New sensing platforms based on nanoscopic devices for probing protein-membrane interactions

A novel nanosized and addressable sensing platform based on membrane coated plasmonic particles for detection of protein adsorption using dark field scattering spectroscopy of single particles has been established. To this end, a detailed analysis of the deposition of gold nanorods on differently functionalized substrates is performed in relation to various factors (such as the pH, ionic strength, concentration of colloidal suspension, incubation time) in order to find the optimal conditions for obtaining a homogenous distribution of particles at the desired surface number density. The possibility of successfully draping lipid bilayers over the gold particles immobilized on glass substrates depends on the careful adjustment of parameters such as membrane curvature and adhesion properties and is demonstrated with complementary techniques such as phase imaging AFM, fluorescence microscopy (including FRAP) and single particle spectroscopy. The functionality and sensitivity of the proposed sensing platform is unequivocally certified by the resonance shifts of the plasmonic particles that were individually interrogated with single particle spectroscopy upon the adsorption of streptavidin to biotinylated lipid membranes. This new detection approach that employs particles as nanoscopic reporters for biomolecular interactions insures a highly localized sensitivity that offers the possibility to screen lateral inhomogeneities of native membranes. As an alternative to the 2D array of gold nanorods, short range ordered arrays of nanoholes in optically transparent gold films or regular arrays of truncated tetrahedron shaped particles are built by means of colloidal nanolithography on transparent substrates. Technical issues mainly related to the optimization of the mask deposition conditions are successfully addressed such that extended areas of homogenously nanostructured gold surfaces are achieved. Adsorption of the proteins annexin A1 and prothrombin on multicomponent lipid membranes as well as the hydrolytic activity of the phospholipase PLA2 were investigated with classical techniques such as AFM, ellipsometry and fluorescence microscopy. At first, the issues of lateral phase separation in membranes of various lipid compositions and the dependency of the domains configuration (sizes and shapes) on the membrane content are addressed. It is shown that the tendency for phase segregation of gel and fluid phase lipid mixtures is accentuated in the presence of divalent calcium ions for membranes containing anionic lipids as compared to neutral bilayers. Annexin A1 adsorbs preferentially and irreversibly on preformed phosphatidylserine (PS) enriched lipid domains but, dependent on the PS content of the bilayer, the protein itself may induce clustering of the anionic lipids into areas with high binding affinity. Corroborated evidence from AFM and fluorescence experiments confirm the hypothesis of a specifically increased hydrolytic activity of PLA2 on the highly curved regions of membranes due to a facilitated access of lipase to the cleavage sites of the lipids. The influence of the nanoscale gold surface topography on the adhesion of lipid vesicles is unambiguously demonstrated and this reveals, at least in part, an answer for the controversial question existent in the literature about the behavior of lipid vesicles interacting with bare gold substrates. The possibility of formation monolayers of lipid vesicles on chemically untreated gold substrates decorated with gold nanorods opens new perspectives for biosensing applications that involve the radiative decay engineering of the plasmonic particles.

Structured antibody surfaces for bio-recognition and a label-free detection of bacteria

