1 in the infection by a broad range of important zoonotic RNA viruses.Ticks are important vectors of infectious emerging diseases and tick-borne phleboviruses represent a growing threat to humans globally. We employed here a high-resolution, label-free mass spectrometry and RNA interference screen approach to reveal the host cell protein GBF1 as a proviral factor, not only for tick-borne phleboviruses, but also for many other important zoonotic RNA viruses. This study lays the basis for the development of innovative antiviral strategies against a broad range of human pathogenic viruses.

The peculiarities of the survival and adaptation of deep-sea organisms raise interest in the study of their metabolites as promising drugs. In this work, the hemolytic, cytotoxic, antimicrobial, and enzyme-inhibitory activities of tentacle extracts from five species of sea anemones (Cnidaria, orders Actiniaria and Corallimorpharia) collected near the Kuril and Commander Islands of the Far East of Russia were evaluated for the first time. The extracts of Liponema brevicorne and Actinostola callosa demonstrated maximal hemolytic activity, while high cytotoxic activity against murine splenocytes and Ehrlich carcinoma cells was found in the extract of Actinostola faeculenta. The extracts of Corallimorphus cf. pilatus demonstrated the greatest activity against Ehrlich carcinoma cells but were not toxic to mouse spleen cells. Sea anemones C. cf. pilatus and Stomphia coccinea are promising sources of antimicrobial and antifungal compounds, being active against Gram-positive bacteria Bacillus subtilis, Staphylococcus aureus, and yeast Candida albicans. Moreover, all sea anemones contain α-galactosidase inhibitors. Peptide mass fingerprinting of L. brevicorne and C. cf. pilatus extracts provided a wide range of peptides, predominantly with molecular masses of 4000–5900 Da, which may belong to a known or new structural class of toxins. The obtained data allow concluding that deep-sea anemones are a promising source of compounds for drug discovery.

Τα πεπτίδια, είτε ως πρωτεϊνικά θραύσματα είτε ως φυσικές οντότητες, χαρακτηρίζονται από την ακολουθία τους και από τα λειτουργικά τους χαρακτηριστικά. Ο σκοπός αυτής της Διδακτορικής Διατριβής είναι η ανάπτυξη μιας μεθοδολογίας εξόρυξης δεδομένων για μοναδικά tags και πεπτιδικά/πρωτεϊνικά χαρακτηριστικά του ανθρώπινου πρωτεώματος καθώς επίσης η ανάλυση και η εφαρμογή αυτών των βιολογικών δεδομένων σε πρωτεϊνες που σχετίζονται με τον καρκίνο. Δημιουργήθηκε μια αποθήκη αρχείων η οποία περιέχει μοριακά βάρη με ακρίβεια 0.01 Da που συνδέονται με τις αντίστοιχες πεπτιδικές ακολουθίες ανθρώπινων πρωτεϊνών της Swiss-Prot βάσης. Αυτές οι πρωτεϊνες διασπάστηκαν εξαντλητικά παρέχοντας ανεξαρτησία στις πεπτιδικές ακολουθίες από άλλες μεθόδους που βασίζονται στην ενζυματική διάσπαση. Από αυτήν την αποθήκη δεδομένων, διαχωρίστηκαν τα μοριακά βάρη που είναι μοναδικά και φτάνουν μέχρι τα 10 kDa καθώς και οι μοναδικές πεπτιδικές ακολουθίες (μέχρι 10 kDa). Στα πλαίσια της αξιοποίησης των δεδομένων εξόρυξης για την ταυτοποίηση των πρωτεϊνών, αναπτύχθηκε μια ευρέως διαθέσιμη διαδικτυακή εφαρμογή όπου γίνεται η αντιστοίχιση των μοριακών βαρών υψηλής ανάλυσης με πεπτίδια και πρωτεϊνες. Μια ακόμη διαδικτυακή εφαρμογή αναπτύχθηκε για να προσφέρει την πληροφορία της μοναδικότητας των μοριακών βαρών και των πεπτιδικών ακολουθιών στο ανθρώπινο πρωτέωμα. Η εφαρμογή μπορεί να αναζητήσει μοναδικά πρωτεϊνικά θραύσματα που προκύπτουν από την εζυματική διάσπαση πρωτεϊνών και να προσφέρει την πληροφορία για όλα τα μοναδικά μοριακά βάρη και τις μοναδικές πεπτιδικές ακολουθίες που περιέχονται σε μια πρωτεϊνη. Πολλές φορές χρειάζεται η μαζική διαχείριση των πεπτιδίων από λίστες. Για το σκοπό αυτό, αναπτύχθηκε ένας web server ο οποίος διαχειρίζεται τις πεπτιδικές λίστες, αναλύοντας τα χαρακτηριστικά των πεπτιδίων και ομαδοποιώντας τα πεπτίδια σύμφωνα μα αυτά τα χαρακτηριστικά, ενώ οπτικοποιείται η ομαδοποίηση με την χρήση ενός java applet. Το PepServe είναι ένα χρήσιμο εργαλείο για την κατανόηση της κατανομής των πεπτιδικών χαρακτηριστικών για ένα σύνολο πεπτιδίων. Τέλος, αναλύθηκαν σύνολα πρωτεϊνών που σχετίζονται με διάφορες περιπτώσεις καρκίνων, για πεπτιδικά χαρακτηριστικά. Αυτή η ανάλυση έχει σκοπό την εύρεση πιθανών προτιμήσεων σε χαρακτηριστικά και την εύρεση μοναδικών tags των πρωτεϊνών που σχετίζονται με καρκίνους. Τα μοναδικά tags μπορούν να χρησιμοποιηθούν στην ανακάλυψη βιοδεικτών και την ανάπτυξη νεων φαρμάκων για την πιο αποτελεσματική διάγνωση και θεραπεία.

Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) has been reported to play critical roles in the proliferation of various cancer cells. However, the roles of LGR5 in brain tumors and the specific intracellular signaling proteins directly associated with it remain unknown. Expression of LGR5 was first measured in normal brain tissue, meningioma, and pituitary adenoma of humans. To identify the downstream signaling pathways of LGR5, siRNA-mediated knockdown of LGR5 was performed in SH-SY5Y neuroblastoma cells followed by proteomics analysis with 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE). In addition, the expression of LGR5-associated proteins was evaluated in LGR5-inhibited neuroblastoma cells and in human normal brain, meningioma, and pituitary adenoma tissue. Proteomics analysis showed 12 protein spots were significantly different in expression level (more than two-fold change) and subsequently identified by peptide mass fingerprinting. A protein association network was constructed from the 12 identified proteins altered by LGR5 knockdown. Direct and indirect interactions were identified among the 12 proteins. HSP 90-beta was one of the proteins whose expression was altered by LGR5 knockdown. Likewise, we observed decreased expression of proteins in the hnRNP subfamily following LGR5 knockdown. In addition, we have for the first time identified significantly higher hnRNP family expression in meningioma and pituitary adenoma compared to normal brain tissue. Taken together, LGR5 and its downstream signaling play critical roles in neuroblastoma and brain tumors such as meningioma and pituitary adenoma.

