Tryptic Digestion Of Proteins Research Articles

Development and application of FD-LC-MS/MS proteomics analysis revealing protein expression and biochemical events in tissues and cells

It is routine to search for and recognized genetic defects in human disorders to provide knowledge for diagnosis, treatment, and protection against diseases. It is also important to investigate and demonstrate the cause of a disease from the proteomic perspective, because intracellular signaling systems depend on protein dynamics. Demonstrating changes in protein levels enables us to understand biochemical events during the initiation and progression of a disease. To understand changes in protein levels in tissues and cells, we have developed a novel proteomics approach, FD-LC-MS/ MS. This consists of fluorogenic derivatization (FD), HPLC separation and detection/quantification of proteins in a biological sample, followed by the isolation and tryptic digestion of target proteins, and then their identification using HPLC and tandem mass spectrometry (MS/MS) with a database-searching algorithm. The method is highly sensitive (femtomole-level detection) through the use of less noisy fluorogenic rather than fluorescence derivatization, and enables precise and comprehensive relative quantitation of protein levels (between-day relative standard deviation of peak heights of ca. 20%) by combining FD with HPLC separation. In this paper, after a simple review of differential profiling using FD-LC-MS/MS, for example the analysis of stimulated vs. unstimulated samples, we introduce the development and application of the FD-LC-MS/MS method for comprehensive differential proteomics of several tissues, including mouse liver, mouse brain, and breast cancer cell lines, to reveal protein levels and biochemical events in tissues and cells.

Sensitive and site-specific identification of carboxymethylated and carboxyethylated peptides in tryptic digests of proteins and human plasma.

Glycation refers to a nonenzymatic post-translational modification formed by the reaction of amino groups and reducing sugars. Consecutive oxidation and degradation can produce advanced glycation end products (AGEs), such as N(ε)-(carboxyethyl)lysine (CEL) and N(ε)-(carboxymethyl)lysine (CML). Although CEL and CML are considered to be markers of arteriosclerosis, diabetes mellitus, and aging, the modified proteins and the exact modification sites are mostly unknown due to their low frequency and a lack of enrichment strategies. Here, we report characteristic fragmentation patterns of CML- and CEL-containing peptides and two modification-specific reporter ions for each modification (CML, m/z 142.1 and 187.1; CEL, m/z 156.1 and 201.1). The protocol allowed sensitive and selective precursor ion scans to detect the modified peptides in complex sample mixtures. The corresponding m/z values identified eight CEL/CML-modification sites in glycated human serum albumin (HSA) by targeted nano-RPC-MS/MS. The same strategy revealed 21 CML sites in 17 different proteins, including modified lysine residues 88 and 396 of human serum albumin, in a pooled plasma sample that was obtained from patients with type 2 diabetes mellitus.

A novel Alaska pollack-derived peptide, which increases glucose uptake in skeletal muscle cells, lowers the blood glucose level in diabetic mice

We found that the tryptic digest of Alaska pollack protein exhibits a glucose-lowering effect in KK-Ay mice, a type II diabetic model. We then searched for glucose-lowering peptides in the digest. Ala-Asn-Gly-Glu-Val-Ala-Gln-Trp-Arg (ANGEVAQWR) was identified from a peak of the HPLC fraction selected based on the glucose-lowering activity in an insulin resistance test using ddY mice. ANGEVAQWR (3 mg kg(-1)) decreased the blood glucose level after intraperitoneal administration. Among its fragment peptides, the C-terminal tripeptide, Gln-Trp-Arg (QWR, 1 mg kg(-1)), lowered the blood glucose level, suggesting that the C-terminal is critical for glucose-lowering activity. QWR also enhanced glucose uptake into C2C12, a mouse skeletal muscle cell line. QWR did not induce the phosphorylation of serine/threonine protein kinase B (Akt) and adenosine monophosphate-activated protein kinase (AMPK). We also demonstrated that QWR lowered the blood glucose level in NSY and KK-Ay, type II diabetic models.