Antibody microarrays are of great research interest because of their potential application as biosensors for high-throughput protein and pathogen screening technologies. In this active area, there is still a need for novel structures and assemblies providing insight in binding interactions such as spherical and annulus-shaped protein structures, e.g. for the utilization of curved surfaces for the enhanced protein-protein interactions and detection of antigens. Therefore, the goal of the presented work was to establish a new technique for the label-free detection of bio-molecules and bacteria on topographically structured surfaces, suitable for antibody binding.rnIn the first part of the presented thesis, the fabrication of monolayers of inverse opals with 10 μm diameter and the immobilization of antibodies on their interior surface is described. For this purpose, several established methods for the linking of antibodies to glass, including Schiff bases, EDC/S-NHS chemistry and the biotin-streptavidin affinity system, were tested. The employed methods included immunofluorescence and image analysis by phase contrast microscopy. It could be shown that these methods were not successful in terms of antibody immobilization and adjacent bacteria binding. Hence, a method based on the application of an active-ester-silane was introduced. It showed promising results but also the need for further analysis. Especially the search for alternative antibodies addressing other antigens on the exterior of bacteria will be sought-after in the future.rnAs a consequence of the ability to control antibody-functionalized surfaces, a new technique employing colloidal templating to yield large scale (~cm2) 2D arrays of antibodies against E. coli K12, eGFP and human integrin αvβ3 on a versatile useful glass surface is presented. The antibodies were swept to reside around the templating microspheres during solution drying, and physisorbed on the glass. After removing the microspheres, the formation of annuli-shaped antibody structures was observed. The preserved antibody structure and functionality is shown by binding the specific antigens and secondary antibodies. The improved detection of specific bacteria from a crude solution compared to conventional “flat” antibody surfaces and the setting up of an integrin-binding platform for targeted recognition and surface interactions of eukaryotic cells is demonstrated. The structures were investigated by atomic force, confocal and fluorescence microscopy. Operational parameters like drying time, temperature, humidity and surfactants were optimized to obtain a stable antibody structure.

Development and applications of hollow fiber flow field-flow fractionation in the bioanalytical field. Studies of aggregation phenomena in complex protein samples

Recent advances in the fast growing area of therapeutic/diagnostic proteins and antibodies - novel and highly specific drugs - as well as the progress in the field of functional proteomics regarding the correlation between the aggregation of damaged proteins and (immuno) senescence or aging-related pathologies, underline the need for adequate analytical methods for the detection, separation, characterization and quantification of protein aggregates, regardless of the their origin or formation mechanism. Hollow fiber flow field-flow fractionation (HF5), the miniaturized version of FlowFFF and integral part of the Eclipse DUALTEC FFF separation system, was the focus of this research; this flow-based separation technique proved to be uniquely suited for the hydrodynamic size-based separation of proteins and protein aggregates in a very broad size and molecular weight (MW) range, often present at trace levels. HF5 has shown to be (a) highly selective in terms of protein diffusion coefficients, (b) versatile in terms of bio-compatible carrier solution choice, (c) able to preserve the biophysical properties/molecular conformation of the proteins/protein aggregates and (d) able to discriminate between different types of protein aggregates. Thanks to the miniaturization advantages and the online coupling with highly sensitive detection techniques (UV/Vis, intrinsic fluorescence and multi-angle light scattering), HF5 had very low detection/quantification limits for protein aggregates. Compared to size-exclusion chromatography (SEC), HF5 demonstrated superior selectivity and potential as orthogonal analytical method in the extended characterization assays, often required by therapeutic protein formulations. In addition, the developed HF5 methods have proven to be rapid, highly selective, sensitive and repeatable. HF5 was ideally suitable as first dimension of separation of aging-related protein aggregates from whole cell lysates (proteome pre-fractionation method) and, by HF5-(UV)-MALS online coupling, important biophysical information on the fractionated proteins and protein aggregates was gathered: size (rms radius and hydrodynamic radius), absolute MW and conformation.

Functional characterisation of in vitro synthesised G-protein coupled receptors in polymersomes