Sesamin is a furofuran-type lignan that is found abundantly in seeds of Sesamum indicum (sesame) and has been widely accepted as a dietary supplement with positive effects on human health. The biological activity of sesamin in human cells and organs has been analysed extensively, although comparatively few studies show biological functions for sesamin in planta. Herein we screened sesamin-binding proteins (SBP) from sesame seedling extracts using sesamin-immobilized nano-beads. In subsequent peptide mass fingerprinting analyses, we identified a SBP, Steroleosin B, which is one of the membrane proteins found in oil bodies. In addition, pull-down assays and saturation transfer difference-nuclear magnetic resonance (STD-NMR) experiments demonstrated that sesamin binds directly to recombinant Steroleosin B in vitro. Finally, ectopic accumulations of sesamin and Steroleosin B in transgenic Arabidopsis thaliana plants induced severe growth defects including suppression of leaf expansion and root elongation. Collectively, these results indicate that sesamin influences tissue development in the presence of Steroleosin B.

The family of staphylococcal superantigen-like proteins (SSLs) have a structure similar to bacterial superantigens but exhibit no superantigenic activity. These exoproteins have recently been shown to disturb the host immune defense system. One family member, SSL5, was reported to bind to human leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) and matrix metalloproteinase-9 (MMP-9) and to interfere with leukocyte trafficking. In the present study, we explored human plasma proteins bound by glutathione S-transferase (GST)-tagged recombinant SSL5 (GST-SSL5) and identified plasma protease C1 inhibitor (C1Inh) as a major SSL5-binding protein based on the results of peptide mass fingerprinting analysis with MALDI-TOFMS. GST-SSL5 was found to attenuate the inhibitory activity of recombinant histidine-tagged C1Inh (C1Inh-His) toward complement C1s. We also observed that the treatment of C1Inh-His with neuraminidase markedly decreased its binding to GST-SSL5. Moreover, C1Inh-His produced by Lec2 mutant cells (deficient in sialic acid biosynthesis) showed much lower binding affinity for SSL5 than that produced by the wild-type CHO-K1 cells, as assessed by pull-down assay. These results suggest that SSL5 binds to C1Inh in a sialic acid-dependent fashion and modulates the host immune defense through perturbation of the complement system in association with S. aureus infection.

Protoiurus kraepelini is a scorpion species found in parts of Turkey and Greece. In this study, the peptide profile of its venomwas determined for the first time. The electrophoretic profile of the crude venom showed a protein distribution from 2 to 130 kDa.MALDI-TOF MS analysis of the venom peptide fraction yielded 27 peptides between 1059 and 4623 Da in mass. Several ion channelblockingand antimicrobial peptides were identified by peptide mass fingerprinting analysis. Cytotoxic and antimicrobial effects of thevenom were also demonstrated on Jurkat cells and Escherichia coli, respectively. As the first peptidomic characterization study on P.kraepelini venom, this report lays the foundation for detailed future studies that may lead to the discovery of novel bioactive peptides.

Drosophila suzukii (Ds) is an invasive pest insect that infests ripening fruit, causing severe economic losses. Control measures based on chemical pesticides are inefficient and undesirable, so biological alternatives have been considered, including native Ds viruses. We previously isolated a strain of La Jolla virus (LJV-Ds-OS20) from Ds in Germany as a candidate biopesticide. Here we characterized the new strain in detail, focusing on the processing of its capsid proteins. We tested LJV growth during Ds development to optimize virus production, and established a laboratory production system using adult flies. This system was suitable for the preparation of virions for detailed analysis. The LJV-Ds-OS20 isolate was cloned by limiting dilution and the complete nucleotide sequence was determined as a basis for protein analysis. The terminal segments of the virus genome were completed by RACE-PCR. LJV virions were also purified by CsCl gradient centrifugation and analyzed by SDS-PAGE and electron microscopy. The capsid proteins of purified LJV virions were resolved by two-dimensional SDS-PAGE for N-terminal sequencing and peptide mass fingerprinting. The N-terminal sequences of VP1 and VP2, together with MS data representing several capsid proteins, allowed us to develop a model for the organization of the LJV structural protein region. This may facilitate the development of new viral strains as biopesticides.

Antimicrobial proteins (AMPs) are small, cationic proteins that exhibit activity against bacteria, viruses, parasites, fungi as well as boost host-specific innate immune responses. Insects produce these AMPs in the fat body and hemocytes, and release them into the hemolymph upon microbial infection. Hemolymph was collected from the bacterially immunized fifth instar larvae of tasar silkworm, Antheraea mylitta, and an AMP was purified by organic solvent extraction followed by size exclusion and reverse-phase high-pressure liquid chromatography. The purity of AMP was confirmed by thin-layer chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The molecular mass was determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry as 14 kDa, and hence designated as AmAMP14. Peptide mass fingerprinting of trypsin-digested AmAMP14 followed by de novo sequencing of one peptide fragment by tandem mass spectrometry analysis revealed the amino acid sequences as CTSPKQCLPPCK. No homology was found in the database search and indicates it as a novel AMP. The minimum inhibitory concentration of the purified AmAMP14 was determined against Escherichia coli, Staphylococcus aureus, and Candida albicans as 30, 60, and 30 µg/ml, respectively. Electron microscopic examination of the AmAMP14-treated cells revealed membrane damage and release of cytoplasmic contents. All these results suggest the production of a novel 14 kDa AMP in the hemolymph of A. mylitta to provide defense against microbial infection.

Objective: To identify proteins associated with the formation of Streptococcus gordonii and Fusobacterium nucleatum biofilms . Material and Methods: Biofilms composed of two bacterial species, S. gordonii and F. nucleatum , were cultured for 1, 4, 7, and 10 days. The presence of both species was confirmed via amplification of the srtA and radD genes using real-time PCR. The concentrations of proteins associated with the biofilms and individual species were quantified using Western blotting. Results: The protein profiles of S. gordonii and F. nucleatum from individual cultures determined using one-dimensional electrophoresis revealed proteins found in S. gordonii and in F. nucleatum. Ct and reciprocal Ct values were determined for the exposed S. gordonii and F. nucleatum biofilms. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein was detected in biofilms and F. nucleatum , whereas HSP40 protein was present only in biofilms after 7 and 10 days of formation . Conclusion: HSP40 was detected only in the formed biofilms; thus, HSP40 is an essential proteins for adhesion.

