Mass spectrometry-based protein profiling strategies for biomarker discovery in liver and inflammatory bowel diseases

The study of protein expression profiles for biomarker discovery in serum and in mammalian cell populations needs the continuous improvement and combination of proteins/peptides separation techniques, mass spectrometry, statistical and bioinformatic approaches. In this thesis work two different mass spectrometry-based protein profiling strategies have been developed and applied to liver and inflammatory bowel diseases (IBDs) for the discovery of new biomarkers. The first of them, based on bulk solid-phase extraction combined with matrix-assisted laser desorption/ionization - Time of Flight mass spectrometry (MALDI-TOF MS) and chemometric analysis of serum samples, was applied to the study of serum protein expression profiles both in IBDs (Crohn’s disease and ulcerative colitis) and in liver diseases (cirrhosis, hepatocellular carcinoma, viral hepatitis). The approach allowed the enrichment of serum proteins/peptides due to the high interaction surface between analytes and solid phase and the high recovery due to the elution step performed directly on the MALDI-target plate. Furthermore the use of chemometric algorithm for the selection of the variables with higher discriminant power permitted to evaluate patterns of 20-30 proteins involved in the differentiation and classification of serum samples from healthy donors and diseased patients. These proteins profiles permit to discriminate among the pathologies with an optimum classification and prediction abilities. In particular in the study of inflammatory bowel diseases, after the analysis using C18 of 129 serum samples from healthy donors and Crohn’s disease, ulcerative colitis and inflammatory controls patients, a 90.7% of classification ability and a 72.9% prediction ability were obtained. In the study of liver diseases (hepatocellular carcinoma, viral hepatitis and cirrhosis) a 80.6% of prediction ability was achieved using IDA-Cu(II) as extraction procedure. The identification of the selected proteins by MALDITOF/ TOF MS analysis or by their selective enrichment followed by enzymatic digestion and MS/MS analysis may give useful information in order to identify new biomarkers involved in the diseases. The second mass spectrometry-based protein profiling strategy developed was based on a label-free liquid chromatography electrospray ionization quadrupole - time of flight differential analysis approach (LC ESI-QTOF MS), combined with targeted MS/MS analysis of only identified differences. The strategy was used for biomarker discovery in IBDs, and in particular of Crohn’s disease. The enriched serum peptidome and the subcellular fractions of intestinal epithelial cells (IECs) from healthy donors and Crohn’s disease patients were analysed. The combining of the low molecular weight serum proteins enrichment step and the LCMS approach allowed to evaluate a pattern of peptides derived from specific exoprotease activity in the coagulation and complement activation pathways. Among these peptides, particularly interesting was the discovery of clusters of peptides from fibrinopeptide A, Apolipoprotein E and A4, and complement C3 and C4. Further studies need to be performed to evaluate the specificity of these clusters and validate the results, in order to develop a rapid serum diagnostic test. The analysis by label-free LC ESI-QTOF MS differential analysis of the subcellular fractions of IECs from Crohn’s disease patients and healthy donors permitted to find many proteins that could be involved in the inflammation process. Among them heat shock protein 70, tryptase alpha-1 precursor and proteins whose upregulation can be explained by the increased activity of IECs in Crohn’s disease were identified. Follow-up studies for the validation of the results and the in-depth investigation of the inflammation pathways involved in the disease will be performed. Both the developed mass spectrometry-based protein profiling strategies have been proved to be useful tools for the discovery of disease biomarkers that need to be validated in further studies.

