Sequencing Of Tryptic Peptides Research Articles

Biochemical and hemostatic description of a thrombin-like enzyme TLBro from Bothrops roedingeri snake venom

Objective: The current study’s objective is to characterize a new throm-bin-like enzyme called TLBro that was obtained from Bothrops roedingeris snake from a biochemical and hemostatic perspective.Methodology: One chromatographic step was used to purify it, producing the serine protease TLBro. Molecular mass was estimated by SDS-PAGE to be between reduced and unreduced by 35 kDa. Tryptic peptide sequencing using Swiss Prot provided the complete amino acid sequence. Expasy.org by conducting a search that is limited to Crotalinae snake serine proteases and displaying a high degree of amino acid sequence.Results: Ser (182) is inhibited by phenylmethylsulfonyl fluoride (PMSF), and TLBro demonstrated the presence of Asp (88) residues. It also deduced the positions of His (43) and Ser (182) in the set of three coordinated amino acids in serine proteases. It was discovered that this substrate had high specificity for BANA, Michaelis-Menten behavior with KM 0 point85 mM and Vmax 1 point89 nmoles -NA/L/min, and high stability between temperatures (15 to 70°C) and pHs (2 point0 to 10 point0). According to doses and incubation times, TLBro degraded fibrin preferentially on the B-chain; additionally, its activities were significantly diminished after preincubation with divalent ions (Zn2 and Cd2). When incubated with PMSF, a particular serine protease inhibitor, enzymatic activities and platelet aggregation were inhibited.Conclusion: The findings revealed distinct structural and functional differences between the serine proteases, adding to the information and assisting in the improvement of the structure-function relationship.

Alzheimer's disease amyloid-β pathology in the lens of the eye

Neuropathological hallmarks of Alzheimer's disease (AD) include pathogenic accumulation of amyloid-β (Aβ) peptides and age-dependent formation of amyloid plaques in the brain. AD-associated Aβ neuropathology begins decades before onset of cognitive symptoms and slowly progresses over the course of the disease. We previously reported discovery of Aβ deposition, β-amyloidopathy, and co-localizing supranuclear cataracts (SNC) in lenses from people with AD, but not other neurodegenerative disorders or normal aging. We confirmed AD-associated Aβ molecular pathology in the lens by immunohistopathology, amyloid histochemistry, immunoblot analysis, epitope mapping, immunogold electron microscopy, quantitative immunoassays, and tryptic digest mass spectrometry peptide sequencing. Ultrastructural analysis revealed that AD-associated Aβ deposits in AD lenses localize as electron-dense microaggregates in the cytoplasm of supranuclear (deep cortex) fiber cells. These Aβ microaggregates also contain αB-crystallin and scatter light, thus linking Aβ pathology and SNC phenotype expression in the lenses of people with AD. Subsequent research identified Aβ lens pathology as the molecular origin of the distinctive cataracts associated with Down syndrome (DS, trisomy 21), a chromosomal disorder invariantly associated with early-onset Aβ accumulation and Aβ amyloidopathy in the brain. Investigation of 1249 participants in the Framingham Eye Study found that AD-associated quantitative traits in brain and lens are co-heritable. Moreover, AD-associated lens traits preceded MRI brain traits and cognitive deficits by a decade or more and predicted future AD. A genome-wide association study of bivariate outcomes in the same subjects identified a new AD risk factor locus in the CTNND2 gene encoding δ-catenin, a protein that modulates Aβ production in brain and lens. Here we report identification of AD-related human Aβ (hAβ) lens pathology and age-dependent SNC phenotype expression in the Tg2576 transgenic mouse model of AD. Tg2576 mice express Swedish mutant human amyloid precursor protein (APP-Swe), accumulate hAβ peptides and amyloid pathology in the brain, and exhibit cognitive deficits that slowly progress with increasing age. We found that Tg2576 trangenic (Tg+) mice, but not non-transgenic (Tg–) control mice, also express human APP, accumulate hAβ peptides, and develop hAβ molecular and ultrastructural pathologies in the lens. Tg2576 Tg+ mice exhibit age-dependent Aβ supranuclear lens opacification that recapitulates lens pathology and SNC phenotype expression in human AD. In addition, we detected hAβ in conditioned medium from lens explant cultures prepared from Tg+ mice, but not Tg– control mice, a finding consistent with constitutive hAβ generation in the lens. In vitro studies showed that hAβ promoted mouse lens protein aggregation detected by quasi-elastic light scattering (QLS) spectroscopy. These results support mechanistic (genotype-phenotype) linkage between Aβ pathology and AD-related phenotypes in lens and brain. Collectively, our findings identify Aβ pathology as the shared molecular etiology of two age-dependent AD-related cataracts associated with two human diseases (AD, DS) and homologous murine cataracts in the Tg2576 transgenic mouse model of AD. These results represent the first evidence of AD-related Aβ pathology outside the brain and point to lens Aβ as an optically-accessible AD biomarker for early detection and longitudinal monitoring of this devastating neurodegenerative disease.