16 p.-6 fig., Clavulanic acid (CLV) and amoxicillin, frequently administered in combination, can be independently involved in allergic reactions. Protein haptenation with β-lactams is considered necessary to activate the immune system. The aim of this study was to assess the suitability of biotinylated analogues of CLV as probes to study protein haptenation by this β-lactam. Two synthetic approaches afforded the labeling of CLV through esterification of its carboxylic group with a biotin moiety, via either direct binding (CLV-B) or tetraethylenglycol linker (CLV-TEG-B). The second analogue offered advantages as solubility in aqueous solution and potential lower steric hindrance for both intended interactions, with the protein and with avidin. NMR reactivity studies showed that both CLV and CLV-TEG-B reacts through β-lactam ring opening by aliphatic amino nitrogen, however with different stability of resulting conjugates. Unlike CLV conjugates, that promoted the decomposition of clavulanate fragment, the conjugates obtained with the CLV-TEG-B remained linked, as a whole structure including biotin, to nucleophile and showed a better stability. This was a desired key feature to allow CLV-TEG-B conjugated protein detection at great sensitivity. We have used biotin detection and mass spectrometry (MS) to detect the haptenation of human serum albumin (HSA) and human serum proteins. MS of conjugates showed that HSA could be modified by CLV-TEG-B. Remarkably, HSA preincubation with CLV excess only reduced moderately the incorporation of CLV-TEG-B, which could be attributed to different protein interferences. The CLV-TEG-B fragment with opened β-lactam was detected bound to the 404–430HSA peptide of the treated protein. Incubation of human serum with CLV-TEG-B resulted in the haptenation of several proteins that were identified by 2D-electrophoresis and peptide mass fingerprinting as HSA, haptoglobin, and heavy and light chains of immunoglobulins. Taken together, our results show that tagged-CLV keeps some of the CLV features. Moreover, although we observe a different behavior in the conjugate stability and in the site of protein modification, the similar reactivity indicates that it could constitute a valuable tool to identify protein targets for haptenation by CLV with high sensitivity to get insights into the activation of the immune system by CLV and mechanisms involved in β-lactams allergy., Work at MJT and MIM laboratory was supported by Instituto de Salud Carlos III (ISCIII) of MICINN (grants cofunded by ERDF: “Una manera de hacer Europa” (PI17/01237, PI18/00095, RETIC ARADYAL RD16/0006/0001 and Euronanomed Program AC19/00082), Miguel Servet I program (CP15/00103) and Sara Borrell program (CD17/00146)), Andalusian Regional Ministry of Health (PI-0179-2014, PE-0172-2018). Work at EP-I laboratory was supported by the Spanish Ministerio de Economía, Industria y Competitividad (CTQ 2016-75870-P), Ministerio de Ciencia y Educación (PID 2019-104293GB-I00), Ministerio de Ciencia e Innovación [Proyectos de I+D+I Programación Conjunta Internacional, EuroNanoMed 2019 (PCI 2019-111825-2)], ISCIII RETIC ARADYAL RD16/0006/0012 and Junta de Andalucía (UMA18-FEDERJA-007). Work at DP-S laboratory was supported by Grants from Agencia Estatal de Investigación, Ministerio de Ciencia e Innovación (MICINN, Spain) and European Regional Development Fund, SAF 2015-68590-R and RTI 2018-097624-B-I00, ISCIII RETIC ARADyAL RD16/0006/0021.

10 pages; International audience; Molluscs were one of the most widely-used natural resources in the past, and their shells are abundant among archaeological findings. However, our knowledge of the variety of shells that were circulating in prehistoric times (and thus their socio-economic and cultural value) is scarce due to the difficulty of achieving taxonomic determination of fragmented and/or worked remains. This study aims to obtain molecular barcodes based on peptide mass fingerprints (PMFs) of intracrystalline proteins, in order to obtain shell identification. Palaeoproteomic applications on shells are challenging, due to low concentration of molluscan proteins and an incomplete understanding of their sequences. We explore different approaches for protein extraction from small-size samples (

Viruses induce profound changes in the cells they infect. Understanding these perturbations will assist in designing better therapeutics to combat viral infection. System-based proteomic assays now provide unprecedented opportunity to monitor large numbers of cellular proteins.This review will describe various quantitative and functional mass spectrometry-based methods, and complementary non-mass spectrometry-based methods, such as aptamer profiling and proximity extension assays, and examples of how each are used to delineate how viruses affect host cells, identify which viral proteins interact with which cellular proteins, and how these change during the course of a viral infection. PubMed was searched multiple times prior to manuscript submissions and revisions, using virus, viral, proteomics; in combination with each keyword. The most recent examples of published works from each search were then analyzed.There has been exponential growth in numbers and types of proteomic analyses in recent years. Continued development of reagents that allow increased multiplexing and deeper proteomic probing of the cell, at quantitative and functional levels, enhancements that target more important protein modifications, and improved bioinformatics software tools and pathway prediction algorithms will accelerate this growth and usher in a new era of host proteome understanding.

All animals, other than Platyhelminthes, produce eggs containing yolk, referred to as "entolecithal" eggs. However, only Neoophora, in the phylum Platyhelminthes, produce "ectolecithal" eggs (egg capsules), in which yolk is stored in the vitelline cells surrounding oocytes. Vitelline cells are derived from vitellaria (yolk glands). Vitellaria are important reproductive organs that may be studied to elucidate unique mechanisms that have been evolutionarily conserved within Platyhelminthes. Currently, only limited molecular level information is available on vitellaria. The current study identified major vitellaria-specific proteins in a freshwater planarian, Dugesia ryukyuensis, using peptide mass fingerprinting (PMF) and expression analyses. Amino acid sequence analysis and orthology analysis via OrthoFinder ver.2.3.8 indicated that the identified major vitellaria-specific novel yolk ferritins were conserved in planarians (Tricladida). Because ferritins play an important role in Fe (iron) storage, we examined the metal elements contained in vitellaria and ectolecithal eggs, using non-heme iron histochemistry, elemental analysis based on inductively coupled plasma mass spectrometry and transmission electron microscopy- energy-dispersive X-ray spectroscopy analysis. Interestingly, vitellaria and egg capsules contained large amounts of aluminum (Al), but not Fe. The knockdown of the yolk ferritin genes caused a decrease in the volume of egg capsules, abnormality in juveniles, and increase in Al content in vitellaria. Yolk ferritins of D. ryukyuensis may regulate Al concentration in vitellaria via their pooling function of Al and protect the egg capsule production and normal embryogenesis from Al toxicity.

