Thomas F. Dyrlund

Spider genomes provide insight into composition and evolution of venom and silk

Spiders are ecologically important predators with complex venom and extraordinarily tough silk that enables capture of large prey. Here we present the assembled genome of the social velvet spider and a draft assembly of the tarantula genome that represent two major taxonomic groups of spiders. The spider genomes are large with short exons and long introns, reminiscent of mammalian genomes. Phylogenetic analyses place spiders and ticks as sister groups supporting polyphyly of the Acari. Complex sets of venom and silk genes/proteins are identified. We find that venom genes evolved by sequential duplication, and that the toxic effect of venom is most likely activated by proteases present in the venom. The set of silk genes reveals a highly dynamic gene evolution, new types of silk genes and proteins, and a novel use of aciniform silk. These insights create new opportunities for pharmacological applications of venom and biomaterial applications of silk.

The Protein Composition of the Digestive Fluid from the Venus Flytrap Sheds Light on Prey Digestion Mechanisms

The Venus flytrap (Dionaea muscipula) is one of the most well-known carnivorous plants because of its unique ability to capture small animals, usually insects or spiders, through a unique snap-trapping mechanism. The animals are subsequently killed and digested so that the plants can assimilate nutrients, as they grow in mineral-deficient soils. We deep sequenced the cDNA from Dionaea traps to obtain transcript libraries, which were used in the mass spectrometry-based identification of the proteins secreted during digestion. The identified proteins consisted of peroxidases, nucleases, phosphatases, phospholipases, a glucanase, chitinases, and proteolytic enzymes, including four cysteine proteases, two aspartic proteases, and a serine carboxypeptidase. The majority of the most abundant proteins were categorized as pathogenesis-related proteins, suggesting that the plants digestive system evolved from defense-related processes. This in-depth characterization of a highly specialized secreted fluid from a carnivorous plant provides new information about the plants prey digestion mechanism and the evolutionary processes driving its defense pathways and nutrient acquisition.

The Human Eye Proteome Project: Perspectives on an emerging proteome

There are an estimated 285 million people with visual impairment worldwide, of whom 39 million are blind. The pathogenesis of many eye diseases remains poorly understood. The human eye is currently an emerging proteome that may provide key insight into the biological pathways of disease. We review proteomic investigations of the human eye and present a catalogue of 4842 nonredundant proteins identified in human eye tissues and biofluids to date. We highlight the need to identify new biomarkers for eye diseases using proteomics. Recent advances in proteomics do now allow the identification of hundreds to thousands of proteins in tissues and fluids, characterization of various PTMs and simultaneous quantification of multiple proteins. To facilitate proteomic studies of the eye, the Human Eye Proteome Project (HEPP) was organized in September 2012. The HEPP is one of the most recent components of the Biology/Disease‐driven Human Proteome Project (B/D‐HPP) whose overarching goal is to support the broad application of state‐of‐the‐art measurements of proteins and proteomes by life scientists studying the molecular mechanisms of biological processes and human disease. The large repertoire of investigative proteomic tools has great potential to transform vision science and enhance understanding of physiology and disease processes that affect sight.

Human cornea proteome: identification and quantitation of the proteins of the three main layers including epithelium, stroma, and endothelium.

Diseases of the cornea are common and refer to conditions like infections, injuries and genetic defects. Morphologically, many corneal diseases affect only certain layers of the cornea and separate analysis of the individual layers is therefore of interest to explore the basic molecular mechanisms involved in corneal health and disease. In this study, the three main layers including, the epithelium, stroma and endothelium of healthy human corneas were isolated. Prior to analysis by LC–MS/MS the proteins from the different layers were either (i) separated by SDS-PAGE followed by in-gel trypsinization, (ii) in-solution digested without prior protein separation or, (iii) in-solution digested followed by cation exchange chromatography. A total of 3250 unique Swiss-Prot annotated proteins were identified in human corneas, 2737 in the epithelium, 1679 in the stroma, and 880 in the endothelial layer. Of these, 1787 proteins have not previously been identified in the human cornea by mass spectrometry. In total, 771 proteins were quantified, 157 based on in-solution digestion and 770 based on SDS-PAGE separation followed by in-gel digestion of excised gel pieces. Protein analysis showed that many of the identified proteins are plasma proteins involved in defense responses.

