Peptide Matches Research Articles

Rational design of glycosaminoglycan binding cyclic peptides using cPEPmatch

Cyclic peptides present a robust platform for drug design, offering high specificity and stability due to their conformationally constrained structures. In this study, we introduce an updated version of the Cyclic Peptide Matching program (cPEPmatch) tailored for the identification of cyclic peptides capable of mimicking protein-glycosaminoglycan (GAG) binding sites. We focused on engineering cyclic peptides to replicate the GAG-binding affinity of antithrombin III (ATIII), a protein that plays a crucial role in modulating anticoagulation through interaction with the GAG heparin. By integrating computational and experimental methods, we successfully identified a cyclic peptide binder with promising potential for future optimization. MD simulations and MM-GBSA calculations were used to assess binding efficacy, supplemented by umbrella sampling to approximate free energy landscapes. The binding specificity was further validated through NMR and ITC experiments. Our findings demonstrate that the computationally designed cyclic peptides effectively target GAGs, suggesting their potential as novel therapeutic agents. This study advances our understanding of peptide-GAG interactions and lays the groundwork for future development of cyclic peptide-based therapeutics.

Comparative analysis of plasma affinity depletion methods: Impact on protein composition and phosphopeptide abundance in human plasma.

Affinity-based protein depletion and TiO2 enrichment methods play a crucial role in detection of low-abundant proteins and phosphopeptides enrichment, respectively. Here, we assessed the effectiveness of HSA/IgG (HU2) and Human 7 (HU7) depletion methods and their impact on phosphopeptides coverage through comparative proteome analysis, utilizing in-solution digestion and nano-LC-Orbitrap mass spectrometry (MS). Our results demonstrated that both HU2 and HU7 affinity depletion significantly decreased high-abundant proteins by 1.5-7.8-fold (p<0.001). A total of 1491 proteins were identified, with 48 proteins showing significant expression in the depleted groups. Notably, cadherin-13, neutrophil defensin 1, APM1, and desmoplakin variant protein were exclusively detected in the HU2/HU7-depleted groups. Furthermore, study on effect of depletion on phosphopeptides revealed an increase in tandem MS spectral counts with notable decrease (∼50%) in peptide spectrum matching in depleted groups, which was attributed to significant reduction in protein counts. Our post translation modification workflow for phosphoproteomics detected 42 phosphorylated peptides, corresponding to 12 phosphoproteins with unique peptide match ≥2 (high false discovery rates confidence). Among them, 10 phosphorylated proteins are highly expressed in depleted groups. Overall, these findings offer valuable insights in selection of protein depletion methods for comprehensive plasma proteomics analysis.

Characterisation and expression analyses of two putative cerato-platanin proteins isolated from Ganoderma boninense Pat.

Cerato-platanin (CP) proteins are small extracellular proteins that play essential roles in the plant-pathogen interactions of some economically important filamentous fungi. At present, there is a lack of information about the roles of CP proteins secreted by Ganoderma boninense, the causal agent of basal stem rot disease in African oil palm, during the early host-pathogen interaction. This work studies the properties and roles of two CP proteins from G. boninense strain PER71. Using bioassay-guided fractionation and nano-LC-MS/MS analysis, we identified peptide matches for G. sinense ZZ0214-1 CP-like and other elicitor proteins in the culture filtrate of G. boninense strain PER71. We found that GbCP2 and GbCP6, two out of nine CP gene models identified in the fungal genome of G. boninense strain PER71, exhibited potential effector properties. We further validated their effector properties by isolating and characterising their coding regions. Interestingly, GbCP6 showed a significant expression level at 11 days post inoculation, indicating its significance in fungal pathogenesis. On the other hand, GbCP2 did not show differential expression at any sampling point, suggesting a different function or role in the pathogenicity of G. boninense. This study identifies GbCP6 as a potential biomarker for early detection of basal stem rot disease in oil palm.

Prevalence and Homology of the Pneumococcal Serine-Rich Repeat Protein at the Global Scale.