Background: DARTS "Drug Affinity Responsive Target Stability" is a novel methodology for identifying and studying protein-ligand interactions. This technique depends on the reduction in the protease susceptibility of the target protein upon drug binding. Objectives: The aim of this study to prove the effectiveness of the method to identify the molecular target proteins of some bioactive endophyte extracts. The basic idea of this research is studying the possibility of the use of Darts technique for the detection of cellular targets for natural extracts before doing chemically analyzed to determine the type of vehicle which they are located. Methodology: To achieve the aim of study, some natural extracts of fungi endophyte were selected for the study, three of which are able to stop the growth of three kinds of cancer cells leukemia and lung cancer and cervical cancer, as demonstrated by tests performed in this study. Results: Then Darts technique was used to identify the cellular targets of these extracts successfully. Possible, as already identified a number of proteins that interact with these extracts within the cell. It was possible for first time by using DARTS followed by MALDI-TOF analysis and peptide mass fingerprinting to identify a number of cellular target molecules for bioactive extracts of endophytes. Conclusion: This will help reduce the number of extracts that go into the process of drug development because the extracts which is not suitable to be excluded from the start without the need to chemically analyzed to determine composition, which helps to reduce the cost and the time period required by the process of drug development in the future. This opens up a new area for the use of this technique in the process of scanning cytometry initial natural extracts without having to analyze it chemically which reduces the effort and expense. Finally, A number of very interesting proteins could be identified that might stimulate the researchers in a future study to the chemical composition of these extracts to exactly identify the active component in each of them.

N-Heterocyclic compounds are widely spread in the biosphere, being constituents of alkaloids, cofactors, allelochemicals, and artificial substances. However, the fate of such compounds including a catabolism of hydroxylated pyridines is not yet fully understood. Arthrobacter sp. IN13 is capable of using 4-hydroxypyridine as a sole source of carbon and energy. Three substrate-inducible proteins were detected by comparing protein expression profiles, and peptide mass fingerprinting was performed using MS/MS. After partial sequencing of the genome, we were able to locate genes encoding 4-hydroxypyridine-inducible proteins and identify the kpi gene cluster consisting of 16 open reading frames. The recombinant expression of genes from this locus in Escherichia coli and Rhodococcus erytropolis SQ1 allowed an elucidation of the biochemical functions of the proteins. We report that in Arthrobacter sp. IN13, the initial hydroxylation of 4-hydroxypyridine is catalyzed by a flavin-dependent monooxygenase (KpiA). A product of the monooxygenase reaction is identified as 3,4-dihydroxypyridine, and a subsequent oxidative opening of the ring is performed by a hypothetical amidohydrolase (KpiC). The 3-(N-formyl)-formiminopyruvate formed in this reaction is further converted by KpiB hydrolase to 3-formylpyruvate. Thus, the degradation of 4-hydroxypyridine in Arthrobacter sp. IN13 was analyzed at genetic and biochemical levels, elucidating this catabolic pathway.

Cellular response against different heavy metal stress differs with the metal. Arsenic and chromium are heavy metals and toxic to living systems. The concentration of these metals in seawater is very low. However, due to their solubility in nature, they actively enter cells via various transport mechanisms and cause damage to the cells. Brevibacterium casei #NIOSBA88, a marine-derived, gram-positive isolate was multi-metal tolerant. Proteomic analysis of this isolate in response to arsenic and chromium resulted in the identification of total 2549 proteins, out of which 880 proteins were found to be commonly expressed at 750 mgL(−1) arsenic and 100 mgL(−1) chromium and in absence of both the metals. In contrast, 533, 212, and 270 proteins were found to be unique in the absence of any metal, 750 mgL(−1) of arsenic and 100 mgL(−1) of chromium respectively. Proteins such as antibiotic biosynthesis monooxygenase, ArsR family transcriptional regulator, cytochrome C oxidase subunit II, and thioredoxin reductase were exclusively expressed only in response to arsenic and chromium. Other proteins like superoxide dismutase, lipid hydroperoxide reductase, and thioredoxin-disulfide reductase were found to be upregulated in response to both the metals. Most of the proteins involved in the normal cell functioning were found to be downregulated. Major metabolic functions affected include amino acid metabolism, carbohydrate metabolism, translation, and energy metabolism. Peptide mass fingerprinting of Brevibacterium casei #NIOSBA88 exposed to arsenic and chromium respectively revealed the deleterious effect of these metals on the bacterium and its strategy to overcome the stress. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s42770-020-00353-7) contains supplementary material, which is available to authorized users.

Success of discovery programs for microbial natural products is dependent on quick and concise discrimination between isolates from diverse environments. However, laboratory isolation and identification of priority genera using current 16S rRNA PCR-based methods are both challenging and time-consuming. An emerging strategy for rapid isolate discrimination is protein fingerprinting via matrix-assisted laser desorption ionization (MALDI) mass spectrometry. Using our in-house environmental isolate repository, we have created a main spectral (MSP) library for the Bruker Biotyper MALDI mass spectrometer that contains 95 entries, including Burkholderia, Caballeronia, Paraburkholderia, and other environmentally related genera. The library creation required the acquisition of over 2,250 mass spectra, which were manually reviewed for quality control and consolidated into a single reference library using a commercial software platform. We tested the effectiveness of the reference library by analyzing 49 environmental isolate strains using two different sample preparation methods. Overall, this approach correctly identified all strains to the genus level provided that suitable reference spectra were present in the MSP library. In this study, we present a fast, accurate method for taxonomic assignment of environmentally derived bacteria from the order Burkholderiales, providing a valuable alternative to traditional PCR-based methods. The MSP library described in the manuscript is available for use. IMPORTANCE The Gram-negative proteobacterial order Burkholderiales has emerged as a promising source of novel natural products in recent years. This order includes the genus Burkholderia and the newly defined genera Caballeronia and Paraburkholderia. However, development of this resource has been hampered by difficulties with rapid and selective isolation of Burkholderiales strains from the environment. Environmental metagenome sequencing has revealed that the potential for natural products is not evenly distributed throughout the microbial world. Thus, large but targeted microbial isolate libraries are needed to effectively explore the chemical potential of natural products. To study these organisms efficiently, methods to quickly identify isolates to the genus level are required. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is already used in clinical settings to reliably identify unknown bacterial pathogens. We have adapted similar methodology using the MALDI Biotyper instrument to rapidly identify environmental isolates of Burkholderia, Caballeronia, and Paraburkholderia for downstream natural product discovery.

