BMC Bioinformatics

BackgroundSurface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI) is a proteomics tool for biomarker discovery and other high throughput applications. Previous studies have identified various areas for improvement in preprocessing algorithms used for protein peak detection. Bottom-up approaches to preprocessing that emphasize modeling SELDI data acquisition are promising avenues of research to find the needed improvements in reproducibility.ResultsWe studied the properties of the SELDI detector intensity response to matrix only runs. The intensity fluctuations and noise observed can be characterized by a natural exponential family with quadratic variance function (NEF-QVF) class of distributions. These include as special cases many common distributions arising in practice (e.g.- normal, Poisson). Taking this model into account, we present a modified Antoniadis-Sapatinas wavelet denoising algorithm as the core of our preprocessing program, implemented in MATLAB. The proposed preprocessing approach shows superior peak detection sensitivity compared to MassSpecWavelet for false discovery rate (FDR) values less than 25%.ConclusionsThe NEF-QVF detector model requires that certain parameters be measured from matrix only spectra, leaving implications for new experiment design at the trade-off of slightly increased cost. These additional measurements allow our preprocessing program to adapt to changing noise characteristics arising from intralaboratory and across-laboratory factors. With further development, this approach may lead to improved peak prediction reproducibility and nearly automated, high throughput preprocessing of SELDI data

Human proteomic profiles in latent and active tuberculosis

Distinguishing patients with active tuberculosis (TB) from those with latent TB is an important clinical problem. The SELDI-TOF MS (Surface Enhanced Laser Desorption Ionisation – Time of Flight Mass Spectrometry) platform allows for high throughput detection of multiple proteins in biological fluids. Proteomic patterns reflecting host-pathogen interaction can be used as a tool to aid our understanding of the Natural History of Tuberculosis. Methods: Plasma samples were collected prospectively in a shanty town in Lima, Peru. Latent and active TB status was defined using the Tuberculin Skin Test (TST), Quantiferon (QFN) assay and TB culture. Crude plasma and fractionated plasma samples were analysed on weak cationic CM10 chip surfaces using a Biomek 3000 Laboratory Automation Workstation. Spectra were generated using a ProteinChip System 4000 Mass spectrometer. Data was analysed using a Support Vector Machine. Results: Samples were collected from 154 patients with active TB, 112 patients with respiratory symptoms suggestive of TB and 151 healthy controls. Multiple peaks differed significantly between active TB patients and unhealthy controls. Trained optimal classifiers discriminate between: i) active TB and unhealthy controls with 84% accuracy (87% sensitivity, 79% specificity) in crude plasma and up to 89% accuracy (90% sensitivity, 88% specificity) in fractionated plasma ii) active TB and latent TB with 89% accuracy (90% sensitivity, 89% specificity) iii) latent TB and no TB in healthy controls with 77% accuracy (67% sensitivity, 84% specificity). Conclusions: SELDI-TOF MS proteomic profiles in combination with trained optimal classifiers accurately discriminate active TB from other respiratory disorders. The classifier for latent TB was not as accurate, but active TB could be discriminated from latent TB