Membrane proteins play an indispensable role in physiological processes. It is, therefore, not surprising that many diseases are based on the malfunction of membrane proteins. Hence membrane proteins and especially G-protein coupled receptors(GPCRs)- the largest subfamily- have become an important drug target. Due to their high selectivity and sensitivity membrane proteins are also feasible for the detection of small quantities of substances with biosensors. Despite this widespread interest in GPCRs due to their importance as drug targets and biosensors there is still a lack of knowledge of structure, function and endogenous ligands for quiet a few of the previously identified receptors.rnBottlenecks in over-expression, purification, reconstitution and handling of membrane proteins arise due to their hydrophobic nature. Therefore the production of reasonable amounts of functional membrane proteins for structural and functional studies is still challenging. Also the limited stability of lipid based membrane systems hampers their application as platforms forrnscreening applications and biosensors.rnIn recent years the in vitro protein synthesis became a promising alternative to gain better yields for expression of membrane proteins in bio-mimetic membrane systems. These expression systems are based on cell extracts. Therefore cellular effects on protein expression are reduced. The open nature of the cell-free expression systems easily allows for the adjustment of reactionrnconditions for the protein of interest. The cell-free expression in the presence of bio-mimetic membrane systems allows the direct incorporation of the membrane proteins and therefore skips the time-consuming purification and reconstitution processes. Amphiphilic block-copolymers emerged as promising alternative for the less stable lipid-based membrane systems. They, likernlipids, form membraneous structures in aqueous solutions but exhibit increased mechanical and chemical stability.rnThe aim of this work was the generation of a GPCR-functionalised membrane system by combining both promising alternatives: in vitro synthesis and polymeric membrane systems. This novel platform should be feasible for the characterisation of the incorporated GPCR. Immunodetection of Dopamine receptor 1 and 2 expressed in diblock- and triblock-polymersomes demonstrated the successful in vitro expression of GPCRs in polymeric membranes. Antibodyrnbinding studies suggested a favoured orientation of dopamine receptors in triblockpolymersomes.rnA dopamine-replacement assay on DRD2-functionalised immobilised triblockpolymersomes confirmed functionality of the receptor in the polymersomes. The altered binding curve suggests an effect of the altered hydrophobic environment presented by the polymer membrane on protein activity.

Proteomics-driven approach for the detection of breast cancer biomarkers

Breast cancer (BC) is the most often diagnosed cancer entity of women worldwide. No molecular biomarkers are usable in the clinical routine for the early detection of BC. Proteomics is one of the dynamic tools for the successful examination of changes on the protein level. In this thesis different proteomics-based investigations were performed for the detection of protein and autoantibody biomarkers in serum samples of BC and healthy (CTRL) subjects. First, protein levels of candidates from previous profiling studies were investigated via antibody-microarray platform. Three proteins were found in distinct levels in both groups: secretoglobin family 1D member 1, alpha-2 macroglobulin and inter-alpha-trypsin inhibitor heavy chain family member 4. The second part was dedicated to the de novo exploration of potentially immunogenic tumor antigens (TA’s) with immunoprecipitation and Western immunoblotting followed by identification over mass spectrometry. Autoantibody levels were verified in individual serum profiling via the protein microarray platform. Two autoantibody’ cohorts (anti-Histone 2B and anti-Recoverin) were found in different levels in both groups. The findings of this PhD thesis underline deregulated serum protein and autoantibody levels in the presence of BC. Further investigations are needed to confirm the results in an independent study population.

Funktionelle Charakterisierung der Protein-Kinase CK2 in der Suppression Th2-vermittelter Immunantworten durch regulatorische T-Zellen