Unconditioned commercial embryo culture media contain a large variety of non-declared proteins: a comprehensive proteomics analysis

STUDY QUESTION Which non-declared proteins (proteins not listed on the composition list of the product data sheet) are present in unconditioned commercial embryo culture media? SUMMARY ANSWER A total of 110 non-declared proteins were identified in unconditioned media and between 6 and 8 of these were quantifiable and therefore represent the majority of the total protein in the media samples. WHAT IS KNOWN ALREADY There are no data in the literature on what non-declared proteins are present in unconditioned (fresh media in which no embryos have been cultured) commercial embryo media. STUDY DESIGN, SIZE, DURATION The following eight commercial embryo culture media were included in this study: G-1 PLUS and G-2 PLUS G5 Series from Vitrolife, Sydney IVF Cleavage Medium and Sydney IVF Blastocyst Medium from Cook Medical and EmbryoAssist, BlastAssist, Sequential Cleav and Sequential Blast from ORIGIO. Two batches were analyzed from each of the Sydney IVF media and one batch from each of the other media. All embryo culture media are supplemented by the manufacturers with purified human serum albumin (HSA 5 mg/ml). The purified HSA (HSA-solution from Vitrolife) and the recombinant human albumin supplement (G-MM from Vitrolife) were also analyzed. PARTICIPANTS/MATERIALS, SETTING, METHODS For protein quantification, media samples were in-solution digested with trypsin and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). For in-depth protein identification, media were albumin depleted, dialyzed and concentrated before sodium dodecyl sulfate polyacrylamide gel electrophoresis. The gel was cut into 14 slices followed by in-gel trypsin digestion, and analysis by LC-MS/MS. Proteins were further investigated using gene ontology (GO) terms analysis. MAIN RESULTS AND THE ROLE OF CHANCE Using advanced mass spectrometry and high confidence criteria for accepting proteins (P < 0.01), a total of 110 proteins other than HSA were identified. The average HSA content was found to be 94% (92-97%) of total protein. Other individual proteins accounted for up to 4.7% of the total protein. Analysis of purified HSA strongly suggests that these non-declared proteins are introduced to the media when the albumin is added. GO analysis showed that many of these proteins have roles in defence pathways, for example 18 were associated with the innate immune response and 17 with inflammatory responses. Eight proteins have been reported previously as secreted embryo proteins. LIMITATIONS, REASONS FOR CAUTION For six of the commercial embryo culture media only one batch was analyzed. However, this does not affect the overall conclusions. WIDER IMPLICATIONS OF THE FINDINGS The results showed that the HSA added to IVF media contained many other proteins and that the amount varies from batch to batch. These variations in protein profiles are problematic when attempting to identify proteins derived from the embryos. Therefore, when studying the embryo secretome and analyzing conditioned media with the aim of finding potential biomarkers that can distinguish normal and abnormal embryo development, it is important that the medium used in the experimental and control groups is from the same batch. Furthermore, the proteins present in unconditioned media could potentially influence embryonic development, gestation age, birthweight and perhaps have subsequent effects on health of the offspring. STUDY FUNDING/COMPETING INTERESTS The study was supported by the Danish Agency for Science, Technology and Innovation. Research at the Fertility Clinic, Aarhus University Hospital is supported by an unrestricted grant from Merck Sharp & Dohme Corp and Ferring. The authors declare no conflicts of interest.

Investigations on collectin liver 1.

Background: The collectin CL-L1 is a pattern recognition molecule of the innate immune system. Results: Several biological parameters are established and furnish a basis for continued investigations. Conclusion: CL-L1 has specificity similar to other collectins, is present as large oligomers in blood from birth, and is associated with MASPs. Significance: A number of novel data supporting a biological role for CL-L1 are presented. Collectins are pattern recognition molecules of the innate immune system showing binding to carbohydrate structures on microorganisms in a calcium-dependent manner. Recently, three novel collectins, collectin liver 1 (CL-L1), collectin kidney 1 (CL-K1 and CL-11), and collectin placenta 1 (CL-P1), were discovered. The roles of these three collectins remain largely unknown. Here, we present a time-resolved immunofluorometric assay for quantification of CL-L1. The concentration of CL-L1 in donor plasma (n = 210) was distributed log-normally with a median value of 3.0 μg/ml (range 1.5–5.5 μg/ml). We observed on average 30% higher concentrations of CL-L1 in plasma as compared with serum. Size analysis by gel-permeation chromatography showed CL-L1 in serum to elute as large 700–800-kDa complexes and smaller 200–300-kDa complexes. CL-L1 showed specific binding to mannose-TSK beads in a Ca2+-dependent manner. This binding could be inhibited by mannose and glucose, but not galactose, indicating that CL-L1 binds via its carbohydrate-recognition domain and has ligand specificity similar to that of mannan-binding lectin. Western blot analysis of CL-L1 showed the presence of several oligomeric forms in serum. Ontogeny studies showed CL-L1 to be present at birth at near adult levels. CL-L1 levels exhibit low variation in healthy adults over a 1-year period. During acute-phase responses, the CL-L1 levels display only minor variations. In serum, CL-L1 was found in complexes with mannan-binding lectin-associated serine proteases, suggesting a role in the lectin pathway of complement activation. The presented data establish a basis for future studies on the biological role of CL-L1.