Biomimetic Materials for Biomedical Applications

Objects with complex shape and functions have always attracted attention and interest. The morphological diversity and complexity of naturally occurring forms and patterns have been a motivation for humans to copy and adopt ideas from Nature to achieve functional, aesthetic and social value. Biomimetics is addressed to the design and development of new synthetic materials using strategies adopted by living organisms to produce biological materials. In particular, biomineralized tissues are often sophisticate composite materials, in which the components and the interfaces between them have been defined and optimized, and that present unusual and optimal chemical-physical, morphological and mechanical properties. Moreover, biominerals are generally produced by easily traceable raw materials, in aqueous media and at room pressure and temperature, that is through cheap process and materials. Thus, it is not surprising that the idea to mimic those strategies proper of Nature has been employed in several areas of applied sciences, such as for the preparation of liquid crystals, ceramic thin films computer switches and many other advanced materials. On this basis, this PhD thesis is focused on the investigation of the interaction of biologically active ions and molecules with calcium phosphates with the aim to develop new materials for the substitution and repair of skeletal tissue, according to the following lines: I. Modified calcium phosphates. A relevant part of this PhD thesis has been addressed to study the interaction of Strontium with calcium phosphates. It was demonstrated that strontium ion can substitute for calcium into hydroxyapatite, causing appreciable structural and morphological modifications. The detailed structural analysis carried out on the nanocrystals at different strontium content provided new insight into its interaction with the structure of hydroxyapatite. At variance with the behaviour of Sr towards HA, it was found that this ion inhibits the synthesis of octacalcium phosphate. However, it can substitute for calcium in this structure up to 15 atom %, in agreement with the increase of the cell parameters observed on increasing ion concentration. A similar behaviour was found for Magnesium ion, whereas Manganese inhibits the synthesis of octacalcium phosphate and it promotes the precipitation of dicalcium phosphate dehydrate. It was also found that Strontium affects the kinetics of the reaction of hydrolysis of α-TCP. It inhibits the conversion from α-TCP to hydroxyapatite. However, the resulting apatitic phase contains significant amounts of Sr2+ suggesting that the addition of Sr2+ to the composition of α-TCP bone cements could be successfully exploited for its local delivery in bone defects. The hydrolysis of α-TCP has been investigated also in the presence of increasing amounts of gelatin: the results indicated that this biopolymer accelerates the hydrolysis reaction and promotes the conversion of α-TCP into OCP, suggesting that its addition in the composition of calcium phosphate cements can be employed to modulate the OCP/HA ratio, and as a consequence the solubility, of the set cement. II. Deposition of modified calcium phosphates on metallic substrates. Coating with a thin film of calcium phosphates is frequently applied on the surface of metallic implants in order to combine the high mechanical strength of the metal with the excellent bioactivity of the calcium phosphates surface layers. During this PhD thesis, thank to the collaboration with prof. I.N. Mihailescu, head of the Laser-Surface-Plasma Interactions Laboratory (National Institute for Lasers, Plasma and Radiation Physics – Laser Department, Bucharest) Pulsed Laser Deposition has been successfully applied to deposit thin films of Sr substituted HA on Titanium substrates. The synthesized coatings displayed a uniform Sr distribution, a granular surface and a good degree of crystallinity which slightly decreased on increasing Sr content. The results of in vitro tests carried out on osteoblast-like and osteoclast cells suggested that the presence of Sr in HA thin films can enhance the positive effect of HA coatings on osteointegration and bone regeneration, and prevent undesirable bone resorption. The possibility to introduce an active molecule in the implant site was explored using Matrix Assisted Pulsed Laser Evaporation to deposit hydroxyapatite nanocrystals at different content of alendronate, a bisphosphonate widely employed in the treatments of pathological diseases associated to bone loss. The coatings displayed a good degree of crystallinity, and the results of in vitro tests indicated that alendronate promotes proliferation and differentiation of osteoblasts even when incorporated into hydroxyapatite. III. Synthesis of drug carriers with a delayed release modulated by a calcium phosphate coating. A core-shell system for modulated drug delivery and release has been developed through optimization of the experimental conditions to cover gelatin microspheres with a uniform layer of calcium phosphate. The kinetics of the release from uncoated and coated microspheres was investigated using aspirin as a model drug. It was shown that the presence of the calcium phosphate shell delays the release of aspirin and allows to modulate its action.

Cross-Talk tra Bifidobacterium e intestino umano: impatto sulle attività health promoting