Pneumococcal pneumonia remains a WHO high-priority disease despite multivalent conjugate vaccines administered in clinical practice worldwide. A protein-based, serotype-independent vaccine has long-promised comprehensive coverage of most clinical isolates of the pneumococcus. Along with numerous pneumococcal surface protein immunogens, the pneumococcal serine-rich repeat protein (PsrP) has been investigated as a potential vaccine target due to its surface exposure and functions toward bacterial virulence and lung infection. Three critical criteria for its vaccine potential - the clinical prevalence, serotype distribution, and sequence homology of PsrP - have yet to be well characterized. Here, we used genomes of 13,454 clinically isolated pneumococci from the Global Pneumococcal Sequencing project to investigate PsrP presence among isolates, distribution among serotypes, and interrogate its homology as a protein across species. These isolates represent all age groups, countries worldwide, and types of pneumococcal infection. We found PsrP present in at least 50% of all isolates across all determined serotypes and nontypeable (NT) clinical isolates. Using a combination of peptide matching and HMM profiles built on full-length and individual PsrP domains, we identified novel variants that expand PsrP diversity and prevalence. We also observed sequence variability in its basic region (BR) between isolates and serotypes. PsrP has a strong vaccine potential due to its breadth of coverage, especially in nonvaccine serotypes (NVTs) when exploiting its regions of conservation in vaccine design. IMPORTANCE An updated outlook on PsrP prevalence and serotype distribution sheds new light on the comprehensiveness of a PsrP-based protein vaccine. The protein is present in all vaccine serotypes and highly present in the next wave of potentially disease-causing serotypes not included in the current multivalent conjugate vaccines. Furthermore, PsrP is strongly correlated with clinical isolates harboring pneumococcal disease as opposed to pneumococcal carriage. PsrP is also highly present in strains and serotypes from Africa, where the need for a protein-based vaccine is the greatest, giving new reasoning to pursue PsrP as a protein vaccine.

Cross-Serological Reaction of Glandless Cottonseed Proteins to Peanut and Tree Nut Allergic IgE

Food allergy is a potentially life-threatening health concern caused by immunoglobulin E (IgE) antibodies that mistakenly recognize normally harmless food proteins as threats. Peanuts and tree nuts contain several seed storage proteins that commonly act as allergens. Glandless cottonseed, lacking the toxic compound gossypol, is a new food source. However, the seed storage proteins in cottonseed may act as allergens. To assess this risk, glandless cottonseed protein extracts were evaluated for IgE binding by peanut and tree nut allergic volunteers. ELISA demonstrated that 25% of 32 samples had significant binding to cottonseed extracts. Immunoblot analysis with pooled sera indicated that IgE recognized a pair of bands migrating at approximately 50 kDa. Excision of these bands and subsequent mass-spectrometric analysis demonstrated peptide matches to cotton C72 and GC72 vicilin and legumin A and B proteins. Further, in silico analysis indicated similarity of the cotton vicilin and legumin proteins to peanut vicilin (Ara h 1) and cashew nut legumin (Ana o 2) IgE-binding epitopes among others. The observations suggest both the cotton vicilin and legumin proteins were recognized by the nut allergic IgE, and they should be considered for future allergen risk assessments evaluating glandless cottonseed protein products.

Partial characterization of purified glycoprotein from nutshell of Arachis hypogea L. towards macrophage activation and leishmaniacidal activity.

Arachis hypogea L. protein fraction-2 (AHP-F2) from the Peanut shell was extracted and characterized and its potent immunomodulatory and anti-leishmanial role was determined in this present study. AHP-F2 was found to be a glycoprotein as the presence of carbohydrates were confirmed by the analysis of high-performance liquid chromatography (HPLC) yielded glucose, galactose, mannose, and xylose. AHP-F2 molecular mass was found to be ∼28kDa as indicated in MALDI-TOF and peptide mass fingerprinting analysis followed by Mascot search. The peptide matches revealed the similarity of the mannose/glucose binding lectin with 71.07% in the BLAST analysis. After that, the 3D structure of the AHP-F2 model was designed and validated by the Ramachandran plot. The immunomodulatory role of AHP-F2 was established in murine peritoneal macrophages as induction of nitric oxide (NO), and stimulation of proinflammatory cytokines (IL-12 and IFN-γ) in a dose-dependent manner was observed. Interestingly, it was also found that AHP-F2 has interacted with the innate immune receptor, toll-like receptors (TLRs) as established in molecular docking as well as mRNA expression. The anti-leishmanial potential of AHP-F2 was revealed with a prominent inhibition of amastigote growth within the murine macrophages with prompt induction of nitrite release. Altogether, the isolated AHP-F2 from Arachis hypogea L. has strong immunomodulatory and anti-leishmanial potential which may disclose a new path to treat leishmaniasis.