A multi-modality approach for enhancing the diagnosis of cholangiocarcinoma

Background: Cholangiocarcinoma (CC) is a malignancy of the bile ducts and mortality is high as patients present too late for curative surgery. In most cases of CC the aetiology is unknown, whilst diagnosis and staging are challenging. The hepatobiliary system excretes carcinogenic toxins and genetic mutations in biliary transporters lead to dysfunction and cholestasis, potentially contributing to cholangiocarcinogenesis. Polymorphisms in the NKG2D receptor have previously been associated with CC in primary sclerosing cholangitis (PSC). Such a role has not been investigated in sporadic CC. CC is difficult to diagnose, particularly in those with PSC. The transition from benign to malignant biliary disease is likely to be reflected in changes to the plasma proteome. However, current plasma biomarkers do not reliably distinguish benign from malignant biliary strictures. Elevation of neutrophil gelatinase-associated lipocalin (NGAL) has been demonstrated in the bile of patients with CC but has not been investigated as a plasma protein biomarker. Staging of CC is inaccurate, with only a minority of operated patients cured. Higher resolution MRI would improve diagnosis and staging. The work presented in this thesis represents a multimodality approach to enhance the diagnosis of CC: Genetic studies: Genetic variation in major biliary transporter proteins, and the NKG2D receptor, were investigated. Single nucleotide polymorphisms (SNPs) in candidate genes were selected using HapMap. DNA from 173 CC patients and 265 healthy controls was genotyped. SNPs in ABCB11, MDR3 and ATP8B1 were nominally associated with altered susceptibility to CC, suggesting a potential role in cholangiocarcinogenesis. The previous association of NKG2D variation with CC in PSC was not replicated in sporadic CC, suggesting a possible difference in pathogenesis. Protein studies: Plasma from subjects with CC, benign disease, and from healthy controls was studied. Two proteomic techniques, liquid chromatography-tandem mass spectrometry (LCMS/ MS) and surfaced enhanced laser desorption ionization time-of-flight MS (SELDITOF MS), were utilised. Differentially expressed proteins were identified where possible. LC-MS/MS fully identified six proteins that were differentially expressed in CC compared to gall stone disease patients. SELDI-TOF MS identified seven m/z peaks that showed significant utility in discriminating CC from PSC controls. An ELISA approach was used to study plasma NGAL levels in CC. Although differentially expressed between CC and healthy control groups, the utility of NGAL in discriminating CC from PSC was limited. Imaging studies: An endoscope-mounted MR coil and intraductal MR detector coil were developed. Quantitative resolution and signal-to-noise-ratio (SNR) testing, and qualitative tissue discrimination appraisal, were undertaken. Sub-0.7mm resolution and excellent SNRs have been demonstrated. High-resolution has been demonstrated in imaged tissue. Imaging with the new devices compares favourably with endoscopic ultrasound imaging

Applications of Mass Spectrometry in Proteomics and Pharmacokinetics

Tremendous technology improvements of the last decades has given mass spectrometry a more and more expanding role in the study of a wide range of molecules: from the identification and quantification of small molecular weight molecules to the structural determination of biomacromolecules. Many are the fields of application for this technique and the various versions of it. In the present study three different applications have been explored. The first application is a pharmacokinetics study of anticancer drug Gemcitabine and its principal metabolite, where the role of the LC-MS/MS is essential both for the selectivity of the detection of the small analytes and the sensitivity enhanced by multi-reaction monitoring experiments. The design of the study involved the collection of several blood samples at selected times and from patients that would have met certain eligibility criteria. The ESI demonstrated to be the most suitable approach and it provided the necessary data to conclude that toxicity of Gemcitabine did not increase when administered at FDR (Fixed Dose Rate) infusion in patients with impaired hepatic function. The second application describes an example of how MS represents a powerful tool in cancer research, from serum profiling study with high resolution MALDITOF and bioinformatic analysis, to the identification of potential biomarker through peak identification. Almost 400 serum sample – homogeneously distributed between biopsy confirmed ovarian cancer and high risk serum samples – were analyzed on a high resolution MALDI-TOF instrument after automated reverse phase magnetic beads separation. The high throughput data have undergone sophisticated bioinformatic procedures that lead to a list of upand down-regulated peaks, although identification studies were possible only for those peaks that showed a good reproducibility. One down-regolated peak has been identified using the LC-MS/MS technique. The identified peak confirmed a basic role of fibrinogen in the ovarian cancer; the other four peaks that have been identified as down-regulated showed an absolutely not satisfactory ionization in electro-spray, therefore further analysis will be performed on these analytes in order to determinate their amino acidic sequence. The most suitable technique seems to be MALDI-TOF/TOF mass spectrometry, since the peptides already showed a good degree of ionization in MALDI. The third and last study belongs to a quite new field, which is the combination of immuno precipitation assays with MALDI-TOF (Immuno Precipitation Mass Spectrometry, IPMS) experiments in order to evaluate the specificity of a series of monoclonal antibodies to specific antigen. The automated assay that has been developed provides structural information about the antigen that binds the monoclonal antibody to be tested and previously conjugated to the surface of magnetic beads, ideal support for robotic automation. IPMS showed its potential as a complementary tool of crucial importance in the selection of the monoclonal antibody for the development of ELISA based assay to be applied in the screening of a consistent number of human specimens for the clinical validation of proteins indicated in literature as potential biomarkers. Mass spectrometry in association with fractionation techniques, such as liquid or magnetic beads chromatography, is a very flexible tool in the cancer research field. Further improvement in the instrumentation and in the technology will bring always more and more results to be confident in