Regulatorische T-Zellen (Tregs) leisten durch ihre suppressiven Eigenschaften einen essenziellen Beitrag zur Aufrechterhaltung der immunologischen Toleranz. Sie verhindern schädliche Immunreaktionen gegen Autoantigene, kommensale Bakterien, sowie harmlose Nahrungsmittel-bestandteile. Gleichzeitig gewährleisten sie die Entwicklung effektiver Immunantworten gegen eindringende Pathogene, wie z.B. Parasiten, Bakterien und Viren. Damit haben Tregs direkten Einfluss auf das Gleichgewicht zwischen Immunität und Toleranz. Fehler in der suppressiven Funktionsweise von Tregs begünstigen daher auf der einen Seite die Entstehung zahlreicher autoimmuner Erkrankungen und Allergien. Auf der anderen Seite können Tregs Immunreaktionen bei chronischen Infektionen reduzieren, sowie die Entstehung effektiver Immunantworten gegen Tumore hemmen. Ihre Beteiligung an der Ätiologie all dieser Krankheiten macht Tregs zu einem bedeutenden potenziellen Zielobjekt, um diese Krankheiten effektiv zu therapieren. Die Erweiterung des Grundwissens um die molekularen Mechanismen der Treg-vermittelten Suppression ist daher ein notwendiger Schritt bei der Entwicklung Treg-basierter Theraphieansätze. 2003 konnte mit Foxp3 ein Transkriptionsfaktor identifiziert werden, der maßgeblich die suppressiven Funktionen von Tregs steuert. Um weiteren Einblick in die der Suppression zugrundeliegenden Signalwege zu erhalten, wurde im Institut für Immunologie ein komparativer Kinomarray durchgeführt, anhand dessen die Casein Kinase 2 (CK2) als eine der aktivsten Kinasen in Tregs identifiziert wurde (Daten freundlicherweise von Prof. Dr. Tobias Bopp bereitgestellt). rnBasierend auf den Ergebnissen des Kinomarrays wurde in dieser Arbeit die Funktion der CK2 in Tregs untersucht. Dabei konnte in in vitro Experimenten die Treg-vermittelte Suppression durch den pharmakologische CK2 Inhibitor DMAT aufgehoben werden. Weil derartige Inhibitoren jedoch nicht absolut spezifisch die Aktivität nur einer Kinase supprimieren, wurden außerdem Mäuse mit konditionalem „knockout“ der CK2β Untereinheit spezifisch in Tregs gekreuzt (CK2βTreg-/- Mäuse). Die Analyse dieser Tiere offenbarte eine essenzielle Beteiligung der CK2 an den suppressiven Funktionen von Tregs. So entwickeln CK2βTreg-/- Mäuse mit zunehmendem Alter Splenomegalien und Lymphadenopathien, von denen in besonderem Maße die Mukosa-assoziierten Lymphknoten betroffen sind. Eine Analyse des Aktivierungsstatus der T-Zellen in den Tieren konnte zudem einen erhöhten Anteil sogenannter Effektor-Gedächtnis T-Zellen aufdecken, die charakteristische Merkmale eines Th2 Phänotyps zeigten. Erhöhte Titer des Antikörperisotyps IgE in den Seren von CK2βTreg-/- Mäusen suggerieren zusätzlich eine fehlerhafte Suppression speziell Th2-vermittelter Immunantworten durch CK2β-defiziente Tregs. In Th2-vermittelten Asthma Experimenten in vivo konnte der Verdacht der fehlerhaften Kontrolle von Th2-Antwort bestätigt werden, wobei zusätzlich aufgedeckt wurde, dass bereits unbehandelte CK2βTreg-/- Mäuse Zeichen einer Entzündungsreaktion in der Lunge aufweisen. Bei der Suche nach den molekularen Ursachen der fehlerhaften Suppression Th2-vermittelter Immunantworten durch CK2β-defiziente Tregs konnten zwei mögliche Erklärungsansätze gefunden werden. Zum einen zeigen CK2β-defiziente Tregs eine verringerte Expression von Foxp3, was, in Analogie zu Ergebnissen der Gruppe von R. Flavell (Wang Y.Y. Nature. 445, 766-770 (2007)), zu einer Konversion von Tregs zu Th2 Zellen und damit zur Entstehung eines Th2-basierten, autoimmunen Phänotyps führt. Des Weiteren weisen CK2β-defiziente Tregs eine reduzierte Expression des Transkriptionsfaktors IRF4 auf, der in Tregs entscheidend für die Kontrolle Th2-basierter Immunreaktionen ist (Zheng Y. Nature. 19; 351-356 (2009)). Die dargelegten Ergebnisse identifizieren die CK2 damit als Kinase, die entscheidend an der Treg-vermittelten Suppression speziell Th2-basierter Immunantworten beteiligt ist. Demnach könnten pharmakologische CK2 Inhibitoren beispielsweise dazu eingesetzt werden, um die Treg-vermittelte Suppression im Rahmen chronischer Parasiten-Infektionen aufzuheben. Die in CK2βTreg-/- Mäusen beobachtete Prävalenz der Funktion der CK2 für Mukosa-assoziierte Organe stellt dabei einen zusätzlichen Vorteil dar, weil systemische Nebenwirkungen, die durch die Blockade der Treg-vermittelte Suppression entstehen, zumindest in nicht-Mukosa-assoziierten Geweben nicht zu erwarten sind.rn