Perovskite for the highly selective enrichment of phosphopeptides

Selective and effective enrichment of phosphopeptides from complex samples is essential in phosphoproteome study by mass spectrometry (MS). In this work, we compared perovskites (MgTiO3, CaTiO3, SrTiO3, BaTiO3 and CaZrO3) with metal oxides (ZrO2 and TiO2) in their capability for the selective enrichment of phosphopeptides. It was found here that perovskites exhibited higher selectivity towards phosphopeptides than commonly used ZrO2 and TiO2, even though they all have high affinity to phosphopeptides. As for perovskites, CaTiO3 exhibited better selectivity for enrichment of phosphopeptides than SrTiO3, MgTiO3, BaTiO3 and CaZrO3, which might be ascribed to their crystal structures and electrophilic abilities. Moreover, to further confirm the performance of CaTiO3, CaTiO3 and TiO2 were applied to the enrichment of phosphopeptides from tryptic digest of proteins of human Jurkat-T cell lysate, respectively. The results showed CaTiO3 has much higher selectivity than TiO2 in the enrichment of phosphopeptides from the complex biological sample. Taken together, here we show that CaTiO3 is an excellent material for the highly selective enrichment of phosphopeptides and it could be potentially used in the large-scale phosphoproteome study.

Development of new multifunctional terpolymer sorbent for proteomics applications.

Determination of the availability of phases for specific separations is an important task achieved by a separation chemist. This becomes vital when the complex samples like biofluids are dealt with in proteome science. The work presented here involves the synthesis and application of terpolymeric sorbent with different functionalizations adopted for the selective enrichment of biomolecules of interest from biological fluids. Synthesis of terpolymer was carried out by the radical polymerization of monomers: methyl acrylate, acrylic acid and vinyl acetate with diethylene glycol dimethacrylate as cross-linking agent, benzoyl peroxide as initiator and chloroform as a porogenic solvent. Characterization was done through Fourier transform infrared spectroscopy, scanning electron microscopy and nitrogen adsorption porosimetry. The polymer was further modified to immobilized metal ion affinity chromatographic material, with immobilized Fe(3+)/La(3+) ions that allowed phosphopeptide enrichment from tryptic digests of standard proteins as well as milk, egg yolk and human serum. Sensitivity of enrichment down to 50 fmol was achieved in the presence of complex protein background as bovine serum albumin. Hydrophobicity was introduced through octadecyl amine, which provides comparable results to ZipTip C18/C4 for desalting of complex mixtures of all caseins. Analysis of the enriched content was performed by Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

Construction of a high-performance magnetic enzyme nanosystem for rapid tryptic digestion.

A magnetic enzyme nanosystem have been designed and constructed by a polydopamine (PDA)-modification strategy. The magnetic enzyme nanosystem has well defined core-shell structure and a relatively high saturation magnetization (Ms) value of 48.3 emu g−1. The magnetic enzyme system can realize rapid, efficient and reusable tryptic digestion of proteins by taking advantage of its magnetic core and biofunctional shell. Various standard proteins (e.g. cytochrome C (Cyt-C), myoglobin (MYO) and bovine serum albumin (BSA)) have been used to evaluate the effectiveness of the magnetic enzyme nanosystem. The results show that the magnetic enzyme nanosystem can digest the proteins in 30 minutes, and the results are comparable to conventional 12 hours in-solution digestion. Furthermore, the magnetic enzyme nanosystem is also effective in the digestion of low-concentration proteins, even at as low as 5 ng μL−1 substrate concentration. Importantly, the system can be reused several times, and has excellent stability for storage. Therefore, this work will be highly beneficial for the rapid digestion and identification of proteins in future proteomics.

Mass Spectrometry-Based Proteomics for Relative Protein Quantification and Biomarker Identification in Primary Human Hepatocytes.