Improving Peptide-Level Mass Spectrometry Analysis via Double Competition.

The analysis of shotgun proteomics data often involves generating lists of inferred peptide-spectrum matches (PSMs) and/or of peptides. The canonical approach for generating these discovery lists is by controlling the false discovery rate (FDR), most commonly through target-decoy competition (TDC). At the PSM level, TDC is implemented by competing each spectrum's best-scoring target (real) peptide match with its best match against a decoy database. This PSM-level procedure can be adapted to the peptide level by selecting the top-scoring PSM per peptide prior to FDR estimation. Here, we first highlight and empirically augment a little known previous work by He et al., which showed that TDC-based PSM-level FDR estimates can be liberally biased. We thus propose that researchers instead focus on peptide-level analysis. We then investigate three ways to carry out peptide-level TDC and show that the most common method ("PSM-only") offers the lowest statistical power in practice. An alternative approach that carries out a double competition, first at the PSM and then at the peptide level ("PSM-and-peptide"), is the most powerful method, yielding an average increase of 17% more discovered peptides at 1% FDR threshold relative to the PSM-only method.

The Role of Testosterone in Spermatogenesis: Lessons From Proteome Profiling of Human Spermatozoa in Testosterone Deficiency.

Testosterone is essential to maintain qualitative spermatogenesis. Nonetheless, no studies have been yet performed in humans to analyze the testosterone-mediated expression of sperm proteins and their importance in reproduction. Thus, this study aimed to identify sperm protein alterations in male hypogonadism using proteomic profiling. We have performed a comparative proteomic analysis comparing sperm from fertile controls (a pool of 5 normogonadic normozoospermic fertile men) versus sperm from patients with secondary hypogonadism (a pool of 5 oligozoospermic hypogonadic patients due to isolated LH deficiency). Sperm protein composition was analyzed, after peptide labelling with Isobaric Tags, via liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) on an LTQ Velos-Orbitrap mass spectrometer. LC-MS/MS data were analyzed using Proteome Discoverer. Criteria used to accept protein identification included a false discovery rate (FDR) of 1% and at least 1 peptide match per protein. Up to 986 proteins were identified and, of those, 43 proteins were differentially expressed: 32 proteins were under-expressed and 11 were over-expressed in the pool of hypogonadic patients compared to the controls. Bioinformatic analyses were performed using UniProt Knowledgebase, and the Gene Ontology Consortium database based on PANTHER. Notably, 13 of these 43 differentially expressed proteins have been previously reported to be related to sperm function and spermatogenesis. Western blot analyses for A-Kinase Anchoring Protein 3 (AKAP3) and the Prolactin Inducible Protein (PIP) were used to confirm the proteomics data. In summary, a high-resolution mass spectrometry-based proteomic approach was used for the first time to describe alterations of the sperm proteome in secondary male hypogonadism. Some of the differential sperm proteins described in this study, which include Prosaposin, SMOC-1, SERPINA5, SPANXB1, GSG1, ELSPBP1, fibronectin, 5-oxoprolinase, AKAP3, AKAP4, HYDIN, ROPN1B, ß-Microseminoprotein and Protein S100-A8, could represent new targets for the design of infertility treatments due to androgen deficiency.

Glyco-Decipher enables glycan database-independent peptide matching and in-depth characterization of site-specific N-glycosylation

Glycopeptides with unusual glycans or poor peptide backbone fragmentation in tandem mass spectrometry are unaccounted for in typical site-specific glycoproteomics analysis and thus remain unidentified. Here, we develop a glycoproteomics tool, Glyco-Decipher, to address these issues. Glyco-Decipher conducts glycan database-independent peptide matching and exploits the fragmentation pattern of shared peptide backbones in glycopeptides to improve the spectrum interpretation. We benchmark Glyco-Decipher on several large-scale datasets, demonstrating that it identifies more peptide-spectrum matches than Byonic, MSFragger-Glyco, StrucGP and pGlyco 3.0, with a 33.5%-178.5% increase in the number of identified glycopeptide spectra. The database-independent and unbiased profiling of attached glycans enables the discovery of 164 modified glycans in mouse tissues, including glycans with chemical or biological modifications. By enabling in-depth characterization of site-specific protein glycosylation, Glyco-Decipher is a promising tool for advancing glycoproteomics analysis in biological research.