Immunofluorescence versus xTAG multiplex PCR for the detection of respiratory picornavirus infections in children

BACKGROUND: Polymerase chain reaction (PCR) is a sensitive tool for detection of respiratory picornaviruses. However, the clinical relevance of picornavirus detection by PCR is unclear. Immunofluorescence (IF), widely used to detect other respiratory viruses, has recently been introduced as a promising detection method for respiratory picornaviruses. OBJECTIVES: To compare the clinical manifestations of respiratory picornavirus infections detected by IF with those of respiratory picornavirus infections detected by xTAG multiplex PCR in hospitalized children. STUDY DESIGN: During a 1-year period, nasopharyngeal aspirates (NPA) from all children hospitalized due to an acute respiratory infection were prospectively analyzed by IF. All respiratory picornavirus positive IF samples and 100 IF negative samples were further tested with xTAG multiplex PCR. After exclusion of children with co-morbidities and viral co-infections, monoinfections with respiratory picornaviruses were detected in 108 NPA of 108 otherwise healthy children by IF and/or PCR. We compared group 1 children (IF and PCR positive, n=84) with group 2 children (IF negative and PCR positive, n=24) with regard to clinical manifestations of the infection. RESULTS: Wheezy bronchitis was diagnosed more often in group 1 than in group 2 (71% vs. 46%, p=0.028). In contrast, group 2 patients were diagnosed more frequently with pneumonia (17% vs. 6%, p=0.014) accompanied by higher levels of C-reactive protein (46mg/l vs. 11mg/l, p=0.009). CONCLUSIONS: Picornavirus detection by IF in children with acute respiratory infection is associated with the clinical presentation of wheezy bronchitis. The finding of a more frequent diagnosis of pneumonia in picornavirus PCR positive but IF negative children warrants further investigation.

Mining for Class-Specific Motifs in Protein Sequence Classification

Background: In protein sequence classification, identification of the sequence motifs or n-grams that can precisely discriminate between classes is a more interesting scientific question than the classification itself. A number of classification methods aim at accurate classification but fail to explain which sequence features indeed contribute to the accuracy. We hypothesize that sequences in lower denominations (n-grams) can be used to explore the sequence landscape and to identify class-specific motifs that discriminate between classes during classification. Discriminative n-grams are short peptide sequences that are highly frequent in one class but are either minimally present or absent in other classes. In this study, we present a new substitution-based scoring function for identifying discriminative n-grams that are highly specific to a class. Results: We present a scoring function based on discriminative n-grams that can effectively discriminate between classes. The scoring function, initially, harvests the entire set of 4- to 8-grams from the protein sequences of different classes in the dataset. Similar n-grams of the same size are combined to form new n-grams, where the similarity is defined by positive amino acid substitution scores in the BLOSUM62 matrix. Substitution has resulted in a large increase in the number of discriminatory n-grams harvested. Due to the unbalanced nature of the dataset, the frequencies of the n-grams are normalized using a dampening factor, which gives more weightage to the n-grams that appear in fewer classes and vice-versa. After the n-grams are normalized, the scoring function identifies discriminative 4- to 8-grams for each class that are frequent enough to be above a selection threshold. By mapping these discriminative n-grams back to the protein sequences, we obtained contiguous n-grams that represent short class-specific motifs in protein sequences. Our method fared well compared to an existing motif finding method known as Wordspy. We have validated our enriched set of class-specific motifs against the functionally important motifs obtained from the NLSdb, Prosite and ELM databases. We demonstrate that this method is very generic; thus can be widely applied to detect class-specific motifs in many protein sequence classification tasks. Conclusion: The proposed scoring function and methodology is able to identify class-specific motifs using discriminative n-grams derived from the protein sequences. The implementation of amino acid substitution scores for similarity detection, and the dampening factor to normalize the unbalanced datasets have significant effect on the performance of the scoring function. Our multipronged validation tests demonstrate that this method can detect class-specific motifs from a wide variety of protein sequence classes with a potential application to detecting proteome-specific motifs of different organisms.