Liquid chromatography-tandem mass spectrometry-based proteomics is a highly sensitive and effective tool to identify and quantify potential biomarkers in repeated dose toxicity studies using primary cell culture systems. In this respect, 8-plex isobaric tag for relative and absolute quantification labeling is the method of choice for relative quantification. After cell lysis and tryptic protein digestion, an individual isobaric tag is added to the amine groups of arginine and lysine. Then, up to eight differentially labeled samples are mixed and analyzed together in a mass spectrometry experiment. During peptide fragmentation in the mass spectrometer, the individual tag intensity of each identified peptide could be detected, reflecting the peptide intensities in the eight samples. The identified peptides are matched to their specific protein using specific search engines and finally to eight individual relative protein quantities. The two-dimensional fractionation of complex peptide mixtures minimizes the possibility of co-fragmentation of peptides from different origin in the mass spectrometer, which leads to a higher number of peptide search matches and therefore to better identification and quantification results.

Abstract 1617: Quantification of pancreatic cancer proteome & phosphorylome: Indicates molecular events likely contributing to cancer & activation status of drug targets

Abstract INTRODUCTION: LC-MS/MS proteomics is an essential technology to help unravel the complex molecular events that lead to and propagate cancer. We have developed an analytical workflow to quantify the expression and phosphorylation status of thousands of proteins simultaneously in frozen resected human pancreatic tumor (T), relative to matched non-tumor (NT) tissue. These measurements enabled us to determine activity of anti-neoplastic drug targets and other signaling proteins. METHODS: Peptides resulting from tryptic digestion of proteins extracted from frozen tissue of pancreatic ductal adenocarcinoma and background pancreas (n=12), were labelled with tandem mass tags (TMT 8-plex), separated by strong cation exchange chromatography, then were analysed by LC-MS/MS directly or first enriched for phosphopeptides using Fe3+ and TiO2, prior to analysis. In-house, commercial and freeware bio-informatics platforms were used to identify relevant biological events from the complex dataset. RESULTS: Of 2,101 proteins identified, 152 demonstrated significant difference in expression between tumor and non-tumor tissue. They included proteins that are known to be up-regulated in pancreatic cancer (e.g. Mucin-1), but the majority were new candidate markers such as homeodomain interacting protein kinase 1 & Myosin light chain kinase. Of the 6,543 unique phosphopeptides identified (6,284 unique phosphorylation sites), 635 showed significant regulation, particularly those from proteins involved in cell migration (Rho GTPase signaling proteins such as Rho guanine nucleotide exchange factors & Serine/threonine-protein kinase MRCKα) as well as proteins involved in disassembly of cell-cell junctions (tight junction, adherens junction) and formation of cell-extracellular matrix (ECM) junctions (focal adhesions). We quantified activator & inhibitory phosphorylation sites on FYN, AKT1, HDAC1&2, GSK3α&β, RAF kinases, MAPKs (p38, ERK1&2), PKCs, Casein Kinases and >20 others, as well as their downstream substrates some of which were often found to be highly modulated (≥ 2 fold) in T versus NT in different cases. CONCLUSION: Application of our LC-MS/MS proteomic workflow to frozen resected human pancreatic T versus NT tissue elucidated molecular events likely contributing to pancreatic cancer in each case, particularly those contributing to cell migration, and in future may help clinicians predict the best targeted anti-cancer therapy bespoke for an individual patient. Citation Format: David Britton, Yoh Zen, Stefan Selzer, Vikram Mitra, Alberto Quaglia, Debashis Sarker, Leandro Castellano, Justin Stebbing, Julia Gee, Rob Nicholson, Nigel Heaton, Ian Pike. Quantification of pancreatic cancer proteome & phosphorylome: Indicates molecular events likely contributing to cancer & activation status of drug targets. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1617. doi:10.1158/1538-7445.AM2014-1617

Tandem Mass Spectral Libraries of Peptides in Digests of Individual Proteins: Human Serum Albumin (HSA)