Bifidobacteria constitute up to 3% of the total microbiota and represent one of the most important healthpromoting bacterial groups of the human intestinal microflora. The presence of Bifidobacterium in the human gastrointestinal tract has been directly related to several health-promoting activities; however, to date, no information about the specific mechanisms of interaction with the host is available. The first health-promoting activities studied in these job was the oxalate-degrading activity. Oxalic acid occurs extensively in nature and plays diverse roles, especially in pathological processes. Due to its highly oxidizing effects, hyper absorption or abnormal synthesis of oxalate can cause serious acute disorders in mammals and be lethal in extreme cases. Intestinal oxalate-degrading bacteria could therefore be pivotal in maintaining oxalate homeostasis, reducing the risk of kidney stone development. In this study, the oxalate-degrading activity of 14 bifidobacterial strains was measured by a capillary electrophoresis technique. The oxc gene, encoding oxalyl-CoA decarboxylase, a key enzyme in oxalate catabolism, was isolated by probing a genomic library of B. animalis subsp. lactis BI07, which was one of the most active strains in the preliminary screening. The genetic and transcriptional organization of oxc flanking regions was determined, unravelling the presence of other two independently transcribed open reading frames, potentially responsible for B. animalis subsp. lactis ability to degrade oxalate. Transcriptional analysis, using real-time quantitative reverse transcription PCR, revealed that these genes were highly induced in cells first adapted to subinhibitory concentrations of oxalate and then exposed to pH 4.5. Acidic conditions were also a prerequisite for a significant oxalate degradation rate, which dramatically increased in oxalate pre-adapted cells, as demonstrated in fermentation experiments with different pH-controlled batch cultures. These findings provide new insights in the characterization of oxalate-degrading probiotic bacteria and may support the use of B. animalis subsp. lactis as a promising adjunct for the prophylaxis and management of oxalate-related kidney disease. In order to provide some insight into the molecular mechanisms involved in the interaction with the host, in the second part of the job, we investigated whether Bifidobacterium was able to capture human plasminogen on the cell surface. The binding of human plasminogen to Bifidobacterium was dependent on lysine residues of surface protein receptors. By using a proteomic approach, we identified six putative plasminogen-binding proteins in the cell wall fraction of three strain of Bifidobacterium. The data suggest that plasminogen binding to Bifidobactrium is due to the concerted action of a number of proteins located on the bacterial cell surface, some of which are highly conserved cytoplasmic proteins which have other essential cellular functions. Our findings represent a step forward in understanding the mechanisms involved in the Bifidobacterium-host interaction. In these job w studied a new approach based on to MALDI-TOF MS to measure the interaction between entire bacterial cells and host molecular target. MALDI-TOF (Matrix Assisted Laser Desorption Ionization-Time of Flight)—mass spectrometry has been applied, for the first time, in the investigation of whole Bifidobacterium cells-host target proteins interaction. In particular, by means of this technique, a dose dependent human plasminogen-binding activity has been shown for Bifidobacterium. The involvement of lysine binding sites on the bacterial cell surface has been proved. The obtained result was found to be consistent with that from well-established standard methodologies, thus the proposed MALDI-TOF approach has the potential to enter as a fast alternative method in the field of biorecognition studies involving in bacterial cells and proteins of human origin.

Methodische Entwicklung der MALDI-TOF-Massenspektrometrie für Grenzbereiche der Polymeranalytik

Zusammenfassung der DoktorarbeitDie MALDI-TOF-Massenspektrometrie (Matrix Assisted Laser Desorption and Ionisation–Time Of Flight) ist in der Lage, Moleküle mit einem Molekulargewicht bis zu mehreren Hunderttausend Da intakt in die Gasphase zu überführen. Dabei wird die Fragmentierung des Analyten stark eingeschränkt bzw. gänzlich vermieden. Diese Methode findet daher zunehmend Verwendung für die Charakterisierung von Biopolymeren und synthetischen Polymeren. Ziel dieser Arbeit war, die MALDI-TOF-Massenspektrometrie zur Charakterisierung von Makromolekülen einzusetzen, bei denen die konventionellen polymeranalytischen Methoden nur unzureichende Informationen oder gar falsche bzw. gar keine Ergebnisse liefern. Mittels einer methodischen Entwicklung der MALDI-TOF-Massenspektrometrie gelang es, die bisherigen Grenzen der Methode zu erweitern und neue Anwendungsbereiche der Polymeranalytik aufzuzeigen. Anhand der erzielten Ergebnisse wurden darüber hinaus neue Erklärungsansätze formuliert, die zu einem besseren Verständnis des noch immer ungeklärten MALDI-Prozesses beitragen können. Besonders vielversprechend sind zum einen die Ergebnisse der Fragmentionenanalyse synthetischer Polymere und zum anderen die Charakterisierung von unlöslichen PAHs (Polycyclic Aromatic Hydrocarbons). Die Möglichkeiten und Aussagekraft der Fragmentionenanalyse wurde an synthetischen Polymeren getestet. Mit Hilfe dieser neuen Technik konnte die komplizierte Endgruppenverteilung einer Polycarbonat-Probe sowie die Zusammensetzung eines Poly-para-phenylenethynylen-b-Polyethylenoxid-Diblock-Copolymers eindeutig bestimmen werden, während die konventionellen MALDI-Massenspektren nur über einen wesentlich geringeren Informationsgehalt verfügten. Auf dem Gebiet der Analytik von unlöslichen PAHs wurde mit der Entwicklung einer neuen MALDI-Probenvorbereitung eine Methode gefunden, die über die PAH Analytik hinaus von großem Nutzen ist. Diese erstmalig angewendete Probenvorbereitung unterscheidet sich von den üblichen MALDI-Probenpräparationen, indem sie auf die Beteiligung eines Lösungsmittels vollkommen verzichtet. Damit konnte speziell ein unlöslicher, zuvor nicht nachweisbarer PAH von ca. 2700 Da mit MALDI eindeutig charakterisiert werden.