Abstract CC01-02: Novel algorithm discovers up to 35% more epitopes translated from non-coding regions in cold tumours

Abstract Currently, most neoantigen pipelines often focus on the detection of neoantigens derived from mutations in the coding regions of the genome. However, in some cancer indications, the number of mutations detectable in tumours can be very low (low tumour mutational burden). This limits the number of actionable neoantigens and results in so-called “cold” tumours. In these cases, non-canonical neoantigens resulting from alterations in non-coding regions of the human genome could represent a high potential alternative for treatment. Indeed, recent research has revealed that previously presumed non-coding regions of the human genome, such as long non-coding RNAs (lncRNAs), can contain translatable small open reading frames (smORFs) generating micropeptides. Some of these micropeptides have already been shown to be involved in cancer development, but these small peptides could also represent a high potential source of non-canonical neoantigens for personalised therapy. Here, we present smORFin, a machine learning algorithm specifically trained to identify smORFs in transcripts and to assess their coding potential. While most tools are focused on longer sequences, smORFin is specifically developed to target small ORFs (&amp;lt;303 nucleotides). Furthermore, smORFin also accounts for smORFs with alternative initiation codons, thereby improving its sensitivity for the detection of novel unannotated smORFs. As a result, the smORFin model reaches a precision of 0.98 and an accuracy of 0.95 on its testing dataset. Using this new prediction tool, a library of human smORFs was assembled, the so-called smORFeome. This library of smORFs, and their associated proteins, was evaluated as a reference for spectrum to peptide matching in mass spectrometry data (MS) analysis. Indeed, the evaluation of seven MS samples revealed and validated the presence of smORFeome-related micropeptides and HLA-Iassociated epitopes originating from smORFs. The impact of mutations in allegedly non-coding regions of tumour genomes and its influence on the neoantigen repertoire, was evaluated through integration of smORFin in a neoantigen identification pipeline targeting lncRNA-derived mutated epitopes; lncRNeos. It was observed that these epitopes only represent a minor fraction of the total neoantigen load. Strikingly, when only focusing on tumours with a low neoantigen load, lncRNeos represented up to 27% of the total neoantigen load. This indicates that for tumours with a low TMB, and therefore with a low neoantigen load, lncRNeos allows to significantly expand the neoantigen repertoire. Biological in vivo/in vitro validation remains necessary to assess the existence, presentation, and actionability of lncRNeos. Citation Format: Lena Pfitzer, Lore Van Oudenhove, Cedric Bogaert, Bruno Fant. Novel algorithm discovers up to 35% more epitopes translated from non-coding regions in cold tumours [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr CC01-02.

Altitude acclimatization via hypoxia-mediated oxidative eustress involves interplay of protein nitrosylation and carbonylation: A redoxomics perspective

AimHypoxia is an important feature of multiple diseases like cancer and obesity and also an environmental stressor to high altitude travelers. Emerging research suggests the importance of redox signaling in physiological responses transforming the notion of oxidative stress into eustress and distress. However, the behavior of redox protein post-translational modifications (PTMs), and their correlation with stress acclimatization in humans remains sketchy. Scant information exists about modifications in redoxome during physiological exposure to environmental hypoxia. In this study, we investigated redox PTMs, nitrosylation and carbonylation, in context of extended environmental hypoxia exposure. MethodsThe volunteers were confirmed to be free of any medical conditions and matched for age and weight. The human global redoxome and the affected networks were investigated using TMT-labeled quantitative proteo-bioinformatics and biochemical assays. The percolator PSM algorithm was used for peptide-spectrum match (PSM) validation in database searches. The FDR for peptide matches was set to 0.01. 1-way ANOVA and Tukey's Multiple Comparison test were used for biochemical assays. p-value<0.05 was considered statistically significant. Three independent experiments (biological replicates) were performed. Results were presented as Mean ± standard error of mean (SEM). Key findingsThis investigation revealed direct and indirect interplay between nitrosylation and carbonylation especially within coagulation and inflammation networks; interlinked redox signaling (via nitrosylation‑carbonylation); and novel nitrosylation and carbonylation sites in individual proteins. SignificanceThis study elucidates the role of redox PTMs in hypoxia signaling favoring tolerance and survival. Also, we demonstrated direct and indirect interplay between nitrosylation and carbonylation is crucial to extended hypoxia tolerance.

The study of stress conditions on growth and proteome of Raoultella planticola: a new emerging pathogen.