This work presents a method for creating a mass spectral library containing tandem spectra of identifiable peptide ions in the tryptic digestion of a single protein. Human serum albumin (HSA(1)) was selected for this purpose owing to its ubiquity, high level of characterization and availability of digest data. The underlying experimental data consisted of ∼3000 one-dimensional LC-ESI-MS/MS runs with ion-trap fragmentation. In order to generate a wide range of peptides, studies covered a broad set of instrument and digestion conditions using multiple sources of HSA and trypsin. Computer methods were developed to enable the reliable identification and reference spectrum extraction of all peptide ions identifiable by current sequence search methods. This process made use of both MS2 (tandem) spectra and MS1 (electrospray) data. Identified spectra were generated for 2918 different peptide ions, using a variety of manually-validated filters to ensure spectrum quality and identification reliability. The resulting library was composed of 10% conventional tryptic and 29% semitryptic peptide ions, along with 42% tryptic peptide ions with known or unknown modifications, which included both analytical artifacts and post-translational modifications (PTMs) present in the original HSA. The remaining 19% contained unexpected missed-cleavages or were under/over alkylated. The methods described can be extended to create equivalent spectral libraries for any target protein. Such libraries have a number of applications in addition to their known advantages of speed and sensitivity, including the ready re-identification of known PTMs, rejection of artifact spectra and a means of assessing sample and digestion quality.

In situ sample processing approach (iSPA) for comprehensive quantitative phosphoproteome analysis.

Current sample preparation protocols for quantitative phosphoproteome analysis are tedious and time-consuming. Here, a facile in situ sample processing approach (iSPA) is developed by using macroporous Ti(IV)-IMAC microspheres as the preparation "beds", where all sample preparation procedures including the enrichment of phosphoproteins, tryptic digestion of proteins, enrichment, and isotope labeling of phosphopeptides are performed in situ sequentially. As a result of the in situ processing design and the seamless procedures, extra steps for desalting and buffer exchanging, which are always required in conventional approaches, are avoided, and the sample loss and contamination could be greatly reduced. Thus, better sensitivity and accuracy for the quantitative phosphoproteome analysis were obtained. This strategy was further applied to differential phosphoproteome analysis of human liver tissues with or without hepatocellular carcinoma (HCC). In total, 8548 phosphorylation sites were confidently quantified from three replicate analyses of 0.5 mg of human liver protein extracts.

One-pot preparation of glutathione–silica hybrid monolith for mixed-mode capillary liquid chromatography based on “thiol-ene” click chemistry

A novel glutathione (GSH)–silica hybrid monolithic column synthesized via a combination of thiol-ene click reaction and one-pot process was described, where thiol-end GSH organic monomer and 2,2-azobisisobutyronitrile (AIBN) were mixed with hydrolyzed tetramethyloxysilane (TMOS) and γ-methacryloxypropyltrimethoxysilane (γ-MAPS) and then introduced into a fused-silica capillary for simultaneous polycondensation and “thiol-ene” click reaction to form the GSH–silica hybrid monolith. The effects of the molar ratio of TMOS/γ-MAPS, the amount of GSH, and the volume of porogen on the morphology, permeability and pore properties of the prepared GSH–silica hybrid monoliths were studied in detail. A uniform monolithic network with high porosity was obtained. A series of test compounds including alkylbenzenes, amides, and anilines were used to evaluate the retention behaviors of the GSH–silica hybrid monolithic column. The results demonstrated that the prepared GSH–silica hybrid monolith exhibited multiple interactions including hydrophobicity, hydrophilicity, as well as cation exchange interaction. The run-to-run, column-to-column and batch-to-batch reproducibilities of the GSH–silica hybrid monolith for phenols’ retention were satisfactory with the relative standard deviations (RSDs) less than 1.3% (n=5), 2.6% (n=3) and 3.2% (n=3), respectively, indicating the effectiveness and practicability of the proposed method. In addition, the GSH–silica hybrid monolith was applied to the separation of nucleotides, peptides and protein tryptic digests, respectively. The successful applications suggested the potential of the GSH–silica hybrid monolith in complex sample analysis.