Sesquiterpene Cyclases from the Basidiomycete Cerrena unicolor

Hundreds of terpenoids have been isolated from Basidiomycota, among them are volatile mono- and sesquiterpenes with amazing sensory qualities, representing a promising alternative to essential oils from endangered plant species. Sesquiterpene synthases (STS) appear to be an abundant class of enzymes in these fungi. The basidiomycete Cerrena unicolor, a known sesquiterpene producer, was in silico screened for sesquiterpene cyclases via homology Basic Local Alignment Search Tool searches. Cyclase genes identified were cloned and heterologously expressed in Escherichia coli Bl21 using pCOLD I as the expression vector. Ten cyclases were successfully produced and purified, and their identity was confirmed using amino acid sequencing of tryptic peptides by nano-liquid chromatography-high resolution-electrospray ionization-tandem mass spectrometry. Gas chromatography/mass spectrometry analysis was applied to characterize these cyclases according to the formation of sesquiterpene hydrocarbons and oxidized terpenoids. Bioinformatic characterization and phylogenetic determination allowed for the classification of these diverse fungal enzymes. A representative single and a multi-product STS, respectively, were further analyzed for their dependency from divalent metal cations as a cofactor for the catalytic activity.

Molecular and biochemical analyses of a novel lectin with MATH domains from Brassica oleracea

A novel Brassica oleracea lectin with two tandem MATH domains in its structure has been characterized. The expression of the lectin is regulated by light and by stimuli related to plant defence response. The coding sequence of the protein has been cloned and a fully functional recombinant protein has been obtained. BOL (Brassica oleracea Lectin) is a novel lectin isolated from Brassica oleracea ssp. Botrytis, composed of a single subunit, non-glycosylated peptide that contains two conserved MATH domains, not previously found in any other characterized lectin. The complete sequence encoding BOL was obtained by PCR using degenerated primers designed according to the N-terminal amino acid sequence and the sequences of various tryptic peptides obtained by the digestion of the purified native protein. The entire cDNA consists of 1053 bp, which encodes a 301-amino acid protein. In silico analysis of the Bol promoter sequence showed several regulatory motifs that could be involved in responses to various stresses, as well as various light regulatory elements. Determination of Bol expression in plant tissues showed higher transcript levels in the leaves than in the stems and the roots. The expression of Bol was regulated by light and by signalling compounds related to stress responses. Specifically, Bol expression was higher in light than in darkness, and was induced by methyl jasmonate, while it decreased in tissues treated with salicylic acid. The full-length BOL coding sequence was cloned, expressed in bacteria, and activity assays were performed to ascertain that the recombinant protein was correctly folded and was able to recognize and bind carbohydrates as the native lectin did. Three-dimensional structure prediction showed that this lectin had a dome-like structure with a closely related topology to l-lectins but with two characteristic MATH domains. Isolation, cloning and recombinant expression of BOL may be helpful to understand its physiological function in the plant and opens the possibility of subsequent investigations about the structural features of this novel lectin.