All bacteria can survive and adapt to different stresses, such as fluctuations in temperature, pH oxidative, and osmotic pressure occurring in their surrounding environments. This study aims to evaluate the effects of a variety of stress conditions on the growth, and proteome ofRaoultella planticolaPTCC 1598.R. planticolacells were exposed to different values of temperatures, sodium chloride, pH, and hydrogen peroxide stresses. Among the stress conditions, oxidative stress, upon exposure to hydrogen peroxide (H2O2) at 4000ppm concentration was selected for proteomics analysis in detail. Approximately, 1400 spots were identified in two-dimensional gel electrophoresis (2-DE). Among the identified spots, 85 spots were repeatable using 2D-Platinum software and eye confirmation and, nine protein spots were differentially expressed. Among nine proteins, six proteins identified successfully with an MASCOT score greater than 40 (p < 0.05) were 2,3-dihydroxybenzoate-2,3-dehydrogenase (oxidoreductase family), hypothetical protein G787-04832, periplasmic D-galactose-binding protein, uridine phosphorylase (glycosyltransferases), a single peptide match to cysteine-binding periplasmic protein, and NADP(H) nitroreductase. All identified proteins showed decreased level expression. Based on the obtained results, we concluded that hydrogen peroxide as an antiseptic compound could affect cell growth and proteomics ofR. planticola. Therefore, we recommend using an antiseptic solution containing H2O2 to prevent the spread ofR. planticola as a newemerging pathogen.

Proteomic analysis of Red Sea Conus taeniatus venom reveals potential biological applications

Background:Diverse and unique bioactive neurotoxins known as conopeptides or conotoxins are produced by venomous marine cone snails. Currently, these small and stable molecules are of great importance as research tools and platforms for discovering new drugs and therapeutics. Therefore, the characterization of Conus venom is of great significance, especially for poorly studied species. Methods:In this study, we used bioanalytical techniques to determine the venom profile and emphasize the functional composition of conopeptides in Conus taeniatus, a neglected worm-hunting cone snail. Results:The proteomic analysis revealed that 84.0% of the venom proteins were between 500 and 4,000 Da, and 16.0% were > 4,000 Da. In C. taeniatus venom, 234 peptide fragments were identified and classified as conotoxin precursors or non-conotoxin proteins. In this process, 153 conotoxin precursors were identified and matched to 23 conotoxin precursors and hormone superfamilies. Notably, the four conotoxin superfamilies T (22.87%), O1 (17.65%), M (13.1%) and O2 (9.8%) were the most abundant peptides in C. taeniatus venom, accounting for 63.40% of the total conotoxin diversity. On the other hand, 48 non-conotoxin proteins were identified in the venom of C. taeniatus. Moreover, several possibly biologically active peptide matches were identified, and putative applications of the peptides were assigned. Conclusion: Our study showed that the composition of the C. taeniatus-derived proteome is comparable to that of other Conus species and contains an effective mix of toxins, ionic channel inhibitors and antimicrobials. Additionally, it provides a guidepost for identifying novel conopeptides from the venom of C. taeniatus and discovering conopeptides of potential pharmaceutical importance.

Identification of immunoreactive proteins in secretions of Leishmania infantum promastigotes: an immunoproteomic approach.

In the Mediterranean region, Leishmania infantum is the main cause of visceral leishmaniasis. Dogs with canine visceral leishmaniasis are an important reservoir of visceral leishmaniasis. Control of canine visceral leishmaniasis could disrupt transmission of visceral leishmaniasis to humans. The secreted antigens of Leishmania promastigotes are potential stimuli of the host immune system. Proteomic techniques facilitate the identification of new protein markers. This study aimed to identify immunoreactive proteins in the secretions of L. infantum promastigotes which could be possible targets for the diagnosis and treatment of canine visceral leishmaniasis and the development of vaccines against the disease. Secretions of L. infantum promastigotes were obtained from the cultivation of 6 × 109 promastigotes in serum- free RPMI-1640 medium during a period of 72 h. After deionization and lyophilization, two-dimensional gel electrophoresis was used for protein separation followed by Western blotting. Thirteen common and repeatable immunoreactive spots were analysed by mass spectrometry. Nine proteins were identified by spectrometry. Most of these proteins were involved in metabolism pathways, survival and pathogenicity of Leishmania parasites. Phospholipase C, immune inhibitor A, chitin-binding protein and a single peptide match to chain A crystal structure of selenomethionine were observed in the secretions of L. infantum promastigotes. The proteins identified in metabolism pathways, survival and pathogenicity of Leishmania parasites are possible targets that could be used for the diagnosis and treatment of canine visceral leishmaniasis and the development of vaccines against the disease in the future.