MS Data Miner: A web-based software tool to analyze, compare, and share mass spectrometry protein identifications

Data processing and analysis of proteomics data are challenging and time consuming. In this paper, we present MS Data Miner (MDM) (http://sourceforge.net/p/msdataminer), a freely available web‐based software solution aimed at minimizing the time required for the analysis, validation, data comparison, and presentation of data files generated in MS software, including Mascot (Matrix Science), Mascot Distiller (Matrix Science), and ProteinPilot (AB Sciex). The program was developed to significantly decrease the time required to process large proteomic data sets for publication. This open sourced system includes a spectra validation system and an automatic screenshot generation tool for Mascot‐assigned spectra. In addition, a Gene Ontology term analysis function and a tool for generating comparative Excel data reports are included. We illustrate the benefits of MDM during a proteomics study comprised of more than 200 LC‐MS/MS analyses recorded on an AB Sciex TripleTOF 5600, identifying more than 3000 unique proteins and 3.5 million peptides.

Proteomics of Fuchs' endothelial corneal dystrophy support that the extracellular matrix of Descemet's membrane is disordered.

Fuchs’ endothelial corneal dystrophy (FECD) is a major corneal disorder affecting the innermost part of the cornea, leading to visual impairment. As the morphological changes in FECD are mainly observed in the extracellular matrix of the Descemet’s membrane/endothelial layer, we determined the protein profiles of diseased and control tissues using two relative quantitation MS methods. The first quantitation method, based on the areas of the extracted ion chromatograms, quantified the 51 and 48 most abundant proteins of the Descemet’s membrane/endothelial layer in patient and control tissues, respectively, of which 10 were significantly regulated. The results indicated that the level of type VIII collagen was unaltered even though the protein previously has been shown to be implicated in familial early-onset forms of the disease. Using the second relative quantitation method, iTRAQ, we identified 22 differentially regulated proteins, many of which are extracellular proteins known to be involved in proper assembly of the basement membrane in other tissues. In total, 26 differentially regulated proteins were identified, of which 6 proteins were regulated in both methods. These results support that the morphological changes observed in FECD are caused in part by an aberrant assembly of the extracellular matrix within the Descemet’s membrane/endothelial layer.

Coagulation Factor XIIIa Substrates in Human Plasma IDENTIFICATION AND INCORPORATION INTO THE CLOT

Background: Coagulation factor XIIIa (FXIIIa) catalyzes cross-linking of Gln and Lys residues during coagulation. Results: A total of 147 FXIIIa substrates were identified in human plasma, and 48 of these were incorporated into the clot. Conclusion: These results indicate that FXIIIa is involved in extensive functionalization of the plasma clot. Significance: We present new insights into roles of FXIIIa in physiological and pathological processes. Coagulation factor XIII (FXIII) is a transglutaminase with a well defined role in the final stages of blood coagulation. Active FXIII (FXIIIa) catalyzes the formation of ϵ-(γ-glutamyl)lysine isopeptide bonds between specific Gln and Lys residues. The primary physiological outcome of this catalytic activity is stabilization of the fibrin clot during coagulation. The stabilization is achieved through the introduction of cross-links between fibrin monomers and through cross-linking of proteins with anti-fibrinolytic activity to fibrin. FXIIIa additionally cross-links several proteins with other functionalities to the clot. Cross-linking of proteins to the clot is generally believed to modify clot characteristics such as proteolytic susceptibility and hereby affect the outcome of tissue damage. In the present study, we use a proteomic approach in combination with transglutaminase-specific labeling to identify FXIIIa plasma protein substrates and their reactive residues. The results revealed a total of 147 FXIIIa substrates, of which 132 have not previously been described. We confirm that 48 of the FXIIIa substrates were indeed incorporated into the insoluble fibrin clot during the coagulation of plasma. The identified substrates are involved in, among other activities, complement activation, coagulation, inflammatory and immune responses, and extracellular matrix organization.

Comparison of two phenotypically distinct lattice corneal dystrophies caused by mutations in the transforming growth factor beta induced (TGFBI) gene

In this study, we investigated whether the phenotypic difference observed between two lattice corneal dystrophy type 1 (LCD type 1) cases caused by either a single A546D substitution or an A546D/P551Q double substitution in TGFBIp (transforming growth factor beta induced protein) can be ascribed to (i) a difference in the proteomes of corneal amyloid deposits, (ii) altered proteolysis of TGFBIp, or (iii) structural changes of TGFBIp introduced by the P551Q amino acid substitution.