Proteomic analysis of biomarkers associated with immunotherapy in murine tumour models

Emergence of proteomics and high-throughput technologies has allowed the identification of protein expression patterns of disease that potentially hold clinical importance in predictive medicine. The analysis of complex data generated by these technologies incorporates the use of computer algorithms for data mining and identification of important protein biomarkers. Such candidate biomarkers can potentially be used for diagnosis, prognosis and monitoring a variety of diseases as well as the prediction of therapy response. Mass spectrometry has been used widely, for the discovery and quantitation of disease associated biomarkers using a variety of samples such as serum and tissue. In particular, matrix assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF MS) has been used to generate proteomic profiles or “fingerprints” from serum to distinguish patients at different clinical stages of disease. Currently, early stage disease is difficult to diagnose in most cancers as current cancer markers have limited sensitivity and specificity. In advanced stage metastatic disease, treatment options are limited, although it is recognised that some patients may benefit from immunotherapy and in particular vaccine therapy. The use of animal models is critical to evaluate the efficacy of immunotherapies and to investigate tumour immunity in general and the mechanisms involved in tumour progression. These models provide an in vivo environment which cannot be reproduced in vitro, which results in more accurate and reliable information on the host response to immunotherapy and the mechanisms involved

Applications of Mass Spectrometry in Proteomics and Pharmacokinetics

Tremendous technology improvements of the last decades has given mass spectrometry a more and more expanding role in the study of a wide range of molecules: from the identification and quantification of small molecular weight molecules to the structural determination of biomacromolecules. Many are the fields of application for this technique and the various versions of it. In the present study three different applications have been explored. The first application is a pharmacokinetics study of anticancer drug Gemcitabine and its principal metabolite, where the role of the LC-MS/MS is essential both for the selectivity of the detection of the small analytes and the sensitivity enhanced by multi-reaction monitoring experiments. The design of the study involved the collection of several blood samples at selected times and from patients that would have met certain eligibility criteria. The ESI demonstrated to be the most suitable approach and it provided the necessary data to conclude that toxicity of Gemcitabine did not increase when administered at FDR (Fixed Dose Rate) infusion in patients with impaired hepatic function. The second application describes an example of how MS represents a powerful tool in cancer research, from serum profiling study with high resolution MALDITOF and bioinformatic analysis, to the identification of potential biomarker through peak identification. Almost 400 serum sample – homogeneously distributed between biopsy confirmed ovarian cancer and high risk serum samples – were analyzed on a high resolution MALDI-TOF instrument after automated reverse phase magnetic beads separation. The high throughput data have undergone sophisticated bioinformatic procedures that lead to a list of upand down-regulated peaks, although identification studies were possible only for those peaks that showed a good reproducibility. One down-regolated peak has been identified using the LC-MS/MS technique. The identified peak confirmed a basic role of fibrinogen in the ovarian cancer; the other four peaks that have been identified as down-regulated showed an absolutely not satisfactory ionization in electro-spray, therefore further analysis will be performed on these analytes in order to determinate their amino acidic sequence. The most suitable technique seems to be MALDI-TOF/TOF mass spectrometry, since the peptides already showed a good degree of ionization in MALDI. The third and last study belongs to a quite new field, which is the combination of immuno precipitation assays with MALDI-TOF (Immuno Precipitation Mass Spectrometry, IPMS) experiments in order to evaluate the specificity of a series of monoclonal antibodies to specific antigen. The automated assay that has been developed provides structural information about the antigen that binds the monoclonal antibody to be tested and previously conjugated to the surface of magnetic beads, ideal support for robotic automation. IPMS showed its potential as a complementary tool of crucial importance in the selection of the monoclonal antibody for the development of ELISA based assay to be applied in the screening of a consistent number of human specimens for the clinical validation of proteins indicated in literature as potential biomarkers. Mass spectrometry in association with fractionation techniques, such as liquid or magnetic beads chromatography, is a very flexible tool in the cancer research field. Further improvement in the instrumentation and in the technology will bring always more and more results to be confident in