Potential Cross-Reactive Immunity to SARS-CoV-2 From Common Human Pathogens and Vaccines.

The recently emerged SARS-CoV-2 causing the ongoing COVID-19 pandemic is particularly virulent in the elderly while children are largely spared. Here, we explored the potential role of cross-reactive immunity acquired from pediatric vaccinations and exposure to common human pathogens in the protection and pathology of COVID-19. To that end, we sought for peptide matches to SARS-CoV-2 (identity ≥ 80%, in at least eight residues) in the proteomes of 25 human pathogens and in vaccine antigens, and subsequently predicted their T and B cell reactivity to identify potential cross-reactive epitopes. We found that viruses subject to pediatric vaccinations do not contain cross-reactive epitopes with SARS-CoV-2, precluding that they can provide any general protection against COVID-19. Likewise, common viruses including rhinovirus, respiratory syncytial virus, influenza virus, and several herpesviruses are also poor or null sources of cross-reactive immunity to SARS-CoV-2, discarding that immunological memory against these viruses can have any general protective or pathological role in COVID-19. In contrast, we found combination vaccines for treating diphtheria, tetanus, and pertussis infectious diseases (DTP vaccine) to be significant sources of potential cross-reactive immunity to SARS-CoV-2. DTP cross-reactive epitopes with SARS-CoV-2 include numerous CD8 and CD4 T cell epitopes with broad population protection coverage and potentially neutralizing B cell epitopes in SARS-CoV-2 Spike protein. Worldwide, children receive several DTP vaccinations, including three-four doses the first year of life and one at 4–6 years of age. Moreover, a low antigenic Tdap dose is also given at ages 9–14. Thereby, children may well be protected from SARS-CoV-2 through cross-reactive immunity elicited by DTP vaccinations, supporting testing in the general population to prevent COVID-19.

Hydroxyproline determination for initial detection of halal-critical food ingredients (gelatin and collagen)

Gelatin and collagen are considered halal-critical ingredients as they are typically derived from either bovine or porcine animals. Current analytical methods for determining the sources of gelatin and collagen suffer from limitations in terms of robustness and false positives in peptide matching. Thus, the aim of this study was to investigate the utility of monitoring hydroxyproline, a signature amino acid for gelatin and collagen, for identifying potentially haram foodstuffs. To determine the hydroxyproline profiles among animal- and plant-based samples, one-way univariate analysis of variance followed by pair-wise comparison was used to establish statistical significance. Multivariate chemometric analysis through principal component analysis revealed a discrete distribution pattern among 59 samples due to hydroxyproline variability. Finally, inter- and intra-laboratory comparisons demonstrated the validity and robustness of hydroxyproline determination according to ISO 17025. Thus, this preliminary identification technique will aid the identification of potentially haram foodstuffs.

A Pangenome Approach for Discerning Species-Unique Gene Markers for Identifications of Streptococcus pneumoniae and Streptococcus pseudopneumoniae.

Correct identifications of isolates and strains of the Mitis-Group of the genus Streptococcus are particularly difficult, due to high genetic similarity, resulting from horizontal gene transfer and homologous recombination, and unreliable phenotypic and genotypic biomarkers for differentiating the species. Streptococcus pneumoniae and Streptococcus pseudopneumoniae are the most closely related species of the clade. In this study, publicly-available genome sequences for Streptococcus pneumoniae and S. pseudopneumoniae were analyzed, using a pangenomic approach, to find candidates for species-unique gene markers; ten species-unique genes for S. pneumoniae and nine for S. pseudopneumoniae were identified. These species-unique gene marker candidates were verified by PCR assays for identifying S. pneumoniae and S. pseudopneumoniae strains isolated from clinical samples. All determined species-level unique gene markers for S. pneumoniae were detected in all S. pneumoniae clinical isolates, whereas fewer of the unique S. pseudopneumoniae gene markers were present in more than 95% of the clinical isolates. In parallel, taxonomic identifications of the clinical isolates were confirmed, using conventional optochin sensitivity testing, targeted PCR-detection for the “Xisco” gene, as well as genomic ANIb similarity analyses for the genome sequences of selected strains. Using mass spectrometry-proteomics, species-specific peptide matches were observed for four of the S. pneumoniae gene markers and for three of the S. pseudopneumoniae gene markers. Application of multiple species-level unique biomarkers of S. pneumoniae and S. pseudopneumoniae, is proposed as a protocol for the routine clinical laboratory for improved, reliable differentiation, and identification of these pathogenic and commensal species.