Plant pathogenic viruses cause major economic losses in many agricultural and horticultural crops. The diagnosis of viruses in the seed and propagative materials assumes greater importance for national quarantine services and is crucial not only for effective implication of disease management strategies but also to restrict the movement of viral inoculum through planting/propagative materials both at the domestic and the international level. However, identification, detection, and characterization of the causal agents of viral diseases are challenging, owing to the low titer and irregular within-plant distribution of viruses. Currently available biological detection techniques are time and resource consuming because of the vector-mediated transmission of several viruses, thus limiting a large-scale and easy-to-use evaluation. Another important constraint for viral diagnosis is that many pathogenic viruses remain latent in planting materials with less population, and in such cases methods of high sensitivity reliability are essential. Several serological and molecular methods have been utilized for the detection and identification of plant viruses, but recent advancements in techniques based on protein and nucleic acid detection have resulted in an efficient and accurate way to diagnose plant virus diseases, for which molecular techniques are expensive. Protein-detection techniques such as serological methods (enzyme-linked immunosorbent assay), immunoblotting, lateral-flow immunochromatographic assay, immunocapture assay, fluorescence polarization immunoassay, chemiluminescence immunoassay, radioimmunoassay, protein fingerprinting technique, and quartz crystal microbalance immunosensors and their modifications, although limited in sensitivity, enable inexpensive, high level of repeatability and reproducibility, and fast parallel diagnosis of many samples, using either polyclonal, monoclonal, or recombinant antibodies.

Ependymoma pediatric brain tumor occurs at approximate frequencies of 10&ndash, 15% in supratentorial and 20&ndash, 30% in posterior fossa regions. These tumors have an almost selective response to surgery and relative and confirmed resistance to radiotherapy and chemotherapic agents, respectively. Alongside histopathological grading, clinical and treatment evaluation of ependymomas currently consider the tumor localization and the genomic outlined associated molecular subgroups, with the supratentorial and the posterior fossa ependymomas nowadays considered diverse diseases. On these grounds and in trying to better understand the molecular features of these tumors, the present investigation aimed to originally investigate the proteomic profile of pediatric ependymoma tissues of different grade and localization by mass spectrometry platforms to disclose potential distinct protein phenotypes. To this purpose, acid-soluble and acid-insoluble fractions of ependymoma tumor tissues homogenates were analyzed by LC-MS following both the top-down and the shotgun proteomic approaches, respectively, to either investigate the intact proteome or its digested form. The two approaches were complementary in profiling the ependymoma tumor tissues and showed distinguished profiles for supratentorial and posterior fossa ependymomas and for WHO II and III tumor grades. Top-down proteomic analysis revealed statistically significant higher levels of thymosin beta 4, 10 kDa heat shock protein, non-histone chromosomal protein HMG-17, and mono-/uncitrullinated forms ratio of the glial fibrillary acidic protein (GFAP) fragment 388&ndash, 432 in supratentorial ependymomas&mdash, the same GFAP fragment as well as the hemoglobin alpha- and the beta-chain marked grade II with respect to grade III posterior fossa ependymomas. Gene ontology classification of shotgun data of the identified cancer and the non-cancer related proteins disclosed protein elements exclusively marking tumor localization and pathways that were selectively overrepresented. These results, although preliminary, seem consistent with different protein profiles of ependymomas of diverse grade of aggressiveness and brain region development and contributed to enlarging the molecular knowledge of this still enigmatic tumor.

Ancient protein analysis is providing new insights into the evolutionary relationships between hominin fossils across the Pleistocene. Protein identification commonly relies on the proteolysis of a protein extract using a single protease, trypsin. As with modern proteome studies, alternative or additional proteases have the potential to increase both proteome size and protein sequence recovery. This could enhance the recovery of phylogenetic information from ancient proteomes. Here we identify 18 novel hominin bone specimens from the Kleine Feldhofer Grotte using MALDI-TOF MS peptide mass fingerprinting of collagen type I. Next, we use one of these hominin bone specimens and three Late Pleistocene Equidae specimens identified in a similar manner and present a comparison of the bone proteome size and protein sequence recovery obtained after using nanoLC-MS/MS and parallel proteolysis using six different proteases, including trypsin. We observe that the majority of the preserved bone proteome is inaccessible to trypsin. We also observe that for proteins recovered consistently across several proteases, protein sequence coverage can be increased significantly by combining peptide identifications from two or more proteases. Our results thereby demonstrate that the proteolysis of Pleistocene proteomes by several proteases has clear advantages when addressing evolutionary questions in palaeoproteomics.SignificanceMaximizing proteome and protein sequence recovery of ancient skeletal proteomes is important when analyzing unique hominin fossils. As with modern proteome studies, palaeoproteomic analysis of Pleistocene bone and dentine samples has almost exclusively used trypsin as its only protease, despite the demonstrated advantages of alternative proteases to increase proteome recovery in modern proteome studies. We demonstrate that Pleistocene bone proteomes can be significantly expanded by using additional proteases beside trypsin, and that this also improves sequence coverage of individual proteins. The use of several alternative proteases beside trypsin therefore has major benefits to maximize the phylogenetic information retrieved from ancient skeletal proteomes.

Abiotic stresses such as drought, heat and salinity are responsible for decelerating agricultural productivity all over the world and the destructive aftermaths are likely to increase further due to changing global climate. Small heat shock proteins (sHsps) are synthesized ubiquitously and play diverse roles in abiotic stress responses of plants. sHsps are reported to be involved in preventing aggregation, stabilizing non-native proteins. In the present study, Hsp17.9 gene from Prosopis cineraria was cloned, characterized and expression was studied in prokaryotic system. In silico analysis was carried out to gain insights in to protein structure and its interaction with other molecules. Overexpression of the gene in Escherichia coli BL21 (DE3) cells was analyzed on SDS–PAGE, followed by western blotting using Anti His-tag antibody and purified by nickel–nitrilo triacetic acid resin based columns. The single purified band was confirmed by peptide mass fingerprinting. Two dimensional protein analysis of bacterial protein showed the presence of overexpressed protein spot at the desired place. Further, the recombinant E. coli cells carrying pET28a-PcHsp17.9 construct were able to tolerate abiotic stresses better than the cells carrying pET28a vector alone. The cloned PcHsp17.9 gene may be useful to generate abiotic stress tolerant transgenic crops for a climate smart agriculture.