Cross-linking of the DNA repair protein O6-alkylguanine DNA alkyltransferase to DNA in the presence of cisplatin

1,1,2,2-cis-diamminedichloroplatinum (II) (cisplatin) is a chemotherapeutic agent widely used in the clinic to treat various cancers. The antitumor activity of cisplatin is generally attributed to its ability to form intrastrand and interstrand DNA-DNA cross-links via sequential platination of two nucleophilic sites within the DNA duplex. However, cisplatin also induces DNA- protein lesions (DPCs) that may contribute to its biological effects due to their ability to block DNA replication and transcription. We previously reported that over 250 nuclear proteins including high mobility group proteins, histone proteins, and elongation factors formed DPCs in human HT1080 cells treated with cisplatin (Ming et al. Chem. Res. Toxicol. 2017, 30, 980–995). Interestingly, cisplatin-induced DNA-protein conjugates were reversed upon heating, by an unknown mechanism. In the present work, DNA repair protein O6-alkylguanine DNA alkyltransferase (AGT) was used as a model to investigate the molecular details of cisplatin-mediated DNA-protein cross-linking and to establish the mechanism of their reversal. We found that AGT is readily cross-linked to DNA in the presence of cisplatin. HPLC-ESI+-MS/MS sequencing of tryptic peptides originating from dG-Pt-AGT complexes revealed that the cross-linking occurred at six sites within this protein including Glu110, Lys125, Cys145, His146, Arg147, and Cys150. Cisplatin-induced Lys-Gua cross-links (1,1-cis-diammine-2-(5-amino-5-carboxypentyl)amino-2-(2'-deoxyguanosine-7-yl)-platinum(II) (dG-Pt-Lys) were detected by HPLC-ESI+-MS/MS of total digests of modified protein in comparison with the corresponding authentic standard. Upon heating, dG-Pt-AGT complexes were subject to platination migration from protein to DNA, forming cis-[Pt(NH3)2{d(GpG)}] cross-links which were detected by HPLC-ESI+-MS/MS. Our results provide a new insight into the mechanism of cisplatin-mediated DNA-protein cross-linking and their dynamic equilibrium with the corresponding DNA-DNA lesions.

Isolation and characterization of cytotoxic and insulin-releasing components from the venom of the black-necked spitting cobra Naja nigricollis (Elapidae).

Four peptides with cytotoxic activity against BRIN-BD11 rat clonal β-cells were purified from the venom of the black-necked spitting cobra Naja nigricollis using reversed-phase HPLC. The peptides were identified as members of the three-finger superfamily of snake toxins by ESI-MS/MS sequencing of tryptic peptides. The most potent peptide (cytotoxin-1N) showed strong cytotoxic activity against three human tumor-derived cell lines (LC50 = 0.8 ± 0.2 μM for A549 non-small cell lung adenocarcinoma cells; LC50 = 7 ± 1 μM for MDA-MB-231 breast adenocarcinoma cells; and LC50 = 9 ± 1 μM for HT-29 colorectal adenocarcinoma cells). However, all the peptides were to varying degrees cytotoxic against HUVEC human umbilical vein endothelial cells (LC50 in the range 2–22 μM) and cytotoxin-2N was moderately hemolytic (LC50 = 45 ± 3 μM against mouse erythrocytes). The lack of differential activity against cells derived from non-neoplastic tissue limits their potential for development into anti-cancer agents. In addition, two proteins in the venom, identified as isoforms of phospholipase A2, effectively stimulated insulin release from BRIN-BD11 cells (an approximately 6-fold increase in rate compared with 5.6 mM glucose alone) at a concentration (1 μM) that was not cytotoxic to the cells suggesting possible application in therapy for Type 2 diabetes.

Antifreeze protein complements cryoprotective dehydration in the freeze-avoiding springtail Megaphorura arctica

The springtail, Megaphorura arctica, is freeze-avoiding and survives sub-zero temperatures by cryoprotective dehydration. At the onset of dehydration there is some supercooling of body fluids, and the danger of inoculative freezing, which would be lethal. To see if the springtails are protected by antifreeze proteins in this pre-equilibrium phase, we examined extracts from cold-acclimated M. arctica and recorded over 3 °C of freezing point depression. Proteins responsible for this antifreeze activity were isolated by ice affinity. They comprise isoforms ranging from 6.5 to 16.9 kDa, with an amino acid composition dominated by glycine (>35 mol%). Tryptic peptide sequences were used to identify the mRNA sequence coding for the smallest isoform. This antifreeze protein sequence has high similarity to one characterized in Hypogastrura harveyi, from a different springtail order. If these two antifreeze proteins are true homologs, we suggest their origin dates back to the Permian glaciations some 300 million years ago.