Identifizierung von T-Zell-erkannten Nierenzellkarzinom-Antigenen

Eine Voraussetzung für die Entwicklung neuer immunmodulatorischer Therapieverfahren ist die Kenntnis immunogener Tumorantigene, die von tumorreaktiven T-Zellen erkannt werden. In der vorliegenden Arbeit wurden tumorreaktive CD8+ zytotoxische T-Lymphozyten (CTL, cytotoxic T-lymphocytes) aus dem Blut eines HLA (human leukocyte antigen)-kompatiblen Fremdspenders generiert. Methodisch wurden hierzu CD8-selektionierte periphere Blutlymphozyten repetitiv mit der klarzelligen Nierenzellkarzinomlinie MZ1851-RCC (RCC, renal cell carcinoma) in einer allogenen gemischten Lymphozyten-Tumorzell Kultur (MLTC, mixed lymphocyte tumor cell culture) stimuliert. Aus den Responderlymphozyten wurden mit Hilfe des Grenzverdünnungsverfahrens klonale zytotoxische T-Zellen generiert und expandiert. Die CTL-Klone wurden anschließend phänotypisch mittels Durchflußzytometrie sowie funktionell mittels HLA-Antikörper-Blockadeexperimenten und Kreuzreaktivitätstests detailliert charakterisiert. Dabei konnte gezeigt werden, daß aus dem Blut eines allogenen gesunden Spenders CD8+ T-Zellen isoliert werden können, welche Reaktivität gegen Nierenzellkarzinome (NZK) aufweisen und über verschiedene HLA-Klasse-I-Allele restringiert sind. Die von den einzelnen CTL-Klonen erkannten Zielstrukturen zeigten entweder ubiquitäre (z.B. HLA-Cw*0704-reaktiver CTL-Klon E77) oder eine tumorspezifische (z.B. HLA-B*0702-restringierter CTL-Klon A4) Gewebeexpression. Zur Identifizierung der natürlich prozessierten Peptidliganden wurden die HLA-B/C-Allele unter Verwendung des monoklonalen Antikörpers B123.2 aus einem zuvor hergestellten Detergenslysat der Nierenzellkarzinomlinie MZ1851-RCC immunchromatographisch aufgereinigt. Aus den so isolierten HLA-Peptid-Komplexen wurden die tumorassoziierten Peptidliganden nach Säureeluation und Filtration abgespalten und über eine „reverse phase“-HPLC (high performance liquid chromatography) fraktioniert. Die Überprüfung der einzelnen HPLC-Fraktionen auf Bioaktivität erfolgte mit den korrespondierenden CTL-Klonen in 51Cr-Zytotoxizitätstests. Dabei wurde eine HPLC-Fraktion identifiziert, die die lytische Funktion des HLA-B*0702-restringierten CTL-Klons A4 auslösen konnte. Die bioaktive HPLC-Fraktion wurde dazu durch eine zweite (second dimension) Kapillar-Flüssigkeitschromatographie (Cap-LC, capillar liquid chromatography) in Subfraktionen geringerer Komplexität aufgetrennt und die darin enthaltenen Peptidepitope durch das MALDI-TOF/TOF (matrix assisted laser desorption/ionization- time of flight/time of flight)-Analyseverfahren sequenziert. Innerhalb dieser HPLC-Fraktion wurden eine Vielzahl von HLA-B/C-assoziierten Peptidliganden erfolgreich sequenziert, was die Effektivität dieser Verfahrenstechnik zur Identifizierung natürlich prozessierter HLA-Klasse-I-bindender Peptide unter Beweis stellt. Leider war es mit dieser Methode bisher nicht möglich, das von CTL-Klon A4 detektierte Peptidepitop zu sequenzieren. Dies liegt möglicherweise in der unzureichenden Konzentration des Peptidepitops in der bioaktiven HPLC-Fraktion begründet. In Folgearbeiten soll nun mit erhöhter Probenmenge beziehungsweise verbesserter Analytik der erneute Versuch unternommen werden, das Zielantigen des CTL-Klons A4 zu identifizieren. Die Kenntnis von Antigenen, die tumorspezifisch exprimiert und von CD8+ CTL aus gesunden Spendern erkannt werden, eröffnet neue therapeutische Möglichkeiten, das spezifische Immunsystem des Stammzellspenders nach allogener Blutstammzelltransplantation gezielt zur Steigerung von Tumorabstoßungsreaktionen (z.B. durch Vakzinierung oder adoptivem T-Zelltransfer) zu nutzen.