Quantification of Pancreatic Cancer Proteome and Phosphorylome: Indicates Molecular Events Likely Contributing to Cancer and Activity of Drug Targets

ObjectiveLC-MS/MS phospho-proteomics is an essential technology to help unravel the complex molecular events that lead to and propagate cancer. We have developed a global phospho-proteomic workflow to determine activity of signaling pathways and drug targets in pancreatic cancer tissue for clinical application.MethodsPeptides resulting from tryptic digestion of proteins extracted from frozen tissue of pancreatic ductal adenocarcinoma and background pancreas (n = 12), were labelled with tandem mass tags (TMT 8-plex), separated by strong cation exchange chromatography, then were analysed by LC-MS/MS directly or first enriched for phosphopeptides using IMAC and TiO2, prior to analysis. In-house, commercial and freeware bioinformatic platforms were used to identify relevant biological events from the complex dataset.ResultsOf 2,101 proteins identified, 152 demonstrated significant difference in abundance between tumor and non-tumor tissue. They included proteins that are known to be up-regulated in pancreatic cancer (e.g. Mucin-1), but the majority were new candidate markers such as HIPK1 & MLCK. Of the 6,543 unique phosphopeptides identified (6,284 unique phosphorylation sites), 635 showed significant regulation, particularly those from proteins involved in cell migration (Rho guanine nucleotide exchange factors & MRCKα) and formation of focal adhesions. Activator phosphorylation sites on FYN, AKT1, ERK2, HDAC1 and other drug targets were found to be highly modulated (≥2 fold) in different cases highlighting their predictive power.ConclusionHere we provided critical information enabling us to identify the common and unique molecular events likely contributing to cancer in each case. Such information may be used to help predict more bespoke therapy suitable for an individual case.

Reinforcement of Bacillus subtilis spores by cross-linking of outer coat proteins during maturation

Resistance characteristics of bacterial endospores towards various environmental stresses such as chemicals and heat are in part attributed to their coat proteins. Heat resistance is developed in a late stage of sporulation and during maturation of released spores. Using our gel-free proteomic approach and LC-FT-ICR-MS/MS analysis we have monitored the efficiency of the tryptic digestion of proteins in the coat during spore maturation over a period of eight days, using metabolically 15N labeled mature spores as reference. The results showed that during spore maturation the loss of digestion efficiency of outer coat and crust proteins synchronized with the increase in heat resistance. This implicates that spore maturation involves chemical cross-linking of outer coat and crust layer proteins leaving the inner coat layer proteins unmodified. It appears that digestion efficiencies of spore surface proteins can be linked to their location within the coat and crust layers. We also attempted to study a possible link between spore maturation and the observed heterogeneity in spore germination.

Enrichment and desalting of tryptic protein digests and the protein depletion using boron nitride

Sample preparation still remains a great challenge in modern bioanalysis and the interest in new efficient solid phase extraction (SPE) materials still remains high. In this work, hexagonal boron nitride (h-BN) is introduced as a new SPE material for the isolation and enrichment of peptides. The h-BN is isoelectronic and structurally similar to graphite. It has remarkable properties including good thermal conductivity, excellent thermal and chemical stability and a better oxidation resistance than graphite. BN attracts increasing interest because of its wide range of applicability. In the present work, the great potential of h-BN, as a new SPE-material, on the enrichment, preconcentration and desalting of tryptic digest of model proteins is demonstrated. A special attention was dedicated to the efficient enrichment of hydrophilic phosphopeptides. Two elution protocols were developed for the enrichment of peptides compatible for subsequent MALDI-MS and ESI-MS analysis. In addition, the recoveries of 5 peptides and 3 phosphopeptides with wide range of pI values utilizing h-BN materials with different surface areas were investigated. 84–106% recovery rate could be achieved using h-BN materials. The results were compared with those obtained using graphite and silica C18 under the same elution conditions, and lower recoveries were obtained. In addition, h-BN was found to have a capability of protein depletion, which is requisite for the peptide profiling.

