Venomics of the Australian eastern brown snake (Pseudonaja textilis): Detection of new venom proteins and splicing variants.

The eastern brown snake is the predominant cause of snakebites in mainland Australia. Its venom induces defibrination coagulopathy, renal failure and microangiopathic hemolytic anemia. Cardiovascular collapse has been described as an early cause of death in patients, but, so far, the mechanisms involved have not been fully identified. In the present work, we analysed the venome of Pseudonaja textilis by combining high throughput proteomics and transcriptomics, aiming to further characterize the components of this venom. The combination of these techniques in the analysis and identification of toxins, venom proteins and putative toxins allowed the sequence description and the identification of the following: prothrombinase coagulation factors, neurotoxic textilotoxin phospholipase A2 (PLA2) subunits and "acidic PLA2", three-finger toxins (3FTx) and the Kunitz-type protease inhibitor textilinin, venom metalloproteinase, C-type lectins, cysteine rich secretory proteins, calreticulin, dipeptidase 2, as well as evidences of Heloderma lizard peptides. Deep data-mining analysis revealed the secretion of a new transcript variant of venom coagulation factor 5a and the existence of a splicing variant of PLA2 modifying the UTR and signal peptide from a same mature protein. The transcriptome revealed the diversity of transcripts and mutations, and also indicates that splicing variants can be an important source of toxin variation.

Pseudechis guttatus venom proteome: Insights into evolution and toxin clustering.

The Australian Elapidae spotted black snake Pseudechis guttatus venom proteome composition was analyzed by high throughput mass spectrometry. The crude venom proteins were decomplexed by 2D-PAGE and in-gel digestion peptides from 66 spot samples and analyzed by tandem mass spectrometry-LC-ESI-ion trap. Protein identification was performed combining PEAKS studio 7.0 and Mascot software. The analysis identified l-amino-acid oxidases, phospholipases A2, metalloproteases, nerve growth factors and ecto-5'-nucleotidases, and for the first time in this venom the components cysteine-rich secretory proteins similar to pseudechetoxin, phospholipase B and transferrin-like protein. The envenomation symptoms are in agreement with the identified components, but the present limitations of database information might impair the detection of toxin families, protein species and still unknown toxins. From the qualitative point of view, the similarity of this venom with the ones from other Pseudechis species could be assigned to recent speciation events. BIOLOGICAL SIGNIFICANCE: Studies on the proteome of Australian Elapidae (Ancanthophiinae) are quite rare. In the present work we performed, using classic proteomic methods, a qualitative and partial analysis of the proteic components of Pseudechis guttatus venom. Although previous studies contributed to the knowledge of the major components of this venom, our study revealed some yet undescribed protein species, as well as new toxins, such as CRiSPs, phospholipase B, transferrin-like protein and ecto 5'-nucleotidase.

Elapid snake venom analyses show the specificity of the peptide composition at the level of genera Naja and Notechis.

Elapid snake venom is a highly valuable, but till now mainly unexplored, source of pharmacologically important peptides. We analyzed the peptide fractions with molecular masses up to 10 kDa of two elapid snake venoms-that of the African cobra, N. m. mossambica (genus Naja), and the Peninsula tiger snake, N. scutatus, from Kangaroo Island (genus Notechis). A combination of chromatographic methods was used to isolate the peptides, which were characterized by combining complimentary mass spectrometric techniques. Comparative analysis of the peptide compositions of two venoms showed specificity at the genus level. Three-finger (3-F) cytotoxins, bradykinin-potentiating peptides (BPPs) and a bradykinin inhibitor were isolated from the Naja venom. 3-F neurotoxins, Kunitz/basic pancreatic trypsin inhibitor (BPTI)-type inhibitors and a natriuretic peptide were identified in the N. venom. The inhibiting activity of the peptides was confirmed in vitro with a selected array of proteases. Cytotoxin 1 (P01467) from the Naja venom might be involved in the disturbance of cellular processes by inhibiting the cell 20S-proteasome. A high degree of similarity between BPPs from elapid and viperid snake venoms was observed, suggesting that these molecules play a key role in snake venoms and also indicating that these peptides were recruited into the snake venom prior to the evolutionary divergence of the snakes.

MALDI imaging on large-scale tissue microarrays identifies molecular features associated with tumour phenotype in oesophageal cancer.