From MALDI-TOF mass spectrometry to soft-landing for electronics

Within this PhD thesis matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been used as a reliable tool for the quantitative characterization of giant molecules, such as alkyl substituted and unsubstituted large polycyclic aromatic hydrocarbons (PAH), which cannot be characterized by conventional analytic techniques due to their lack of solubility. The use of the MALDI solvent-free technique for the sample preparation and the application of the standard addition method have allowed the quantitative characterization of synthetic PAH mixtures. The knowledge, acquired by studying these representative systems, has been then transferred to the quantitative analyses of complex and slightly soluble natural PAH mixtures, such as mesophase pitch. Moreover, the possibility to ionize intractable and insoluble molecules via mass spectrometry has been recognized to be not only a powerful analytical method, but also to represent a unique change to handle giant aromatic systems and to deposit them on a surface for further investigations, in a process, which is defined as “soft-landing”. Within this novel deposition technique, ions of the desired analytes or analyte mixtures are generated by means of an MS ionization source, discriminated by their different mass to charge ratios via a mass analyzer and landed with retention of their structure on a desired surface. This soft-deposition is guaranteed by the use of decelerating potentials, which have in this work been recognized to influence the final packing of the analyte molecules reaching the landing surface. For a more detailed study of the electrical field action on disc-like and rod-like molecules, soft-landing-independent experiments have been additionally carried out. As a result unidirectionally ordered films of the analyte molecules have been obtained due to the application of an external electrical strength. This versatile alignment technique has then been used for obtaining ordered layers of semiconducting materials for the fabrication of organic field effect transistors (OFET) with improved performances.

Entwicklung und Evaluierung von Methoden zur zeitnahen Identifizierung weinrelevanter Mikroorganismen