Analysis of glycoside hydrolases from oat (Avena sativa) seedling extract.

The abundance and the diversity of oligo- and polysaccharides provide a wide range of biological roles attributed either to these carbohydrates or to their relevant enzymes, i.e., the glycoside hydrolases (GHs). The biocatalysis by these families of enzymes is highly attractive for the generation of products used in potential applications, e.g., pharmaceuticals and food industries. It is thus very important to extract and characterize such enzymes, particularly from plant tissues. In this study, we characterized novel sequences of class I chitinases from seedlings extract of the common oat (Avena sativa L.) using proteomics and sequence-structure-function analysis. These enzymes, which belong to the GH19 family of protein, were extracted from oat and identified using SDS-PAGE, trypsin digestion, LC-MS-MS, and sequence-structure-function analysis. The amino acid sequences of the oat tryptic peptides were used to identify cDNAs from the Avena sativa databases of the expressed sequence tags (ESTs) and transcriptome shotgun assembly (TSA). Based upon the Avena sativa sequences of ESTs and TSA, at least 4 predicted genes that encoded oat class I chitinases were identified and reported. The structural characterization of the oat sequences of chitinases provided valuable insights to the context.

Abstract 603: Differential Proteoform Regulation in Cardiac Aging

Background: The aging heart is prone to diseases and injuries, and is associated with changes in gene expression and alternative splicing. The goal of our study is to identify changes in protein isoforms in cardiac aging and explore their roles in age-related processes. Methods: Left ventricles (LV) from young (12 weeks) and old (78 weeks) C57BL/6J mice (male and female) were collected and stored at -80 °C until use. LV proteins were extracted, reduced, and alkylated. Proteins were digested with trypsin or chymotrypsin and the tryptic or chymotryptic peptides were desalted using C18 spin columns followed by injection into an Easy-nLC 1200 liquid chromatography system coupled to a high-resolution Q-Exactive HF Hybrid Quadrupole-Orbitrap mass spectrometer for LC-MS/MS analysis. To quantify isoform expression, MS spectra were searched against a custom peptide isoform database generated from a publicly available ENCODE RNAseq dataset (accession ENCSR000BYQ) on normal C57BL/6J heart (8 weeks) through an in-house computational workflow. Protein isoform expression was compared between the young and old heart samples. Results: Our experiments identified over 1,200 proteins (FDR < 0.05) in young and aged mouse LV tissue, including 65 isoform-specific tryptic and chymotryptic peptide sequences originating from 45 alternatively spliced genes that are not currently documented in the commonly utilized protein sequence database SwissProt. The detected protein isoforms include TMED2, TOM1 and TACC2. Our preliminary analysis suggests widespread proteome changes between young and aged hearts, which may have implication on energetics and fibrotic changes in cardiac aging. Conclusion: We report the first large-scale survey on the expression of canonical and alternative protein isoforms in the aging LV proteome. The identification of age-associated alternative isoforms suggests a potential mechanism for protein function regulation in the aging heart and may have implications for age-related disease processes.

Engineering the acceptor substrate specificity in the xyloglucan endotransglycosylase TmXET6.3 from nasturtium seeds (Tropaeolum majus L.).