Determination of free and total valproic acid in human plasma by capillary electrophoresis with contactless conductivity detection

A new approach for the determination of free and total valproic acid in small samples of 140 μL human plasma based on capillary electrophoresis with contactless conductivity detection is proposed. A dispersive liquid-liquid microextraction technique was employed in order to remove biological matrices prior to instrumental analysis. The free valproic acid was determined by isolating free valproic acid from protein-bound valproic acid by ultrafiltration under centrifugation of 100 μL sample. The filtrate was acidified to turn valproic acid into its protonated neutral form and then extracted. The determination of total valproic acid was carried out by acidifying 40 μL untreated plasma to release the protein-bound valproic acid prior to extraction. A solution consisting of 10 mM histidine, 10 mM 3-(N-morpholino)propanesulfonic acid and 10 μM hexadecyltrimethylammonium bromide of pH 6.5 was used as background electrolyte for the electrophoretic separation. The method showed good linearity in the range of 0.4-300 μg/mL with a correlation coefficient of 0.9996. The limit of detection was 0.08 μg/mL, and the reproducibility of the peak area was excellent (RSD=0.7-3.5%, n=3, for the concentration range from 1 to 150 μg/mL). The results for the free and total valproic acid concentration in human plasma were found to be comparable to those obtained with a standard immunoassay. The corresponding correlation coefficients were 0.9847 for free and 0.9521 for total valproic acid.

Assessment of estrogenic exposure in brown trout (Salmo trutta) in a Swiss midland river: integrated analysis of passive samplers, wild and caged fish, and vitellogenin mRNA and protein

This field study examined the vitellogenin (VTG) biomarker response under conditions of low and fluctuating activities of environmental estrogenicity. The present study was performed on immature brown trout (Salmo trutta) exposed to the small river Luetzelmurg, which is located in the prealpine Swiss midland region and receives effluents from a single sewage treatment plant (STP). To understand better factors influencing the relationship between estrogenic exposure and VTG induction, we compared VTG levels in caged (stationary) and feral (free-ranging) fish, VTG levels in fish from up- and downstream of the STP, and two different methods for quantifying VTG (enzyme-linked immunosorbent assay vs real-time reverse transcription-polymerase chain reaction), and we used passive samplers (polar organic chemical integrative sampler [POCIS]) to integrate the variable, bioaccumulative estrogenic load in the river water over time. The POCIS from the downstream site contained approximately 20-fold higher levels of bioassay-derived estrogen equivalents than the POCIS from the upstream site. In feral fish, this site difference in estrogenic exposure was reflected in VTG protein levels but not in VTG mRNA. In contrast, in caged fish, the site difference was evident only for VTG mRNA but not for VTG protein. Thus, the outcome of VTG biomarker measurements varied with the analytical detection method (protein vs mRNA) and with the exposure modus (caged vs feral). Our findings suggest that for environmental situations with low and variable estrogenic contamination, a multiple-assessment approach may be necessary for the assessment of estrogenic exposure in fish.

Rapid measurement of S-100B serum protein levels by Elecsys S100 immunoassay in patients undergoing carotid artery stenting or endarterectomy

OBJECTIVES:: This study was designed to apply the rapid Elecsys(R) S100 immunoassay for real-time measurement of S100 protein serum levels indicating acute brain damage in patients undergoing carotid artery stenting (CAS) or endarterectomy (CEA). DESIGN AND METHODS:: Data of 14 CAS patients were compared to those of 43 CEA and 14 control patients undergoing coronary angiography (CA). S100 serum levels were measured by the full-automatic Elecsys(R) S100 immunoassay and compared to those obtained by the well-established LIA-mat(R) S100 system. RESULTS:: In contrast to CAS and CA patients, median S100 serum levels of CEA patients significantly increased to 0.24 ng/mL before declamping, but subsequently returned to baseline. Three CEA patients with neurological deficits showed sustained elevated S100 levels 6 h after extubation. Absolute S100 values were not significantly different between the two methods. Bland-Altman plot analyses displayed a good agreement, mostly indicating slightly smaller values applying the Elecsys(R) S100 system. CONCLUSIONS:: The Elecsys(R) S100 system appears to be suitable for rapid real-time detection of neurological deficits in patients undergoing CAS and CEA. Persistent elevations of Elecsys(R) S100 levels during CEA were associated with prolonged neurological disorders, whereas transient increases seem to represent impaired blood-brain barrier integrity without neurological deficits.