AIMS: Matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) and tissue microarray (TMA) technologies were jointly utilized to search for molecular features associated with clinicopathological parameters in oesophageal cancer. METHODS AND RESULTS: Two TMAs from formalin-fixed tissue samples, including 300 adenocarcinomas and 177 squamous cell carcinomas with clinical follow-up data, were analysed. MALDI-MSI analysis revealed 72 distinct mass per charge (m/z) signals associated with tumour cells, 48 of which were found in squamous cell carcinomas only, and 12 of which were specific for adenocarcinomas. In adenocarcinomas, six signals were linked to early-stage (pT1-T2) tumours (two signals) and the presence (one signal) or absence (three signals) of lymph node metastasis. In squamous cell carcinomas, 24 signals were strongly linked to different phenotypic features, including tumour stage (four signals), histological grade (four signals), and lymph node metastasis (three signals). CONCLUSIONS: The high number of m/z signals that were found to be significantly linked to one or more phenotypic features of oesophageal cancer highlights the power of MALDI-MSI in the analysis of high-density TMAs. The data also emphasise substantial biological differences between adenocarcinomas and squamous cell carcinomas.

One- and two-dimensional high-performance thin-layer chromatography as an alternative analytical tool for investigating polyphenol-protein interactions.

INTRODUCTION: Polyphenols and simple phenolic compounds are able to react with other food constituents during processing and storage. In the past, it has been shown that their reaction with proteins can lead to changes of the technofunctional or even physiological properties of both compound classes. However, identification of specific binding sites of small molecules within a protein sequence (and the corresponding conformational position) is still challenging. OBJECTIVE: Investigating the reaction between different food proteins and phenolic compounds in alkaline medium with one- and two-dimensional high-performance thin-layer chromatography (HPTLC) coupled to matrix-assisted laser desorption/ionisation (MALDI) with time-of-flight (TOF) MS for analysing the peptide profiles after tryptic digestion. METHODS: After modification with phenolic compounds, protein derivatives were digested and peptides were separated with one- and two-dimensional HPTLC. Peptide profiles were detected with visible and UV wavelengths as well as with fluorescamine, ninhydrin and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid staining. In order to perform mass spectrometric measurements, peptides separated in the first dimension were analysed by MALDI/TOF/MS. RESULTS: Results show that the phenolic acids applied in this study show different specificity and susceptibility when modifying proteins resulting in changes of the peptide profiles, peptide quantity, polarity, UV-activity, radical-scavenging activity and molecular mass. CONCLUSION: One- and two-dimensional HPTLC supported by mass spectrometric detection represents an innovative, alternative tool for investigating and understanding polyphenol-protein interactions. This approach enables the identification of binding sites inside the protein chain and contributes to understanding the mechanism of polyphenol-protein interactions in vitro and in vivo.

MALDI mass spectrometric imaging based identification of clinically relevant signals in prostate cancer using large-scale tissue microarrays.

To identify molecular features associated with clinico-pathological parameters and TMPRSS2-ERG fusion status in prostate cancer, we employed MALDI mass spectrometric imaging (MSI) to a prostate cancer tissue microarray (TMA) containing formalin-fixed, paraffin-embedded tissues samples from 1,044 patients for which clinical follow-up data were available. MSI analysis revealed 15 distinct mass per charge (m/z)-signals associated to epithelial structures. A comparison of these signals with clinico-pathological features revealed statistical association with favorable tumor phenotype such as low Gleason grade, early pT stage or low Ki67 labeling Index (LI) for four signals (m/z 700, m/z 1,502, m/z 1,199 and m/z 3,577), a link between high Ki67LI for one signal (m/z 1,013) and a relationship with prolonged time to PSA recurrence for one signal (m/z 1,502; p = 0.0145). Multiple signals were associated with the ERG-fusion status of our cancers. Two of 15 epithelium-associated signals including m/z 1,013 and m/z 1,502 were associated with detectable ERG expression and five signals (m/z 644, 678, 1,044, 3,086 and 3,577) were associated with ERG negativity. These observations are in line with substantial molecular differences between fusion-type and non-fusion type prostate cancer. The signals observed in this study may characterize molecules that play a role in the development of TMPRSS2-ERG fusions, or alternatively reflect pathways that are activated as a consequence of ERG-activation. The combination of MSI and large-scale TMAs reflects a powerful approach enabling immediate prioritization of MSI signals based on associations with clinico-pathological and molecular data.

MALDI imaging in human skin tissue sections: focus on various matrices and enzymes.