Proteomics and ecotoxicoldgy:.marine invertebrates and endocrine disrupting chemicals

The key problem faced by environmental scientists is to predictand recognize the damaging effects of chemical pollutants on natural biota. The aim of this thesis was to evaluate the potential for proteomics in ecotoxicology and environmental risk assessment (ERA), with the hypothesis that proteomic technologies (i.e. ProteinChip technology in combination with SELDI TOF MS) could be a useftil supplement to existing methods of environmental assessment, by providing a sensitive, non-invasive, rapid multi-endpoint assessment of effects of anthropogenic chemicals on organism in vivo. Three invertebrate species, Mytilus edulis, Hyas araneus and Strongylocentrotus droebachiensis was exposed to natural and anthropogenic chemicals in laboratory and field studies. Results revealed that proteomics was a sensitive endpoint, as all exposure regimes significantly affected protein expression. It was shown that plasma protein expression profiles contained information that was compound, dose, site, species and gender-specific. Regarding the latter; male and female organisms responded differently to all exposures both quantitatively (e.g. in terms of number of affected protein species) and qualitatively (e.g. in terms of tj^e of affected protein species). Furthermore, genders have shown opposite responses following the same exposure regime. Equally, species-specific responses were observed. Moreover, exposing organisms to graded levels of contamination under controlled laboratory conditions and in the field revealed that different subsets of proteomes were affected at different levels of exposure. This finding represents an opportunity for appljdng proteomics for both prognostic (e.g. early warning of potential adverse effects or assessment of recovery) and diagnostic purposes. Moreover, those protein features that were changed by all exposure concentrations showed complex dose-response relationships, including both linear and various types of biphasic response-curves. In summary, results from the present study indicate that proteomics have the potential to be a useftil tool in ERA. For example, identification of key molecules could elucidate mechanism of action related to mixture effects, gender and species-specific susceptibility to environmental pollutants as well as dose-response relationships at low doses. Furthermore, key proteins (i.e. putative biomarkers) could, be purified and coupled to e.g. a biosensor for automated monitoring.School of Biological Sciences; International Research Institute of Stavanger (IRIS-BIOMILJ0

Tilstedeværelsen av en akutt fase-reaksjon hos lam med eksperimentell klassisk skrapesjuke indikerer et skifte mot en pro-inflammatorisk tilstand i det kliniske endestadiet