The plant Polygonum tinctorium produces the secondary metabolite indican (indoxyl-β-D-glucoside), a precursor of the blue dye indigo. P. tinctorium synthesizes indican through the actions of the UDP-glucosyltransferase (UGT), indican synthase. Herein, we partially purified an indican synthase from the leaves and subsequently performed peptide mass fingerprinting analysis. Consequently, we identified a fragment that was homologous to a UDP-glucosyltransferase 72B (UGT72B) family member. We named it PtIgs (P. tinctoriumindoxyl-β-D-glucoside synthase) and obtained the full-length cDNA using rapid amplification of the cDNA ends. The primary structure of PtIGS, which PtIgs encoded, showed high identity with indican synthases (ItUGT1 and ItUGT2) from Indigofera tinctoria (Inoue et al., 2017). Moreover, in expression analyses of P. tinctorium, PtIGS mRNA was virtually found only in the leaves, was most highly expressed in the 1st leaves, and decreased with leaf age. Because PtIGS expression tended to reflect indican contents and synthesis activities, we concluded that PtIGS functions as an indican synthase in plant cells. To examine intracellular localization of PtIGS, crude leaf extracts were separated into cytosol and microsome fractions, and found PtIGS in the cytosol and in microsome fractions. Furthermore, microsomal PtIGS was soluble in the presence of detergents and urea and was strongly associated with membranes. Finally, we confirmed endoplasmic reticulum (ER) membrane localization of PtIGS using ultracentrifugation with a sucrose density gradient. These data suggest that PtIGS interacts with some kind of proteins on ER membranes to certainly carry out a delivery of substrate.

Knowledge of zooplankton community structure is essential to understand ecosystem functioning. Therefore, accurate species identification is a crucial step in plankton studies. Yet, with the current array of methodologies, species richness is still largely underestimated and high-resolution distribution patterns remain unknown. A novel, cost-effective and rapid alternative to morphological or genetic approaches is the identification of species by proteomic fingerprinting based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The present study aims to demonstrate the applicability of proteomic fingerprinting for large-scale identification and quantification of the pelagic copepod family Spinocalanidae. Therefore, MALDI-TOF MS was applied to discriminate, identify and quantify adult and juvenile planktonic copepods of the ecologically important deep-sea copepod family Spinocalanidae from stratified depth samples (0–1000 m) at two stations in the eastern tropical Atlantic. Spectral peak data from 1178 spinocalanid specimens of all developmental stages were compared with a reference library of previous proteomic fingerprints of genetically and morphologically identified spinocalanid species. A comparison with the library yielded 25 valid species clusters, which were used to create fine-scale vertical distribution profiles with a high species and stage resolution. Further, we were able to show vertical habitat partitioning among cryptic species and developmental stages, which has major consequences for our understanding of niche partitioning of the mesopelagic realm. Thus, proteomic fingerprinting holds great potential in accelerating and improving community analysis, providing a powerful tool to obtain new insights into biodiversity processes, phylogeographic patterns and mechanisms of vertical distribution.

Authentication of meat is a global concern owing to meat adulteration with pork; however, no study, to date, has established a method to identify species-specific peptide markers for ascertaining adulterated meat. The present study aimed to describe chemometrics-assisted shotgun proteomics, which has recently been reported to establish potential peptide markers for non-Halal pork among Halal beef and chicken meat. To identify the potential peptide markers through peptide mass fingerprinting (PMF) analysis, chemometric analysis comprised principal component analysis and orthogonal partial least square-discriminant analysis, both of which proved statistically useful. Subsequently, through targeted tandem liquid chromatography-mass spectrometry (LC-MS) analysis, the primary structure of the identified peptide markers was revealed using the de novo identification approach. A decoy, randomised and concatenated database search program comprising MS-Fit and MS-Tag was used for PMF and targeted tandem LC-MS analysis, respectively. Results obtained using both PMF and targeted tandem LC-MS analysis complemented one another, indicating that these methods can yield consistent results to identify peptide markers together with chemometric analysis.

The present review article reports the most innovative methods to detect proteins in historical and archeological samples as well as to characterize proteins used as binders in artworks. Difficulties to ascribe proteins to a certain animal species are often due to post-translational modifications originated by chemical or microbial deterioration during aging. Combining different techniques such as peptide mass fingerprinting and tandem mass spectrometry can solve some of these problems and also allow discrimination between taxonomically related species like sheep and goat. The most studied proteins in bones and textile samples are osteocalcin, collagen and keratin, whereas egg yolk and white proteins, casein and collagen are the most relevant for binders used in old paintings. With the suitable approaches (immune-based methods, DOT-blot, etcaEuro broken vertical bar) it is also possible to obtain in situ characterization or analyze the samples directly in the museum laboratories, with the advantage of avoiding artwork damage and expensive external commitments. Recent cutting-edge strategies allowed detection of proteinaceous infection markers that, for instance, were used to establish the cause of death of old Inca mummies and also proved the presence of Yersinia pestis in old documents dating from the period in 17th century in which the plague ravaged Europe.

β-Xylosidase plays an important role in xylan degradation by relieving the end product inhibition of endo-xylanase caused by xylo-oligosaccharides. β-Xylosidase has a wide range of applications in food, feed, paper and pulp, pharmaceutical industries and in bioconversion of lignocellulosic biomass. Hence, in the present study focused on purification, biochemical characterization and partial sequencing of purified β-xylosidase from xylanolytic strain Aspergillus niger ADH-11. Acetone precipitation followed by GPC using Sephacryl S-200 yielded 20.59-fold purified β-xylosidase with 58.30% recovery. SDS-PAGE analysis of purified β-xylosidase relieved a monomeric subunit with a molecular weight 120.48kDa. Kinetic parameters of purified β-xylosidase viz Km, Vmax, Kcat and catalytic efficiency were assessed. Purified β-xylosidase was additionally active on p-nitrophenyl-β-d-glucopyranoside substrate also. Moreover, peptide mass fingerprinting analysis support our biochemical studies and showed that the purified protein is a novel β-xylosidase with β-glucosidase activity and belongs to the bi-functional GH3 superfamily. Besides, tolerance of purified β-xylosidase towards glucose and xylose was also assessed.