The knowledge of substrate specificity of XET enzymes is important for the general understanding of metabolic pathways to challenge the established notion that these enzymes operate uniquely on cellulose-xyloglucan networks. Xyloglucan xyloglucosyl transferases (XETs) (EC 2.4.1.207) play a central role in loosening and re-arranging the cellulose-xyloglucan network, which is assumed to be the primary load-bearing structural component of plant cell walls. The sequence of mature TmXET6.3 from Tropaeolum majus (280 residues) was deduced by the nucleotide sequence analysis of complete cDNA by Rapid Amplification of cDNA Ends, based on tryptic and chymotryptic peptide sequences. Partly purified TmXET6.3, expressed in Pichia occurred in N-glycosylated and unglycosylated forms. The quantification of hetero-transglycosylation activities of TmXET6.3 revealed that (1,3;1,4)-, (1,6)- and (1,4)-β-D-glucooligosaccharides were the preferred acceptor substrates, while (1,4)-β-D-xylooligosaccharides, and arabinoxylo- and glucomanno-oligosaccharides were less preferred. The 3D model of TmXET6.3, and bioinformatics analyses of identified and putative plant xyloglucan endotransglycosylases (XETs)/hydrolases (XEHs) of the GH16 family revealed that H94, A104, Q108, K234 and K237 were the key residues that underpinned the acceptor substrate specificity of TmXET6.3. Compared to the wild-type enzyme, the single Q108R and K237T, and double-K234T/K237T and triple-H94Q/A104D/Q108R variants exhibited enhanced hetero-transglycosylation activities with xyloglucan and (1,4)-β-D-glucooligosaccharides, while those with (1,3;1,4)- and (1,6)-β-D-glucooligosaccharides were suppressed; the incorporation of xyloglucan to (1,4)-β-D-glucooligosaccharides by the H94Q variant was influenced most extensively. Structural and biochemical data of non-specific TmXET6.3 presented here extend the classic XET reaction mechanism by which these enzymes operate in plant cell walls. The evaluations of TmXET6.3 transglycosylation activities and the incidence of investigated residues in other members of the GH16 family suggest that a broad acceptor substrate specificity in plant XET enzymes could be more widespread than previously anticipated.

Comparative proteomics of Helicobacter pylori strains reveals geographical features rather than genomic variations.

Helicobacter pylori, a pathogen of various gastric diseases, has many genome sequence variants. Thus, the pathogenesis and infection mechanisms of the H.pylori-driven gastric diseases have not been elucidated. Here, we carried out a large-scale proteome analysis to profile the heterogeneity of the proteome expression of 7 H.pylori strains by using an LC/MS/MS-based proteomics approach combined with a customized database consisting of nonredundant tryptic peptide sequences derived from full genome sequences of 52 H.pylori strains. The nonredundant peptide database enabled us to identify more peptides in the database search of MS/MS data compared with a simply merged protein database. Using this approach, we carried out proteome analysis of genome-unknown strains of H.pylori at as large a scale as genome-known ones. Clustering of the H.pylori strains using proteome profiling slightly differed from the genome profiling and more clearly divided the strains into two groups based on the isolated area. Furthermore, we identified phosphorylated proteins and sites of the H.pylori strains and obtained the phosphorylation motifs located in the N-terminus that are commonly observed in bacteria.

ComPIL 2.0: An Updated Comprehensive Metaproteomics Database.

We designed a metaproteomic analysis method (ComPIL) to accommodate the ever-increasing number of sequences against which experimental shotgun proteomics spectra could be accurately and rapidly queried. Our objective was to create these large databases for the analysis of complex metasamples with unknown composition, including those derived from human, animal, and environmental microbiomes. The amount of high-throughput sequencing data has substantially increased since our original database was assembled in 2014. Here, we present a rebuild of the ComPIL libraries comprised of updated publicly disseminated sequence data as well as a modified version of the search engine ProLuCID-ComPIL optimized for querying experimental spectra. ComPIL 2.0 consists of 113 million protein records and roughly 4.8 billion unique tryptic peptide sequences and is 2.3 times the size of our original version. We searched a data set collected on a healthy human gut microbiome proteomic sample and compared the results to demonstrate that ComPIL 2.0 showed a substantial increase in the number of unique identified peptides and proteins compared to the first ComPIL version. The high confidence of protein identification and accuracy demonstrated by the use of ComPIL 2.0 may encourage the method's application for large-scale proteomic annotation of complex protein systems.

Optimization of carbamylation conditions and study on the effects on the product ions of carbamylation and dual modification of the peptide by Q-TOF MS.