Hefen der Gattung Saccharomyces und Milchsäurebakterien sind bei der Weinbereitung von besonderer Bedeutung. Neben der alkoholischen Gärung sind Hefen an der Ausbildung von Aromastoffen beteiligt. Milchsäurebakterien spielen eine Rolle beim biologischen Säureabbau (malolaktische Fermentation), können jedoch aufgrund ihrer Stoffwechseleigenschaft weitere Aromamodifikationen bewirken. Die Zusammensetzung der mikrobiellen Flora zu verschiedenen Zeitpunkten der Weinbereitung hat einen direkten Einfluss auf die Qualität der Weine, welche sich sowohl positiv als auch negativ verändern kann. Daher ist die zuverlässige Identifizierung und Differenzierung verschiedener Mikroorganismen auf Art- aber auch Stamm-Ebene während der Vinifikation von Bedeutung.rnDer erste Teil dieser Arbeit beschäftigte sich mit der Differenzierung von Hefearten der Gattung Saccharomyces, welche mit Hilfe konventioneller Methoden nicht eindeutig identifiziert werden können. Unter Verwendung des DNA-Fingerprintverfahrens Specifically Amplified Polymorphic DNA (SAPD)-PCR sowie der Matrix-Assisted-Laser-Desorption/Ionization-Time-Of-Flight-Mass-Spectrometry (MALDI-TOF-MS) war eine Differenzierung dieser taxonomisch sehr nah verwandten Arten möglich. Weiterhin konnten interspezifische Hybridstämme detektiert werden. In diesem Zusammenhang wurde der Hybridcharakter des Stammes NCYC 3739 (S. cerevisiae x kudriavzevii) entdeckt. Um die Elternspezies eines Hybridstamms zuverlässig zu bestimmen, sind weiterführende Genanalysen notwendig. Hierzu konnte eine Restriktionsfragmentlängenpolymorphismus (RFLP)-Analyse verschiedener genetischer Marker erfolgreich herangezogen werden.rnIm Rahmen dieser Arbeit wurde weiterhin ein Schnellidentifizierungssystem zum Nachweis weinrelevanter Milchsäurebakterien entwickelt. Mit Hilfe der Sequence Characterized Amplified Region (SCAR)-Technik konnten artspezifische Primer generiert werden, welche auf der Grundlage charakteristischer Fragmente der SAPD-PCR abgeleitet wurden. Durch die Anwendung dieser Primer in einer Multiplex-PCR-Reaktion war die Detektion verschiedener, einerseits häufig in Wein vorkommender und andererseits potentiell an der Ausbildung von Weinfehlern beteiligter Milchsäurebakterien-Arten möglich. Die ermittelte Nachweisgrenze dieser Methode lag mit 10^4 - 10^5 Zellen/ml im Bereich der Zelltiter, die in Most und Wein anzutreffen sind. Anhand der Untersuchung verschiedener Weinproben von Winzern in Rheinhessen wurde die Praxistauglichkeit dieser Methode demonstriert. rnUm die gesamten Milchsäurebakterien-Population im Verlauf der Weinbereitung zu kontrollieren, kann die Denaturierende Gradienten-Gelelektrophorese herangezogen werden. Hierzu wurden in dieser Arbeit Primer zur Amplifikation eines Teilbereichs des rpoB-Gens abgeleitet, da dieses Gen eine Alternative zur 16S rDNA darstellt. Die DNA-Region erwies sich als geeignet, um zahlreiche weinrelevante Milchsäurebakterien-Arten zu differenzieren. In einigen ersten Versuchen konnte gezeigt werden, dass diese Methode für eine praktische Anwendung in Frage kommt.rnOenococcus oeni ist das wichtigste Milchsäurebakterien während der malolaktischen Fermentation und wird häufig in Form kommerzieller Starterkulturen eingesetzt. Da verschiedene Stämme unterschiedliche Eigenschaften aufweisen können, ist es von Bedeutung, die Identität eines bestimmten Stammes zweifelsfrei feststellen zu können. Anhand der Analyse verschiedener O. oeni-Stämme aus unterschiedlichen Weinbaugebieten konnte gezeigt werden, dass sowohl die nested SAPD-PCR als auch die MALDI-TOF-MS genügend Sensitivität aufweisen, um eine Unterscheidung auf Stamm-Ebene zu ermöglichen, wobei die mittels nSAPD-PCR ermittelten Distanzen der Stämme zueinander mit deren geographischer Herkunft korrelierte.rnDie in der vorliegenden Arbeit entwickelten Methoden können dazu beitragen, den Prozess der Weinherstellung besser zu kontrollieren und so eine hohe Qualität des Endproduktes zu gewährleisten.rn

'Pergularain e I'--a plant cysteine protease with thrombin-like activity from Pergularia extensa latex

Pergularain e I, a cysteine protease with thrombin-like activity, was purified by ion exchange chromatography from the latex of Pergularia extensa. Its homogeneity was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), native PAGE and reverse-phase high-performance liquid chromatography (RP-HPLC). The molecular mass of pergularain e I by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) was found to be 23.356 kDa and the N-terminal sequence is L-P-H-D-V-E. Pergularain e I is a glycoprotein containing approximately 20% of carbohydrate. Pergularain e I constituted 6.7% of the total protein with a specific activity of 9.5 units/mg/min with a 2.11-fold increased purity. Proteolytic activity of the pergularain e I was completely inhibited by iodoacetic acid (IAA). Pergularain e I exhibited procoagulant activity with citrated plasma and fibrinogen similar to thrombin. Pergularain e I increases the absorbance of fibrinogen solution in concentration-dependent and time-dependent manner. At 10 microg concentration, an absorbance of 0.48 was reached within 10 min of incubation time. Similar absorbance was observed when 0.2 NIH units of thrombin were used. Thrombin-like activity of pergularain e I is because of the selective hydrolysis of A alpha and B beta chains of fibrinogen and gamma-chain was observed to be insusceptible to hydrolysis. Molecular masses of the two peptide fragments released from fibrinogen due to the hydrolysis by pergularain e I at 5-min incubation time were found to be 1537.21 and 1553.29 and were in close agreement with the molecular masses of 16 amino acid sequence of fibrinopeptide A and 14 amino acid sequence of fibrinopeptide B, respectively. Prolonged fibrinogen-pergularain e I incubation releases additional peptides and their sequence comparison of molecular masses of the released peptides suggested that pergularain e I hydrolyzes specifically after arginine residues.