Human parechoviruses (HPeV) are picornaviruses with a highly-ordered RNA genome contained within icosahedrally-symmetric capsids. Ordered RNA structures have recently been shown to interact with capsid proteins VP1 and VP3 and facilitate virus assembly in HPeV1. Using an assay that combines reversible cross-linking, RNA affinity purification and peptide mass fingerprinting (RCAP), we mapped the RNA-interacting regions of the capsid proteins from the whole HPeV1 virion in solution. The intrinsically-disordered N-termini of capsid proteins VP1 and VP3, and unexpectedly, VP0, were identified to interact with RNA. Comparing these results to those obtained using recombinantly-expressed VP0 and VP1 confirmed the virion binding regions, and revealed unique RNA binding regions in the isolated VP0 not previously observed in the crystal structure of HPeV1. We used RNA fluorescence anisotropy to confirm the RNA-binding competency of each of the capsid proteins’ N-termini. These findings suggests that dynamic interactions between the viral RNA and the capsid proteins modulate virus assembly, and suggest a novel role for VP0.

Background The highly pathogenic avian influenza viruses of the H5 subtype, such as the H5N1 viral strains or the novel H5N8 and H5N2 reassortants, are of both veterinary and public health concern worldwide. To combat these viruses, monoclonal antibodies (mAbs) against H5 hemagglutinin (HA) play a significant role. These mAbs are effective diagnostic and therapeutic agents and powerful tools in vaccine development and basic scientific research. The aim of this study was to obtain diagnostically valuable mAbs with broad strain specificity against H5-subtype AIVs. Results We applied the hybridoma method to produce anti-HA mAbs. The cloning and screening procedures resulted in the selection of 7 mouse hybridoma cell lines and their respective antibody clones. Preliminary immunoreactivity studies showed that these newly established mAbs, all of the IgG1 isotype, had high specificity and broad-range activities against the H5 HAs. However, these studies did not allow for a clear distinction among the selected antibodies and mAb-secreting hybridoma clones. To differentiate the analyzed mAbs and determine the exact number of hybridoma clones, peptide mapping of the Fc and Fab fragments was performed using a Matrix-Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF/TOF) mass spectrometer. Detailed analyses of the acquired MS and MS/MS spectra confirmed that the Fc fragments constituted highly conserved species- and isotype-immunoglobulin components, whereas the Fab fragments exhibited considerable variation in the sequences that determine antibody specificity. This approach enabled unambiguous characterization of the selected mAbs according to their peptide composition. As a result, 6 different clones were distinguished. Conclusions Our work provided a unique panel of anti-H5 HA mAbs, which meets the demand for novel, high-specificity analytical tools for use in serologic surveillance. Applications of these mAbs in areas other than diagnostics are also possible. Moreover, we demonstrated for the first time that peptide mapping of antibody fragments with mass spectrometry is an efficient method for the differentiation of antibody clones and relevant antibody-producing cell lines. The method may be successfully used to characterize mAbs at the protein level. Electronic supplementary material The online version of this article (10.1186/s12985-017-0886-2) contains supplementary material, which is available to authorized users.

Rajamannar Thennati,1 Sanjay Kumar Singh,1 Nitin Nage,1 Yena Patel,1 Sandip Kumar Bose,1 Vinod Burade,2 Ranjit Sudhakar Ranbhor1 1Sun Pharmaceutical Industries Limited, 2Sun Pharmaceutical Advanced Research Center, Vadodara, Gujarat, India Introduction: Regulatory agencies recommend a stepwise approach for demonstrating biosimilarity between a proposed biosimilar and reference biological product emphasizing for functional and structural characterization to trace if there is any difference which may impact safety and efficacy. We studied the comparative structural and biological attributes of recombinant human chorionic gonadotropin (rhCG), SB005, with reference product, Ovidrel® and Ovitrelle®. Recombiant hCG was approved in 2000 by the US Food and Drug Administration for the induction of final follicular maturation, early luteinization in infertile women as part of assisted reproductive technology program. It is also indicated for the induction of ovulation and pregnancy in ovulatory infertile patients whose cause of infertility is not due to ovarian failure. Materials and methods: Primary structure was studied by intact mass analysis, peptide fingerprinting, peptide mass fingerprinting and sequence coverage analysis. Higher order structure was studied by circular dichroism, ultraviolet-visible spectroscopy, fluorescence spectroscopy, and disulfide bridge analysis. Different isoforms of reference product and SB005 were identified using capillary isoelectric focusing and capillary zone electrophoresis. Glycosylation was studied by N-glycan mapping using LC-ESI-MS, point of glycosylation, released glycan analysis using ultra performance liquid chromatography and sialic acid analysis. Product related impurities such as oligomer content analysis and oxidized impurities were studied using size exclusion chromatography and reverse phase high performance liquid chromatography, respectively. Biological activity in term of potency of reference product and SB005 was studied by in vivo analysis.Results and Conclusion: In this study we have compared analytical similarity of recombinant rhCG (SB005) produced at Sun Pharmaceuticals with the reference product with respect to its primary, higher order structure, isoforms, charge variants, glycosylation, sialyation pattern, pharmacodynamic and in vivo efficacy. Our studies show that the in house produced rhCG has a high degree of structural and functional similarity with the reference product available in the market. Keywords: human chorionic gonadotropin, rhCG, biosimilarity, characterization, comparability, Ovidrel®, Ovitrelle®&nbsp

For vaccine development, 3D-structure determination, direct fluorescent labelling, and numerous other studies, homogeneous virus preparations of high purity are essential. Working with human rhinoviruses (RVs), members of the picornavirus family and the main cause of generally mild respiratory infections, we noticed that our routine preparations appeared highly pure on analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), exclusively showing the four viral capsid proteins (VPs). However, the preparations turned out to contain substantial amounts of contaminating material when analyzed by orthogonal analytical methods including capillary zone electrophoresis, nano electrospray gas-phase electrophoretic mobility molecular analysis (nES GEMMA), and negative stain transmission electron microscopy (TEM). Because these latter analyses are not routine to many laboratories, the above contaminations might remain unnoticed and skew experimental results. By using human rhinovirus serotype A2 (RV-A2) as example we report monolithic anion-exchange chromatography (AEX) as a last polishing step in the purification and demonstrate that it yields infective, highly pure, virus (RV-A2 in the respective fractions was confirmed by peptide mass fingerprinting) devoid of foreign material as judged by the above criteria.