Myeloid-related protein 8/14 complex is released by monocytes and granulocytes at the site of coronary occlusion: a novel, early, and sensitive marker of acute coronary syndromes

AIMS: We investigated whether myeloid-related protein 8/14 complex (MRP8/14) expressed by infiltrating monocytes and granulocytes may represent a mediator and early biomarker of acute coronary syndromes (ACS). METHODS AND RESULTS: Immunohistochemistry of coronary thrombi was done in 41 ACS patients. Subsequently, levels of MRP8/14 were assessed systemically in 75 patients with ACS and culprit lesions, with stable coronary artery disease (CAD), or with normal coronary arteries. In a subset of patients, MRP8/14 was measured systemically and at the site of coronary occlusion. Macrophages and granulocytes, but not platelets stained positive for MRP8/14 in 76% of 41 thrombi patients. In ACS, local MRP8/14 levels [22.0 (16.2-41.5) mg/L] were increased when compared with systemic levels [13.4 (8.1-14.7) mg/L, P = 0.03]. Systemic levels of MRP8/14 were markedly elevated [15.1 (12.1-21.8) mg/L, P = 0.001] in ACS when compared with stable CAD [4.6 (3.5-7.1) mg/L] or normals [4.8 (4.0-6.3) mg/L]. Using a cut-off level of 8 mg/L, MRP8/14 but not myoglobin or troponin, identified ACS presenting within 3 h from symptom onset. CONCLUSION: In ACS, MRP8/14 is markedly expressed at the site of coronary occlusion by invading phagocytes. The occurrence of elevated MRP8/14 in the systemic circulation prior to markers of myocardial necrosis makes it a prime candidate for the detection of unstable plaques and management of ACS.

Compositional protein analysis of high density lipoproteins in hypercholesterolemia by shotgun LC-MS/MS and probabilistic peptide scoring

A protein of a biological sample is usually quantified by immunological techniques based on antibodies. Mass spectrometry offers alternative approaches that are not dependent on antibody affinity and avidity, protein isoforms, quaternary structures, or steric hindrance of antibody-antigen recognition in case of multiprotein complexes. One approach is the use of stable isotope-labeled internal standards; another is the direct exploitation of mass spectrometric signals recorded by LC-MS/MS analysis of protein digests. Here we assessed the peptide match score summation index based on probabilistic peptide scores calculated by the PHENYX protein identification engine for absolute protein quantification in accordance with the protein abundance index as proposed by Mann and co-workers (Rappsilber, J., Ryder, U., Lamond, A. I., and Mann, M. (2002) Large-scale proteomic analysis of the human spliceosome. Genome Res. 12, 1231-1245). Using synthetic protein mixtures, we demonstrated that this approach works well, although proteins can have different response factors. Applied to high density lipoproteins (HDLs), this new approach compared favorably to alternative protein quantitation methods like UV detection of protein peaks separated by capillary electrophoresis or quantitation of protein spots on SDS-PAGE. We compared the protein composition of a well defined HDL density class isolated from plasma of seven hypercholesterolemia subjects having low or high HDL cholesterol with HDL from nine normolipidemia subjects. The quantitative protein patterns distinguished individuals according to the corresponding concentration and distribution of cholesterol from serum lipid measurements of the same samples and revealed that hypercholesterolemia in unrelated individuals is the result of different deficiencies. The presented approach is complementary to HDL lipid analysis; does not rely on complicated sample treatment, e.g. chemical reactions, or antibodies; and can be used for projective clinical studies of larger patient groups.