Lipid remodelling of glycosylphosphatidylinositol (GPI) glycoconjugates in procyclic-form trypanosomes: biosynthesis and processing of GPIs revisited

The African trypanosome, Trypanosoma brucei, has been used as a model to study the biosynthesis of GPI (glycosylphosphatidylinositol) anchors. In mammalian (bloodstream)-form parasites, diacyl-type GPI precursors are remodelled in their lipid moieties before attachment to variant surface glycoproteins. In contrast, the GPI precursors of insect (procyclic)-form parasites, consisting of lyso-(acyl)PI (inositol-acylated acyl-lyso-phosphatidylinositol) species, remain unaltered before protein attachment. By using a combination of metabolic labelling, cell-free assays and complementary MS analyses, we show in the present study that GPI-anchored glycoconjugates in T. congolense procyclic forms initially receive tri-acylated GPI precursors, which are subsequently de-acylated either at the glycerol backbone or on the inositol ring. Chemical and enzymatic treatments of [3H]myristate-labelled lipids in combination with ESI-MS/MS (electrospray ionization-tandem MS) and MALDI-QIT-TOF-MS3 (matrix-assisted laser-desorption ionization-quadrupole ion trap-time-of-flight MS) analyses indicate that the structure of the lipid moieties of steady-state GPI lipids from T. congolense procyclic forms consist of a mixture of lyso-(acyl)PI, diacyl-PI and diacyl-(acyl)PI species. Interestingly, some of these species are myristoylated at the sn-2 position. To our knowledge, this is the first demonstration of lipid remodelling at the level of protein- or polysaccharide-linked GPI anchors in procyclic-form trypanosomes.

Glycomic analysis of human mast cells, eosinophils and basophils

In allergic diseases such as asthma, eosinophils, basophils and mast cells, through release of preformed and newly generated mediators, granule proteins and cytokines, are recognized as key effector cells. While their surface protein phenotypes, mediator release profiles, ontogeny, cell trafficking and genomes have been generally explored and compared, there has yet to be any thorough analysis and comparison of their glycomes. Such studies are critical to understand the contribution of carbohydrates to the induction and regulation of allergic inflammatory responses and are now possible using improved technologies for detecting and characterizing cell-derived glycans. We thus report here the application of high-sensitivity mass spectrometric-based glycomics methodologies to the analysis of N-linked glycans derived from isolated populations of human mast cells, eosinophils and basophils. The samples were subjected to matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) screening analyses and MALDI-TOF/TOF sequencing studies. Results reveal substantive quantities of terminal N-acetylglucosamine containing structures in both the eosinophil and the basophil samples, whereas mast cells display greater relative quantities of sialylated terminal epitopes. For the first time, we characterize the cell surface glycan structures of principal allergic effector cells, which by interaction with glycan-binding proteins (e.g. lectins) have the possibility to dictate cellular functions, and might thus have important implications for the pathogenesis of inflammatory and allergic diseases.

A new variant of Actinobacillus pleuropneumoniae serotype 3 lacking the entire apxII operon

Actinobacillus pleuropneumoniae is an important respiratory pathogen causing pleuropneumonia in pig. The species is genetically characterized by the presence of 4 RTX (Repeats in the Structural ToXin) toxin genes: apxI, apxII, and apxIII genes are differentially present in various combinations among the different serotypes, thereby defining pathogenicity; the apxIV gene is present in all serotypes. Polymerase chain reaction (PCR)-based apx gene typing is done in many veterinary diagnostic laboratories, especially reference laboratories. The present report describes the isolation of atypical A. pleuropneumoniae from 4 independent cases from 2 countries. All isolates were beta-nicotinamide adenine dinucleotide (beta-NAD) dependent and nonhemolytic but showed strong co-hemolysis with the sphingomyelinase of Staphylococcus aureus on sheep blood agar. Classical biochemical tests as well as Matrix-assisted laser desorption ionization time-of-flight mass spectrometry and sequence-based analysis (16S ribosomal RNA [rRNA] and rpoB genes) identified them as A. pleuropneumoniae. Apx-toxin gene typing using 2 different PCR systems showed the presence of apxIV and only the apxIII operon (apxIIICABD). None of the apxI or apxII genes were present as confirmed by Southern blot analysis. The 16S rRNA and rpoB gene analyses as well as serotype-specific PCR indicate that the isolates are variants of serotype 3. Strains harboring only apxIV and the apxIII operon are possibly emerging types of A. pleuropneumoniae and should therefore be carefully monitored for epidemiological reasons.