The green colonial microalga Botryococcus braunii produces large amounts of liquid hydrocarbons that can be converted into transportation fuels. Colony cells are held together by a complex extracellular matrix (ECM) made up of a cross-linked long-chain hydrocarbon network around which liquid hydrocarbons are stored, a retaining wall for holding hydrocarbons within the cross-linked hydrocarbon network, and a polysaccharide fibrillar sheath radiating from the retaining wall and surrounding the entire colony. Analysis of “shells” shed from cell apical regions during cell division and containing the retaining wall and polysaccharide fibers shows association of a single protein where the fibers meet the retaining wall, suggesting involvement of this protein in polysaccharide fiber formation. Here we use peptide mass fingerprinting and bioinformatics to identify this protein called p oly s accharide a ssociated p rotein (PSAP). PSAP does not show similarity to any protein in databases, but contains several Proline-rich domains. Staining studies confirm PSAP as a glycoprotein, and mass spectrometry analysis identified ten N -linked glycosylation sites comprising seven different glycans containing mainly mannose and N -acetylglucosamine. Three of these glycans also contain fucose, with one of these glycans being unusual since it also contains arabinose. Additionally, four hydroxyproline residues have short O -linked glycans of mainly arabinose and galactose, with one also containing a 6-deoxyhexose. PSAP secretion and localization to shell material is confirmed using western blot analysis and microscopy. These studies indicate PSAP contains unique glycans and suggest its involvement in ECM polysaccharide fiber biosynthesis.

Snake venom Kunitz-type proteins are well known to inhibit serine proteases but a few studies have also shown matrix metalloproteases (MMPs) inhibition. In view of the fact that MMPs and snake venom metalloproteases (SVMPs) have similar catalytic site, inhibition of SVMP activity by Kunitz-type proteins remains to be studied. Recent proteomic studies of Naja naja (N. naja) venom revealed the abundance of Kunitz-type proteins. In this regard, present study aimed at purification of a protease inhibitor from N. naja venom that inhibits the toxicity of SVMPs rich Echis carinatus (E. carinatus) venom. N. naja venom effectively inhibited E. carinatus venom-induced hemorrhage. Purification of the active principle responsible for anti-hemorrhagic effect was achieved by fractionation of N. naja venom in three successive chromatographic steps. SDS-PAGE revealed that purified anti-hemorrhagic protein (NNAh) has an apparent molecular mass of ∼44 kDa and single peak in RP-HPLC demonstrated its homogeneity. NNAh also inhibited myonecrosis induced by E. carinatus venom and reduced activity of creatine kinase in NNAh treated animal sera substantiated the anti-myonecrotic effect. Hemorrhage and myonecrosis inhibitory effects of NNAh were further supported by inhibition of E. carinatus venom-mediated gelatinolysis and collagenolysis. NNAh falls into the category of Kunitz-type serine protease inhibitor as determined by peptide mass fingerprinting and shown to be a strong inhibitor of chymotrypsin. Collectively our data signify that NNAh is a Kunitz-type chymotrypsin inhibitor which also inhibited metalloprotease activities of E. carinatus venom. In future, complete sequence of NNAh and peptide region(s) responsible for inhibition will assist to deduce the mechanism of action.

Proteases play an indispensable role in the life cycles of several life-threatening organisms such as the ones causing malaria, cancer and AIDS. A targeted blockade of these enzymes could be an efficient approach for drug modeling against these causative agents. Our study was directed towards the extraction and characterization of a protease inhibitor having activity against Chikungunya virus (CHIKV). A protein-based protease inhibitor (PI) in Streptomyces griseoincarnatus HK12 with anti-viral activity against CHIKV was revealed when screened against two major proteases, papain and trypsin. The PI was efficiently extracted at 60 % ammonium sulfate saturation and purified by ion-exchange chromatography (CM-Sepharose) at 300 mM NaCl elution followed by SDS-PAGE (10 %). The protein was characterized by denaturing SDS-PAGE, reverse zymography, and MALDI-TOF peptide mass fingerprinting. The protein-based PI was studied to have a high molecular weight of 66−70 kDA. The PI was tested to supress the supress cytopathic effects (CPE) exerted by the clinically isolated virus in BHK21 cells. This was used as a measure to determine the antiviral activity. The PI exerted significant effects with an effective concentration calculated as EC50 11.21 μg/mL. The protein was found to be reported as the first of its kind which also stands out to be the first a natural protease inhibitor against the treatment of the chikungunya virus.

Bacteriocins are gaining utmost importance in antimicrobial and chemotherapy due to their diverse structure and activity. This study centres on magainin-2 like bacteriocin with anticancer action, produced by Bacillus safensis strain SDG14 isolated from gut of marine fish Sardinella longiceps. The purified bacteriocin designated as BpSl14 was thermostable and pH tolerant. The molecular weight of BpS114 was estimated to be 6061.2 Da using MALDI-ToF MS. The partial primary sequence was elucidated by peptide mass fingerprinting using MALDI MS/MS. The tertiary structure of the partial sequence was similar to that of two magainin-2 α-helices joined together by extended indolicidin. The BpSl14 protein inhibited the cells of lung carcinoma, one of the deadliest cancers. Docking studies conducted with DR5 and TGF-β, two of the most prominent apoptotic receptors in adenocarcinoma, also proved the anti-apoptotic action of BpSl14.

Natural peptides are emerging as a leading alternative to conventional drugs and antibiotics, owing to their remarkable potency, better stability and less toxicity. Such peptides encompass numerous healing properties such as antimicrobial, anti-inflammatory, immunomodulatory, etc. Though plant- derived peptides have been widely studied for their therapeutic benefits, however, fungal peptides are still lesser explored. Ganoderma lucidum, a highly medicinal oriental mushroom comprises a vast array of phytoconstituents, namely flavonoids, phenolics, terpenoids, polysaccharides, proteins, glycolipids, etc and hence, is being used since several decades in traditional Chinese medicine (TCM) for its various ameliorative effects e.g. anti-inflammatory, antimicrobial, anti-proliferative and antioxidant properties. This study presents the isolation and characterization of antibacterial peptide fractions from fruiting body (GLF) and mycelium (GLM) of Indian G. lucidum. Representative amide bonds were identified in the fractions using established standard techniques. Peptide mass fingerprinting and HPLC confirmed the presence of cationic and hydrophobic amino acids in the peptide fractions which are known to be major structural features of antimicrobial peptides. Secondary structure prediction showed abundance of α-helices and random coils in GLF and GLM fractions respectively. The fractions exhibited appreciable antioxidant potential. Besides, these also possessed substantial antibacterial activity against Escherichia coli and Salmonella typhi wherein it was observed that generation of reactive oxygen species and induction of intracellular protein leakage within the bacterial cells were the possible mechanisms of inhibitory action.