Detection of surfactant proteins A and D in human tear fluid and the human lacrimal system

PURPOSE: To evaluate the expression and presence of surfactant protein (SP) A and SP-D in the lacrimal apparatus, at the ocular surface, and in tears in healthy and pathologic states. METHODS: Expression of mRNA for SP-A and SP-D was analyzed by RT-PCR in healthy lacrimal gland, conjunctiva, cornea, and nasolacrimal ducts as well as in a spontaneously immortalized conjunctival epithelial cell line (HCjE; IOBA-NHC) and a SV40-transfected cornea epithelial cell line (HCE). Deposition of SP-A and SP-D was determined by Western blot, dot blot, and immunohistochemistry in healthy tissues, in tears, aqueous humor, and in sections of different corneal abnormalities (keratoconus, herpetic keratitis, and Staphylococcus aureus-based ulceration). Cell lines were stimulated with different cytokines and bacterial components and were analyzed for the production of SP-A and SP-D by immunohistochemistry. RESULTS: The presence of SP-A and SP-D on mRNA and protein levels was evidenced in healthy lacrimal gland, conjunctiva, cornea, and nasolacrimal duct samples. Moreover, both proteins were present in tears but were absent in aqueous humor. Immunohistochemistry revealed the production of both peptides by acinar epithelial cells of the lacrimal gland and epithelial cells of the conjunctiva and nasolacrimal ducts, whereas goblet cells revealed no reactivity. Healthy cornea revealed weak reactivity on epithelial surface cells only. In contrast, SP-A and SP-D revealed strong reactivity in patients with herpetic keratitis and corneal ulceration surrounding lesions and in several immigrated defense cells. Reactivity in corneal epithelium and endothelium was also seen in patients with keratoconus. Cell culture experiments revealed that SP-A and SP-D are produced by both epithelial cell lines without and after stimulation with cytokines and bacterial components. CONCLUSIONS: These results show that SP-A, in addition to SP-D, is a peptide of the tear film. Based on the known direct and indirect antimicrobial effects of collectins, the surfactant-associated proteins A and D seem to be involved in several ocular surface diseases.

Inflammation and NO(X)-induced nitration: assay for 3-nitrotyrosine by HPLC with electrochemical detection

The identification of 15N-labeled 3-nitrotyrosine (NTyr) by gas chromatography/mass spectroscopy in protein hydrolyzates from activated RAW 264.7 macrophages incubated with 15N-L-arginine confirms that nitric oxide synthase (NOS) is involved in the nitration of protein-bound tyrosine (Tyr). An assay is presented for NTyr that employs HPLC with tandem electrochemical and UV detection. The assay involves enzymatic hydrolysis of protein, acetylation, solvent extraction, O-deacetylation, and dithionite reduction to produce an analyte containing N-acetyl-3-aminotyrosine, an electrochemically active derivative of NTyr. We estimate the level of protein-bound NTyr in normal rat plasma to be approximately 0-1 residues per 10(6) Tyr with a detection limit of 0.5 per 10(7) Tyr when > 100 nmol of Tyr is analyzed and when precautions are taken to limit nitration artifacts. Zymosan-treated RAW 264.7 cells were shown to have an approximately 6-fold higher level of protein-bound NTyr compared with control cells and cells treated with N(G)-monomethyl-L-arginine, an inhibitor of NOS. Intraperitoneal injection of F344 rats with zymosan led to a marked elevation in protein-bound NTyr to approximately 13 residues per 10(6) Tyr, an approximately 40-fold elevation compared with plasma protein of untreated rats; cotreatment with N(G)-monomethyl-L-arginine inhibited the formation of NTyr in plasma protein from blood and peritoneal exudate by 69% and 53%, respectively. This assay offers a highly sensitive and quantitative approach for investigating the role of reactive byproducts of nitric oxide in the many pathological conditions and disease states associated with NO(X) exposure such as inflammation and smoking.