Matrix-assisted laser/desorption ionization (MALDI) mass-spectrometric imaging (MSI), also known as MALDI imaging, is a powerful technique for mapping biological molecules such as endogenous proteins and peptides in human skin tissue sections. A few groups have endeavored to apply MALDI-MSI to the field of skin research; however, a comprehensive article dealing with skin tissue sections and the application of various matrices and enzymes is not available. Our aim is to present a multiplex method, based on MALDI-MSI, to obtain the maximum information from skin tissue sections. Various matrices were applied to skin tissue sections: (1) 9-aminoacridine for imaging metabolites in negative ion mode; (2) sinapinic acid to obtain protein distributions; (3) α-cyano-4-hydroxycinnamic acid subsequent to on-tissue enzymatic digestion by trypsin, elastase, and pepsin, respectively, to localize the resulting peptides. Notably, substantial amounts of data were generated from the distributions retrieved for all matrices applied. Several primary metabolites, e.g. ATP, were localized and subsequently identified by on-tissue postsource decay measurements. Furthermore, maps of proteins and peptides derived from on-tissue digests were generated. Identification of peptides was achieved by elution with different solvents, mixing with α-cyano-4-hydroxycinnamic acid, and subsequent tandem mass spectrometry (MS/MS) measurements, thereby avoiding on-tissue MS/MS measurements. Highly abundant peptides were identified, allowing their use as internal calibrants in future MALDI-MSI analyses of human skin tissue sections. Elastin as an endogenous skin protein was identified only by use of elastase, showing the high potential of alternative enzymes. The results show the versatility of MALDI-MSI in the field of skin research. This article containing a methodological perspective depicts the basics for a comprehensive comparison of various skin states.

Antigen-specificity of oligoclonal abnormal protein bands in multiple myeloma after allogeneic stem cell transplantation.

Myeloma patients may develop oligoclonal immunoglobulins, so-called abnormal protein bands (APB), after stem cell transplantation. APB do not correspond to the patient's paraprotein and confer a good prognosis. We set out to investigate whether such APB represent a humoral anti-myeloma immune response by screening immunoglobulins of 15 myeloma patients after allogeneic stem cell transplantation and a control group of healthy donors for reactivity with myeloma protein extracts. While the immunoglobulins of healthy donors did not react with myeloma protein extracts, patient-derived immunoglobulins showed variable levels of interaction, depending on the presence of APB on immunofixation. Most commonly, we detected interactions with heat-shock proteins, followed by neutral alpha-glucosidase, alpha-enolase and vimentin, as well as proliferating cell nuclear antigen and MAGEA4. More than 80% of targets were upregulated in myeloma. Heat-shock protein 60 (HSP60) was subsequently evaluated as an exemplary antigen. We found that HSP60 was aberrantly displayed on the surface of primary myeloma cells. Indeed, patient-derived APB-containing immunoglobulins recognized surface HSP60 suggesting that this antigen becomes accessible to the immune system after aberrant membrane exposition. We conclude that immunoglobulin fractions with APB recognize recurrent myeloma antigens and that this humoral response may contribute to the more favorable prognosis in patients with APB.

Venom peptide analysis of Vipera ammodytes meridionalis (Viperinae) and Bothrops jararacussu (Crotalinae) demonstrates subfamily-specificity of the peptidome in the family Viperidae.

Snake venom peptidomes are valuable sources of pharmacologically active compounds. We analyzed the peptidic fractions (peptides with molecular masses < 10,000 Da) of venoms of Vipera ammodytes meridionalis (Viperinae), the most toxic snake in Europe, and Bothrops jararacussu (Crotalinae), an extremely poisonous snake of South America. Liquid chromatography/mass spectrometry (LC/MS), direct infusion electrospray mass spectrometry (ESI-MS) and matrix-assisted desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) were applied to characterize the peptides of both snake venoms. 32 bradykinin-potentiating peptides (BPPs) were identified in the Crotalinae venom and their sequences determined. 3 metalloproteinase inhibitors, 10 BPPs and a Kunitz-type inhibitor were observed in the Viperinae venom peptidome. Variability in the C-terminus of homologous BPPs was observed, which can influence the pharmacological effects. The data obtained so far show a subfamily specificity of the venom peptidome in the Viperidae family: BPPs are the major peptide component of the Crotalinae venom peptidome lacking Kunitz-type inhibitors (with one exception) while the Viperinae venom, in addition to BPPs, can contain peptides of the bovine pancreatic trypsin inhibitor family. We found indications for a post-translational phosphorylation of serine residues in Bothrops jararacussu venom BPP (S[combining low line]QGLPPGPPIP), which could be a regulatory mechanism in their interactions with ACE, and might influence the hypotensive effect. Homology between venom BPPs from Viperidae snakes and venom natriuretic peptide precursors from Elapidae snakes suggests a structural similarity between the respective peptides from the peptidomes of both snake families. The results demonstrate that the venoms of both snakes are rich sources of peptides influencing important physiological systems such as blood pressure regulation and hemostasis. The data can be used for pharmacological and medical applications.