Antimicrobial peptide (AMP) from Zingiber zerumbet rhizomes with inhibitory effect on Pythium myriotylum secretory proteases and zoospore viability

Protease mediated proteolysis has been widely implicated in virulence of necrotrophic fungal pathogens. This is counteracted in plants by evolving new and effective antimicrobial peptides (AMP) that constitute important components of innate immune system. Peptide extraction from rhizome of Zingiber zerumbet was optimized using ammonium sulphate (50-80% w/v) and acetone (60 and 100% v/v) with maximal protein recovery of 1.2 ± 0.4mg/g obtained using 100% acetone. Evaluation of inhibitory potential of Z. zerumbet rhizome protein extract to prominent hydrolases of necrotrophic Pythium myriotylum revealed maximal inhibition of proteases (75.8%) compared to other hydrolytic enzymes. Protein was purified by Sephacryl S200HR resin resulting in twofold purification and protease inhibition of 84.4%. Non-reducing polyacrylamide gel electrophoresis (PAGE) of the fractions yielded two bands of 75kDa and 25kDa molecular size. Peptide mass fingerprint of the protein bands using matrix assisted laser desorption/ionization (MALDI)-time of flight (TOF) mass spectroscopy (MS) and subsequent MASCOT searches revealed peptide match to methylesterase from Arabidopsis thaliana (15%) and to hypothetical protein from Oryza sativa (98%) respectively. Further centrifugal filter purification using Amicon Ultra (10,000MW cut-off) filter, yielded a prominent band of 25kDa size. Concentration dependent inhibition of zoospore viability by Z. zerumbet AMP designated as ZzAMP was observed with maximal inhibition of 89.5% at 4µg protein and an IC50 value of 0.59µg. Studies are of particular relevance in the context of identifying the molecules involved in imparting below ground defense in Z. zerumbet as well in development of AMPs as potential candidate molecules for control of necrotrophic pathogens of agricultural relevance.

Oligopeptides for Immunotherapy Approaches in Ovarian Cancer Treatment.

Anti-ovarian cancer vaccines based on minimal immune determinants uniquely expressed in ovarian cancer biomarkers appear to promise a high level of sensitivity and specificity for ovarian cancer immunodiagnostics, immunoprevention, and immunotherapy. Using the Pir Peptide Match program, three ovarian cancer biomarkers - namely, sperm surface protein Sp17, WAP four-disulfide core domain protein 2, and müllerian-inhibiting substance - were searched for unique peptide segments not shared with other human proteins. Then, the unique peptide segments were assembled to define oligopeptides potentially usable as synthetic ovarian cancer antigens. This study describes a methodology for constructing ovarian cancer biomarkerderived oligopeptide constructs that might induce powerful, specific, and non-crossreactive immune responses against ovarian cancer.

Genome annotation improvements from cross-phyla proteogenomics and time-of-day differences in malaria mosquito proteins using untargeted quantitative proteomics.

The malaria mosquito, Anopheles stephensi, and other mosquitoes modulate their biology to match the time-of-day. In the present work, we used a non-hypothesis driven approach (untargeted proteomics) to identify proteins in mosquito tissue, and then quantified the relative abundance of the identified proteins from An. stephensi bodies. Using these quantified protein levels, we then analyzed the data for proteins that were only detectable at certain times-of-the day, highlighting the need to consider time-of-day in experimental design. Further, we extended our time-of-day analysis to look for proteins which cycle in a rhythmic 24-hour (“circadian”) manner, identifying 31 rhythmic proteins. Finally, to maximize the utility of our data, we performed a proteogenomic analysis to improve the genome annotation of An. stephensi. We compare peptides that were detected using mass spectrometry but are ‘missing’ from the An. stephensi predicted proteome, to reference proteomes from 38 other primarily human disease vector species. We found 239 such peptide matches and reveal that genome annotation can be improved using proteogenomic analysis from taxonomically diverse reference proteomes. Examination of ‘missing’ peptides revealed reading frame errors, errors in gene-calling, overlapping gene models, and suspected gaps in the genome assembly.