Classical scrapie in sheep is a transmissible and fatal neurodegenerative disease caused by the self-replicating and infectious prion protein, PrPSc, which is a conformational variant of the normal cellular prion protein, PrPC. The prion protein is a highly conserved glycoprotein encoded by the PRNP gene and therefore within the same host both PrPC and PRPSC have the same unique amino acid sequence and they only differ in their three-dimensional folded structure. Specific mutations at codons 136, 154 and 171 of the PRNP gene leads to single amino acid substitutions, and the most common polymorphisms give rise to five possible alleles and 15 PRNP genotypes found in sheep. The different alleles are highly associated with levels of susceptibility to classical scrapie, where A136R154R171 allele provides high genetic resistance and V136R154Q171 allele results in highly susceptible animals. On the basis of this association between PRNP genotype and susceptibility, many EU MSs have implemented national breeding for resistance programme with the aim of increasing distribution of ARR allele and reducing the distribution of VRQ allele. For almost 20 years, the EU TSE regulation has required surveillance within each country to establish prevalence of prion diseases and the different PRNP genotypes. Classical scrapie has a widespread distribution and incidence rate fluctuates due to the complex interaction between prion and host factors, and prevalence can only be estimated by ante mortem testing through active and passive surveillance. Transmission between sheep occurs through direct and indirect contact, and PrPSc can remain infective in the environment for years. The most common route of infection is the oral route, and infected animals can excrete PrPSc through foetal membranes and fluids, saliva, urine, faeces, and milk. Pathogenesis is highly influenced by PRNP genotype, as animals of the most susceptible genotypes have the most effective uptake of PrPSc across small intestine followed by an extensive dissemination and involvement of the SLOs, and an early neuroinvasion with spread of PrPSc within the CNS. The susceptible genotypes will contribute the most to spread of infectivity and environmental contamination. This work describes the results from experimental classical scrapie where homozygous VRQ lambs were inoculated orally at birth with homogenated brain material from either healthy sheep or from natural cases of classical scrapie. This resulted in a worst-case scenario type of classical scrapie with sudden onset of severe clinical signs at 22 wpi followed by a rapid deterioration and euthanasia at 23 wpi. Serum samples were collected at regular intervals and tissue samples from brain and liver were sampled at post mortem examination. Proteomic examinations of serum revealed a downregulation of several protein peaks during the pre-symptomatic incubation period in the scrapie affected group compared to the control group, and a shift to upregulation of protein peaks onwards from 22 wpi. Genomic examinations of serum samples showed a slight downregulation IL1B and TLR4 at 16 wpi, followed by a change at 22 wpi with upregulation of genes encoding TLRs, C3 and APPs. Genomic examination of liver and brain tissues showed an alteration in gene expression of APPs in accordance with an APR. Serum analyses of different APPs showed increased levels of the positive APPs and a reduced concentration of negative APPs. These findings are indicative of a shift from anti-inflammatory to pro-inflammatory systemic innate immune response that coincide with the onset of debilitating clinical disease. In neurodegenerative diseases, the innate immune response in the CNS has a key role in both onset and progression of disease and resolution of inflammation. The accumulation of PrPSc in the CNS has been associated with a chronic activation of the innate immune response, pro-inflammatory activation of microglia, neuroinflammation, and neurodegeneration. The diseases phenotype registered in this work is a result of PRNP genotype, and time and dose of inoculation, which can occur naturally if the right circumstances are in place. New-born homozygous VRQ lambs from an infected dam can get infected at birth. These cases could develop a similar disease progression as described in this work, resulting in an efficient and fast uptake and widespread peripheral and central dissemination of PrPSc, and clinical disease at a young age. These cases would present as a diagnostic challenge and easily missed as classical scrapie. Due to their young age, these cases would not be sampled through active surveillance. If incubation period extends commercial lifespan, these lambs would be slaughtered for human consumption, and due to their PRNP genotype, prions would enter the food chain. Control of classical scrapie can probably not be achieved by absence of infectivity, but absence of clinical disease is possible through breeding for resistance which will provide flock immunity to classical scrapie.Klassisk skrapesyke hos sau er en overførbar og dødelig nevrodegenerativ sykdom forårsaket av det selvrepliserende og smittsomme prionproteinet, PrPSc, som er en variant av det normale cellulære prionproteinet, PrPC. Prionproteinet er et glykoprotein som er kodet for av PRNP-genet. Dette betyr at PrPC og PRPSC hos samme verten, har den samme unike aminosyresekvensen og det er kun den tredimensjonale strukturen som skiller dem. Spesifikke mutasjoner ved kodonene 136, 154 og 171 i PRNP-genet fører til substitusjoner av enkelte aminosyrer, og de vanligste polymorfismer gir opphav til fem mulige alleler, og 15 PRNP-genotyper hos sau. De forskjellige allelene er assosiert med nivå av mottakelighet for klassisk skrapesyke, og A136R154R171-allel fører til genetisk resistens, og V136R154Q171-allel gir høy mottagelighet. På bakgrunn av denne sammenhengen mellom PRNP-genotype og mottakelighet, har mange EU medlemsland innført nasjonale avlsprogram som har mål om å øke utbredelsen av ARR-allel, og samtidig en reduksjon av VRQ-allel. I snart 20 år har EUs TSE-regelverk krevd nasjonale overvåkingsprogram for å bestemme forekomsten av prionsykdommer og kartlegge utbredelsen av de forskjellige PRNP-genotypene. Klassisk skrapesyke er utbredt, men forekomsten vil variere med bakgrunn i det komplekse samspillet mellom prionprotein og vertsfaktorer. Prevalens kan estimeres gjennom ante mortem testing i forbindelse med aktivt og passivt overvåkingsprogram. Smitteoverføring mellom sau skjer ved direkte og indirekte kontakt, og PrPSc er smittsomt i flere år i miljøet. Den vanligste infeksjonsveien er gjennom oralt inntak, og dyr kan skille ut smittsomt PrPSc via fosterhinner og væsker, spytt, urin, feces og melk, og nivå er styrt av PRNP genotype.Research Council of Norwa