Modified peptides fragmented by collision-induced dissociation can offer additional sequence information, which is beneficial for the de novo sequencing of peptides. Here, the model peptide VQGESNDLK was carbamylated. The optimal conditions were as follows: temperature of 90℃, pH of 7, and the time of 60 min. Then, we studied the b- and y-series ions of the native, carbamylated, and dual-modified peptides. The results were as follows. The short carbamylated peptides (≤10 amino acid residues) produced more b-series ions (including b1 ion). The long carbamylated peptides (>10 amino acid residues) produced additional b1 ion but fewer y-series ions (especially in the high-mass region). The short dual-modified peptides produced more b-series ions (including b1 ion) and more y-series ions, and their peptide sequence coverage was almost 100%. The long dual-modified peptides produce b1 ion and more y-series ions, and their peptide sequence coverage was nearly above 90%. Therefore, both carbamylation and the dual modification method could be used to identify the N-terminal amino acid, and the dual modification method was also excellent for the de novo sequencing of the tryptic peptides.

Abstract P1-01-08: DPYSL3 modulates proliferation and migration in claudin-low breast cancer

Abstract Cancer is often driven by deregulated protein kinases pathways including breast cancer. To profile the kinome at the protein level we are developing a kinase pulldown (KiP) proteomic platform that uses multiple kinase inhibitors conjugated to sepharose beads for protein kinase profiling. Mass spectrometry-base tryptic peptide sequencing is used to identify and quantify the kinome in an unbiased manner. To date, we have profiled and analyzed the kinomes of more than 50 breast cancer PDX model systems that include all five breast cancer subtypes. In these analyses we have begun to explore the kinome interactome because we have observed that KiP also pulls down breast cancer subtype specific kinase interacting proteins that may represent new biological insights. For example we have consistently identified dihydropyrimidinase-like 3 (DPYSL3) in p21-activated kinase (PAK) inhibitor KiP interactome in the claudin-low WHIM12 PDX model. DPYSL3 is an intracellular phosphoprotein known to play a role in cell migration and metastasis. However it remains to be seen if DPYSL3 functions as a suppressor or a promoter of metastasis. We therefore generated DPYSL3 knock-down cell lines to evaluate function in claudin-low breast cancer. Proliferation levels of DPYSL3 depleted Claudin-low WHIM12 cells (DPYSL3-) were lower than those of control WHIM 12 (DPYSL3+) cells. In contrast, migration levels of DPYSL3- WHIM12 were greater than those of control cells, suggesting a pivotal role for DPYSL3 in several cellular physiologies. Additionally the activity of a PAK inhibitor (FRAX597) on migration appeared to require DPYSL3, as the inhibitory effect of FRAX579 on migration in WHIM12 was lost when DPYSL3 expression was depleted. Additionally we find that only the long isoform of DPYSL3, not a short isoform, interacts with PAK to regulate migration, suggesting that alternative splicing of DPYSL3 may an important to the control of migration. Furthermore Snail is also negatively regulated DPYSL3 with elevated expression in DYPSL3- cells. In conclusion, the study of the kinome interactome in breast cancer PDX identified DPYSL3 as regulator of the PAK family of kinases in a claudin low breast cancer. DPYSL3 is necessary for PAK to promote migration and may play a pivotal role in complex cellular transitions from a proliferative state versus an EMT/cell migratory state. Citation Format: Matsunuma R, Chan DW, Kim B-J, Singh P, Han A, Malovannaya A, Perou CM, Ellis MJ. DPYSL3 modulates proliferation and migration in claudin-low breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-01-08.

De Novo Sequencing of Tryptic Phosphopeptides Using Matrix-Assisted Laser Desorption/Ionization Based Tandem Mass Spectrometry with Hydrogen Atom Attachment.

