Targeted tandem mass spectrometry strategies to quantify proteins biomarkers of inflammatory diseases

Abstract Mass spectrometry is a sensitive technique used to detect, identify and quantitate molecules based on their mass-to-charge (m/z) ratio in simple and complex mixtures. Originally developed almost 100 years ago to measure elemental atomic weights and the natural abundance of specific isotopes, MS was first used in the biological sciences to trace heavy isotopes through biological systems. The following PhD project was involved in the application of advanced methods of mass spectrometry in Multiple Reaction Monitoring ion mode (MRM) into different metabolomics and proteomics research areas. For the most part, the studies conducted over the past three years have had a single common thread: development and application of MRM mass spectrometry methods for the identification and quantification of targeted metabolites and/or proteins involved in inflammatory processes. It is clear that, for each project, we started from the study of the different biological matrices to find the most effective extraction strategy for the target analytes and subsequent steps have covered in-depth literature studies to identify the best condition to perform chromatographic separation and the subsequent optimization of instrumental parameters. The three main areas of application explored in these years concerned the following three points: 1. MRM/MS analysis of metabolites In collaboration with Prof. Greco, Prof. Auricchio and Prof. Ruoppolo of the Department of Translational Medical Sciences and Department of Molecular Medicine and Medical Biotechnology of the University of Naples Federico II. The project provided for the development of a method that allow the investigation of the lipidomic profile of genetically predisposed children to celiac disease in order to identify potential molecular biomarkers for disease prediction, first of all, in genetically predisposed patients but also for subjects whose clinical history is unknown. Serum samples of two cohorts: 23 children who became coeliac and 23 not yet (used as control), do share a similar genetic background, since they come from families with one celiac proband and bear the specific HLA haplotype (DQ2 or DQ8). It does appear that the genetic profile may not explain fully the great differences found between the two cohorts. The developed MRM method allowed to monitor and quantify 83 different classes of analytes and allowed us to identify some classes of lipids as putative molecular biomarkers by comparing the results obtained from the analysis of the samples of serum collected at 4 months, before introduction of gluten, at 12 months, with the introduction of gluten in the diet of the child and a t> 12 months for children who have been diagnosed with celiac disease. 2. MRM/MS analysis of proteins. A peculiar feature of a MRM method is the ability to monitor multiple precursor ion-product ion transitions. This greatly increase the selectivity and specificity of the analysis and this represents a huge advantage in the proteomic field because each target analyte, in this case peptide, it can be identified within complex mixtures (such as biological fluids) by monitoring transitions closely related to its own amino acid sequence. Different biological aspects were investigated: - The project, in collaboration with Prof. Piccoli and Dr. Arciello of the Department of Chemical Sciences of the University of Naples Federico II, involved the development and optimization of an MRM method for the quantification of proteins involved in inflammatory processes: TNF -α, INF-γ, IL-8 and IL-10 in THP-I cell samples. In particular, differentiated cells have been treated with LPS, a well-known endotoxin, to stimulate the onset of an inflammatory process. A time course analysis was performed on differentiated and stimulated cells with LPS for 2h, 4h, 6h, 9h and 24h. These analyses allowed to monitor the variation in protein expression during the whole inflammatory process, in both acute and late phase of the inflammation and the obtained data are consistent with published works. Quantitative analysis was conducted using the external standard method. In order to increase the selectivity and the specificity of the method, for each target protein, two or three peptides have been identified thanks to the aid of bioinformatic software which have a unique amino acid sequence and can be used as a stoichiometric representation of the protein in the quantitative analysis. - Project in collaboration with prof. Francisco Blanco and Dr. Cristina Ruiz-Romero of the INIBIC Biomedical Research Institute of A Coruña, Spain. At this research institute I spent six months for the foreign period of the PhD. In these months I have worked on both the optimization of a SIS-MRM/MS method for the quantification of proteins involved in rheumatoid arthritis (RA) pathogenesis and the application of this method to a cohort of 80 serum samples of subjects whom RA has been diagnosed. Quantitative analysis was performed using the internal standard