Proteomic and peptidomic study of proteolysis in quarter milk after infusion with lipoteichoic acid from Staphylococcus aureus

Mastitic milk is associated with increased bovine protease activity, such as that from plasmin and somatic cell enzymes, which cause proteolysis of the caseins and may reduce cheese yield and quality. The aim of this work was to characterize the peptide profile resulting from proteolysis in a model mastitis system and to identify the proteases responsible. One quarter of each of 2 cows (A and B) was infused with lipoteichoic acid from Staphylococcus aureus. The somatic cell counts of the infused quarters reached a peak 6h after infusion, whereas plasmin activity of those quarters also increased, reaching a peak after 48 and 12h for cow A and B, respectively. Urea-polyacrylamide gel electrophoretograms of milk samples of cow A and B obtained at different time points after infusion and incubated for up to 7 d showed almost full hydrolysis of beta- and alpha(S1)-casein during incubation of milk samples at peak somatic cell counts, with that of beta-casein being faster than that of alpha(S1)-casein. Two-dimensional gel electrophoretograms of milk 6h after infusion with the toxin confirmed hydrolysis of beta- and alpha(S1)-casein and the appearance of lower-molecular-weight products. Peptides were subsequently separated by reversed-phase HPLC and handmade nanoscale C(18) columns, and identified by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. Twenty different peptides were identified and shown to originate from alpha(s1)- and beta-casein. Plasmin, cathepsin B and D, elastase, and amino- and carboxypeptidases were suggested as possible responsible proteases based on the peptide cleavage sites. The presumptive activity of amino- and carboxypeptidases is surprising and may indicate the activity of cathepsin H, which has not been reported in milk previously.

A mass spectrometric multicenter study supports classification of preeclampsia as heterogeneous disorder

The diagnostic value of affinity-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis to distinguish preeclampsia (PE) from matched controls was tested in a multicenter setting.

Mass spectrometry-based analytical tools for the molecular protein characterization of human plasma lipoproteins

Lipoproteins are a heterogeneous population of blood plasma particles composed of apolipoproteins and lipids. Lipoproteins transport exogenous and endogenous triglycerides and cholesterol from sites of absorption and formation to sites of storage and usage. Three major classes of lipoproteins are distinguished according to their density: high-density (HDL), low-density (LDL) and very low-density lipoproteins (VLDL). While HDLs contain mainly apolipoproteins of lower molecular weight, the two other classes contain apolipoprotein B and apolipoprotein (a) together with triglycerides and cholesterol. HDL concentrations were found to be inversely related to coronary heart disease and LDL/VLDL concentrations directly related. Although many studies have been published in this area, few have concentrated on the exact protein composition of lipoprotein particles. Lipoproteins were separated by density gradient ultracentrifugation into different subclasses. Native gel electrophoresis revealed different gel migration behaviour of the particles, with less dense particles having higher apparent hydrodynamic radii than denser particles. Apolipoprotein composition profiles were measured by matrix-assisted laser desorption/ionization-mass spectrometry on a macromizer instrument, equipped with the recently introduced cryodetector technology, and revealed differences in apolipoprotein composition between HDL subclasses. By combining these profiles with protein identifications from native and denaturing polyacrylamide gels by liquid chromatography-tandem mass spectrometry, we characterized comprehensively the exact protein composition of different lipoprotein particles. We concluded that the differential display of protein weight information acquired by macromizer mass spectrometry is an excellent tool for revealing structural variations of different lipoprotein particles, and hence the foundation is laid for the screening of cardiovascular disease risk factors associated with lipoproteins.

Prevalence and risk factors for carriage of multi-drug resistant Staphylococci in healthy cats and dogs

We investigated the distribution of commensal staphylococcal species and determined the prevalence of multi-drug resistance in healthy cats and dogs. Risk factors associated with the carriage of multi-drug resistant strains were explored. Isolates from 256 dogs and 277 cats were identified at the species level using matrix-assisted laser desorption ionisation-time of flight mass spectrometry. The diversity of coagulase-negative Staphylococci (CNS) was high, with 22 species in dogs and 24 in cats. Multi-drug resistance was frequent (17%) and not always associated with the presence of the mecA gene. A stay in a veterinary clinic in the last year was associated with an increased risk of colonisation by multi-drug resistant Staphylococci (OR = 2.4, 95% CI: 1.1˜5.2, p value LRT = 0.04). When identifying efficient control strategies against antibiotic resistance, the presence of mechanisms other than methicillin resistance and the possible role of CNS in the spread of resistance determinants should be considered.