Phosphorylation is the most abundant protein modification, and tandem mass spectrometry (MS/MS) with radical-based fragmentation techniques has proven to be a promising method for phosphoproteomic applications, owing to its ability to determine phosphorylation sites on proteins. The radical-induced fragmentation technique involves the attachment or abstraction of hydrogen to peptides in an ion trap mass spectrometer, in a process called hydrogen attachment/abstraction dissociation (HAD), which has only been recently developed. In the present investigation, we have analyzed model phosphopeptides and phosphoprotein digests using HAD-MS/MS, combined with matrix-assisted laser desorption/ionization (MALDI), in order to demonstrate the usefulness of the HAD-MS/MS-based analytical method. The tryptic peptides were categorized as arginine- and lysine-terminated peptides, and MALDI HAD-MS/MS is found to facilitate the sequencing of arginine-terminated tryptic peptides, because of the selective observation of C-terminal side fragment ions. In contrast, MALDI HAD-MS/MS of lysine-terminated tryptic peptides produced both N- and C-terminal side fragments, such that the mass spectra were complex. The guanidination of peptide converted lysine into homoarginine, which facilitated the interpretation of MALDI HAD-MS/MS mass spectra. The present method was useful for de novo sequencing of tryptic phosphopeptides.

The Sarda Sheep Host Fecal Proteome.

The first characterization of the sheep fecal microbiota was recently reported, as obtained by using a multi meta-omic approach. Here, the mass spectra generated by single-run LC/high-resolution MS in the context of that study were reanalyzed using a host-specific database, in order to gain insights for the first time into the host fecal proteome of healthy Sarda sheep. On the whole, 5349 non-redundant tryptic peptide sequences were identified, belonging to 1046 different proteins. The "core" fecal proteome (common to all animals) comprised 431 proteins, mainly related to biological processes as immune response and proteolysis. Proteins involved in the immune/inflammatory response and peptidases were specifically investigated. This dataset provides novel insights into the repertoire of proteins secreted in the sheep intestinal lumen, and constitutes the basis for future shotgun and targeted proteomics studies aimed at monitoring changes in the sheep fecal proteome in response to production variables, infectious/inflammatory states, and variations in the gut microbiota. Data are available via ProteomeXchange with identifier PXD006145.

Immunocontraceptive target repertoire defined by systematic identification of sperm membrane alloantigens in a single species.

Sperm competence in animal fertilization requires the collective activities of numerous sperm-specific proteins that are typically alloimmunogenic in females. Consequently, sperm membrane alloantigens are potential targets for contraceptives that act by blocking the proteins’ functions in gamete interactions. Here we used a targeted proteomics approach to identify the major alloantigens in swine sperm membranes and lipid rafts, and thereby systematically defined the repertoire of these sperm-specific proteins in a single species. Gilts with high alloantibody reactivity to proteins in sperm membranes or lipid rafts produced fewer offspring (73% decrease) than adjuvant-only or nonimmune control animals. Alloantisera recognized more than 20 potentially unique sperm membrane proteins and five sperm lipid raft proteins resolved on two-dimensional immunoblots with or without prior enrichment by anion exchange chromatography. Dominant sperm membrane alloantigens identified by mass spectrometry included the ADAMs fertilin α, fertilin ß, and cyritestin. Less abundant alloantigens included ATP synthase F1 β subunit, myo-inositol monophosphatase-1, and zymogen granule membrane glycoprotein-2. Immunodominant sperm lipid raft alloantigens included SAMP14, lymphocyte antigen 6K, and the epididymal sperm protein E12. Of the fifteen unique membrane alloantigens identified, eleven were known sperm-specific proteins with uncertain functions in fertilization, and four were not previously suspected to exist as sperm-specific isoforms. De novo sequences of tryptic peptides from sperm membrane alloantigen “M6” displayed no evident homology to known proteins, so is a newly discovered sperm-specific gene product in swine. We conclude that alloimmunizing gilts with sperm membranes or lipid rafts evokes formation of antibodies to a relatively small number of dominant alloantigens that include known and novel sperm-specific proteins with possible functions in fertilization and potential utility as targets for immunocontraception.

"Bottom-up" in situ proteomic differentiation of human and non-human haemoglobins for forensic purposes by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry.