Selective enrichment of phosphopeptides with aspartic acid based immobilized metal ion affinity chromatography materials

A natural amino acid, aspartic acid, was modified to bind to silica microspheres with a click chemistry method (-Click Asp).The modified Click Asp microspheres were further modified by incorporating Fe3+ ions, resulting in Fe3+ and Click Asp based immobilized ion affinity chromatography (IMAC)(Fe3+-Click Asp).The prepared materials were characterized using Fourier transform infrared spectrometry, X-ray photoelectron spectrometry, and scanning electron microscopy, and it was confirmed that Fe3+-Click Asp was successfully prepared.Subsequently, the Fe3+-Click Asp microspheres were used to enrich phosphopeptides from tryptic digests of proteins and milk.It was found that the phosphopeptides could be selectively enriched with this material.This study describes a novel material and enrichment method for phosphoproteomics.

Mass Spectrometry Analysis Coupled with de novo Sequencing Reveals Amino Acid Substitutions in Nucleocapsid Protein from Influenza A Virus

Amino acid substitutions in influenza A virus are the main reasons for both antigenic shift and virulence change, which result from non-synonymous mutations in the viral genome. Nucleocapsid protein (NP), one of the major structural proteins of influenza virus, is responsible for regulation of viral RNA synthesis and replication. In this report we used LC-MS/MS to analyze tryptic digestion of nucleocapsid protein of influenza virus (A/Puerto Rico/8/1934 H1N1), which was isolated and purified by SDS poly-acrylamide gel electrophoresis. Thus, LC-MS/MS analyses, coupled with manual de novo sequencing, allowed the determination of three substituted amino acid residues R452K, T423A and N430T in two tryptic peptides. The obtained results provided experimental evidence that amino acid substitutions resulted from non-synonymous gene mutations could be directly characterized by mass spectrometry in proteins of RNA viruses such as influenza A virus.

Differentiation of Cronobacter spp. by tryptic digestion of the cell suspension followed by MALDI-TOF MS analysis

Intact cell MALDI-TOF mass spectrometry is a rapid tool for the identification and classification of microorganisms, now widely used even in clinical laboratories. However, its distinctive power is not sufficient for some closely-related species. The genus Cronobacter, formerly known as Enterobacter sakazakii, contains such species. In this work, a new method for the differentiation of five Cronobacter species is presented involving the tryptic digestion of cytoplasmatic proteins followed by MALDI mass spectrometry analysis. A database was developed for use in Bruker Biotyper software including 52 reference spectra and tested on a set of 45 samples with an overall accuracy of about 80%. The possibility of measurement automation and the short time and low cost requirements of this method compared to those of biochemical tests or PCR methods make it a supplementary option to intact cell MALDI, providing additional information about the differentiation of problematic species.

Development of a novel imidazolium‐based aromatic quaternary ammonium tag: Synthesis and application to the efficient analysis of cysteinyl‐peptides by mass spectrometry

Chemical derivatization is a very promising technique for improving analysis of peptides by mass spectrometry (MS). In this study, a novel kind of imidazolium-based aromatic quaternary ammonium tag, 1-[3-[(2-iodo-1-oxoethyl)amino]propyl]-3-butylimidazolium bromide (IPBI), designed with strong gas-phase basicity and a permanent positive charge, was firstly synthesized and further used for derivatization of cysteinyl-peptides with improved ionization efficiency and higher charge states. Both the model peptides and tryptic digests of proteins were used to evaluate the effect of IPBI derivatization on the MS performance of the derivatized peptides, and the results were further compared with the commonly used iodoacetamide (IAA) tag. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)-MS and electrospray ionization (ESI)-MS were used to evaluate the ionization efficiency and charge states of the derivatized peptides. With model peptides as samples, a nearly 100% derivatization efficiency and superior stability were achieved via IPBI derivatization. By further analysis of both standard peptides and tryptic protein digests, the ionization efficiency and charge states of IPBI-derivatized peptides could be remarkably improved. For example, for protein bovine serum albumin, compared with the commercial available IAA tag, the identification efficiency of cysteinyl-peptides was increased about 67% by combining with IPBI derivatization. The results indicated that the novel tag is an effective derivatization reagent for cysteinyl-peptide identification. We hope it could be further used for high-efficiency cysteinyl-peptide identification in proteome research, especially those with low abundance and poor ionization efficiency.