Post-translational modifications of the gamma-subunit affect intracellular trafficking and complex assembly of GlcNAc-1-phosphotransferase.

GlcNAc-1-phosphotransferase plays a key role in the generation of mannose 6-phosphate, a recognition marker essential for efficient transport of lysosomal hydrolases to lysosomes. The enzyme complex is composed of six subunits (α(2)ß(2)γ(2)). The α- and ß-subunits are catalytically active, whereas the function of the γ-subunit is still unclear. We have investigated structural properties, localization, and intracellular transport of the human and mouse γ-subunits and the molecular requirements for the assembly of the phosphotransferase complex. The results showed that endogenous and overexpressed γ-subunits were localized in the cis-Golgi apparatus. Secreted forms of γ-subunits were detectable in media of cultured cells as well as in human serum. The γ-subunit contains two in vivo used N-glycosylation sites at positions 88 and 115, equipped with high mannose-type oligosaccharides. (35)S pulse-chase experiments and size exclusion chromatography revealed that the majority of non-glycosylated γ-subunit mutants were integrated in high molecular mass complexes, failed to exit the endoplasmic reticulum (ER), and were rapidly degraded. The substitution of cysteine 245 involved in dimerization of γ-subunits impaired neither ER exit nor trafficking through the secretory pathway. Monomeric γ-subunits failed, however, to associate with other GlcNAc-1-phosphotransferase subunits. The data provide evidence that assembly of the GlcNAc-1-phosphotransferase complex takes place in the ER and requires dimerization of the γ-subunits.

Stereotypical chronic lymphocytic leukemia B-cell receptors recognize survival promoting antigens on stromal cells.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world. Survival of CLL cells depends on their close contact with stromal cells in lymphatic tissues, bone marrow and blood. This microenvironmental regulation of CLL cell survival involves the stromal secretion of chemo- and cytokines as well as the expression of adhesion molecules. Since CLL survival may also be driven by antigenic stimulation through the B-cell antigen receptor (BCR), we explored the hypothesis that these processes may be linked to each other. We tested if stromal cells could serve as an antigen reservoir for CLL cells, thus promoting CLL cell survival by stimulation through the BCR. As a proof of principle, we found that two CLL BCRs with a common stereotyped heavy chain complementarity-determining region 3 (previously characterized as "subset 1") recognize antigens highly expressed in stromal cells--vimentin and calreticulin. Both antigens are well-documented targets of autoantibodies in autoimmune disorders. We demonstrated that vimentin is displayed on the surface of viable stromal cells and that it is present and bound by the stereotyped CLL BCR in CLL-stroma co-culture supernatant. Blocking the vimentin antigen by recombinant soluble CLL BCR under CLL-stromal cell co-culture conditions reduces stroma-mediated anti-apoptotic effects by 20-45%. We therefore conclude that CLL BCR stimulation by stroma-derived antigens can contribute to the protective effect that the stroma exerts on CLL cells. This finding sheds a new light on the understanding of the pathobiology of this so far mostly incurable disease.

Application of displacement chromatography for the analysis of a lipid raft proteome.

Defining membrane proteomes is fundamental to understand the role of membrane proteins in biological processes and to find new targets for drug development. Usually multidimensional chromatography using step or gradient elution is applied for the separation of tryptic peptides of membrane proteins prior to their mass spectrometric analysis. Displacement chromatography (DC) offers several advantages that are helpful for proteome analysis. However, DC has so far been applied for proteomic investigations only in few cases. In this study we therefore applied DC in a multidimensional LC-MS approach for the separation and identification of membrane proteins located in cholesterol-enriched membrane microdomains (lipid rafts) obtained from rat kidney by density gradient centrifugation. The tryptic peptides were separated on a cation-exchange column in the displacement mode with spermine used as displacer. Fractions obtained from DC were analyzed using an HPLC-chip system coupled to an electrospray-ionization ion-trap mass spectrometer. This procedure yielded more than 400 highly significant peptide spectrum matches and led to the identification of more than 140 reliable protein hits within an established rat kidney lipid raft proteome. The majority of identified proteins were membrane proteins. In sum, our results demonstrate that DC is a suitable alternative to gradient elution separations for the identification of proteins via a multidimensional LC-MS approach.