The detection and identification of human blood on crime-related items are of particular relevance to many investigations because shed blood can provide evidence of violent contact between individuals. However, for any detection and identification technique, specificity is a critical performance characteristic to assess; that is, whether the technique has the capability to differentiate between human blood (which usually is of relevance to a criminal investigation) and non-human blood (which usually would not be associated with a crime but may be detected incidentally). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) approaches using "top-down" (detection of intact proteins) and "bottom-up" (detection of tryptic peptide markers) were used to detect and identify haemoglobin in blood from humans and from a range of Australian native mammals; the technique could be carried out directly on blood stains without the need to extract proteins (i.e., in situ measurement). Imaging of haemoglobin was achieved in bloodied fingermarks, including those that had been enhanced using two "industry standard" fingermark enhancement processes. Differentiation of intact haemoglobin proteins in human and non-human blood using "top-down" MALDI-TOF-MS was difficult. However, in situ "bottom-up" approaches using tandem mass spectrometry (MS/MS) and de novo sequencing of tryptic digest peptides allowed unambiguous differentiation. Imaging mass spectrometry of human haemoglobin, even when it was mixed with animal blood, was achieved in bloodied fingermarks that had been enhanced using two common processes (staining with Amido Black or dusted with magnetic powder) and "lifted" using adhesive tape. The MALDI-TOF-MS-based in situ "bottom-up" proteomic methodology described here shows great promise for the detection of human blood and even imaging of blood in bloodied fingermarks. The approach is sensitive, can differentiate between human blood and that from many animals (including several Australian native animals), and can be implemented after traditional crime scene fingermark enhancement techniques have been carried out.

Farnesyl biliverdins IXα are novel ligands of biliproteins from moths of the Noctuoidea superfamily: A chemosystematic view of the Lepidoptera

Bilins, derived from biliverdin IXα, are known from animals, plants and microorganisms, where they play vital roles as light-absorbing pigments. Bilins occur also in many insects. Recently, we discovered in insects a novel structural type of bilins with a farnesyl substituent at pyrrole ring A of biliverdin IXα. The first of these unusual bilins with a molecular mass of 852 (C48H60O10N4) was identified in Cerura vinula, subsequently in Spodoptera littoralis; both species are members of the Noctuoidea superfamily of moths. From an evolutionary point of view, it was of interest to examine other species and families of this monophyletic clade. Here, we show that other moths species in this clade (three Notodontidae species, one Erebidae species, and one Noctuidae species) have farnesylated biliverdins IXα that are present as a mixture of three bilins, differing by the number of oxygen atoms (O8-10). These bilins are associated with typical hemolymph storage proteins, which were identified by mass spectroscopic sequencing of tryptic peptides as arylphorins (a class of 500-kDa hexamerins) in the Notodontidae and Erebidae families, and as 350-kDa very high-density lipoproteins in the Noctuidae family. Circular dichroism spectroscopy revealed that the bilins adopt opposite conformations in complex with the two different classes of proteins. At present, farnesylated biliverdins and IXα-isomers of bilins in general are known only from species of the Noctuoidea clade; the sister clades of Bombycoidea and Papilionoidea synthesise the IXγ-isomer of biliverdin and derivatives thereof.

The Primary Structure of β(I)-Chain of Hemoglobin from Snake Sindhi Krait (Bungarus sindanus sindanus).

The amino acid sequence of β(I)-globin chain from Sindhi Krait (Bungarus sindanus sindanus) was determined to study the molecular evolution among snakes. The hemoglobin was isolated from the red blood cells and was analyzed by ion-exchange chromatography (IEX). The crude globin was subjected to reversed phased-high performance liquid chromatography (RP-HPLC) using C4 column. The N-terminal sequences of intact globin chains and tryptic peptides were determined by Edman degradation in a pulsed liquid gas phase sequencer using an online Phenylthiohydantoin analyzer. Sindhi Krait is expected to express three hemoglobin components that are composed of β(II), β(I), α(D) and α(A)-globin chains, as apparent by IEX, RP-HPLC and N-terminal sequence analyses. Sequence alignment and phylogenetic analyses of β(I) globin chain from Sindhi Krait showed closest relationship with β(I) globin chain from Rattlesnake, Water snake and Indigo snake. Interestingly, comparison of primary sequence of β(I) globin chain of Sindhi Krait with human β chain revealed 63% similarity along with the retention of all heme contact points. Variations among the two sequences were prominent at αβ contact points and in regions directly not important for function.