Quantitative phosphoproteomic analysis of host responses in human lung epithelial (A549) cells during influenza virus infection

The emergence of antiviral drug-resistant influenza viruses highlights the need for alternative therapeutic strategies. Elucidation of host factors required during virus infection provides information not only on the signaling pathways involved but also on the identification of novel drug targets. RNA interference screening method had been utilized by several studies to determine these host factors; however, proteomics data on influenza host factors are currently limited.In this study, quantitative phosphoproteomic analysis of human lung cell line (A549) infected with 2009 pandemic influenza virus A (H1N1) virus was performed. Phosphopeptides were enriched from tryptic digests of total protein of infected and mock-infected cells using a titania column on an automated purification system followed by iTRAQ labeling. Identification and quantitative analysis of iTRAQ-labeled phosphopeptides were performed using LC–MS/MS.We identified 366 phosphorylation sites on 283 proteins. Of these, we detected 43 upregulated and 35 downregulated proteins during influenza virus infection. Gene ontology enrichment analysis showed that majority of the identified proteins are phosphoproteins involved in RNA processing, immune system process and response to infection. Host–virus interaction network analysis had identified 23 densely connected subnetworks. Of which, 13 subnetworks contained proteins with altered phosphorylation levels during by influenza virus infection. Our results will help to identify potential drug targets that can be pursued for influenza antiviral drug development.

Discovering innovative drugs and diagnostic tools to control haemostasis by venomics of the Vipera a. ammodytes snake

Snake venoms are a rich source of proteins acting on the haemostatic system, i.e. platelet aggregation, coagulation cascade and fibrinolysis. Many of these components have already been proved as useful for developing novel agents for treatment of vascular thromboembolic disorders (1). The venom of Vipera a. ammodytes (Vaa) snake causes different clinical disorders in man, the most pronounced of which are clotting disorders and haemorrhage. We have used a systematic proteomic approach to explore haemostatic activity of Vaa venom. With a two dimensional gel electrophoresis of the raw venom we obtained 208 discrete protein spots of molecular masses ranging from 10 to 70 kDa. Spots were excised from the gel and tryptic digests of proteins analysed by mass spectrometry. Identified proteins belong to 13 different families, including those that have a high potential to affect haemostasis, i.e. serine and metalloproteinases, phospholipases, C-type lectins and disintegrins. Our intent has however not been only to detect venom components acting on haemostasis but also to find components affecting particular parts of haemostatic system critical in some pathologies. In this way we have been targeting novel-pharmacological leads to develop pathology-specific therapeutics. For this reason in the first step the whole venom was fractionated by gel filtration and fractions were tested in vitro for their effect on different blood clotting factors, blood coagulation, and platelet aggregation and agglutination. In this screen we have detected several inhibitors of platelet aggregation and agglutination, probably acting on different platelet receptors and/or their ligands. Other components affected the process of blood coagulation, either activating different steps of this process or inhibiting it. In the second step, we have purified components in the venom fractions with haemostatic activity to homogeneity using various chromatographic technics in order to identify those responsible for effects at the molecular level. Some of them were present in rather small amounts. Thus the venom gland cDNA library from Vipera a. ammodytes was constructed, that will enable isolation of specific nucleotide sequences and synthesis of proteins using recombinant production.