method: stable isotope labelled standard peptides (SIS) were used. The target peptides belong to proteins that were statistically significant (p <0.05) in previous experiments of 8-plex iTRAQ and large-scale proteomics. RA diagnosis is complex and nowadays it is carried out by putting together radiographic data, DAS 28 and serum parameters such as C-reactive protein, rheumatoid factor (RF) and anti-citrullinated antibodies (ACPA) levels. The main problem in diagnostics is incurring false positives, as in the case of RF that shows the same trend also in other diseases like: chronic hepatitis, chronic viral infections, leukaemia, dermatomyositis, mononucleosis, scleroderma, Hashimoto's thyroiditis, systemic lupus erythematosus and Sjögren's syndrome. First step involved the development of the method, the validation of the selected transitions and the choice of the concentration of labelled peptides to be added to the real samples to obtain the best signal/noise ratio. Reverse calibration curves for each heavy target peptide were realized and analytical parameters Detection limit (LOD), Quantification limit (LOQ) and linearity range were calculated. The developed method was used for the identification and quantification of the 10 target proteins in a cohort of 80 samples of sera from RA patients subdivided into 4 sub-groups based on rheumatoid factor (RF) and anti-citrullinated antibodies (ACPA) values. The four sub-groups were: RF-/ACPA-, RF-/ACPA+, RF+/ACPA- and RF+/ACPA+. Data analysis allowed to select some of the proteins monitored for the subsequent steps of method validation by using complementary techniques such as ELISA immunoassay. 3. MRM/MS analysis for discovery. - An interesting application of mass spectrometry in MRM ion mode was in forensic field for the identification of biomarker proteins of biological fluids. This project was carried out in collaboration with Ten.Col. Peluso of the RIS department of the Carabinieri in Rome. The basic idea was to exploit the potential of mass spectrometry in MRM ion mode to identify the nature of biological traces found at a crime scene from which the DNA was extracted. In this way it is possible to conduct complementary investigations on both the identity of the suspect and the sequence of events that lead to the crime. Presumptive and confirmatory tests are needed to be absolutely sure of the identity of the biological fluid found at a crime scene. These tests suffer from limitations due, above all, to poor specificity and to the necessity to conduct cascade tests to evaluate the nature of the trace. The developed MRM method allows to overcome these limitations as it allows to discriminate between four biological matrices: blood, saliva, seminal fluid and urine through a single analysis and through a single sample treatment, which involves hydrolysis with trypsin, to carry out the extraction DNA and the subsequent proteomic analysis. - Project in collaboration with Prof Nicosia from Department of Molecular Medicine and Medical Biotechnologies of the University of Naples Federico II for the identification of proteins in HCT116 cells (human colorectal tumour cells) deriving from alternative splicing processes and which could be closely related to the onset and progression of this type of cancer. Previous studies performed on mRNA have shown that such alternative splicing phenomena lead to proteins that have a mutated sequence to C-ter. This sequence was used for setting up of the MRM / MS method thanks to the high specificity and selectivity that derives from the use of mass spectrometry in this mode, but above all the high sensitivity to which it is possible to arrive (amol/μL). A method was developed to identify the presence of 5 mutated proteins using the mutated C-ter aminoacidic sequence as molecular target and it was possible to verify the presence of these mutated sequences confirming the translation of these proteins. Finally, for the method validation HT 29 and LS147T cell lines were used. The results seem promising, in all the selected cell lines the mutated proteins were detected. Further developments concern the implementation of the MRM / MS method developed with the use of isotopically labelled peptides to validate and perform quantitative analysis. Results obtained in the present PhD thesis show the broad applicability of the MRM/MS methodology. This strategy was effective both during the discovery phase and for the quantitative analysis of metabolites and proteins, demonstrating high sensitivity, selectivity and specificity. The chance to simultaneously analyse a panel of numerous analytes allows to optimize analysis times and costs. Finally, the "multiple" ability of this method allows all methods to be implemented by inserting the characteristic transitions of heavy isotopically labelled standards that have the same chemical and physical properties of the target molecules but different m/z ratio. The use of an internal standard is fundamental to evaluate the matrix effect, the efficiency of an extraction methodology and the identification of an analyte in complex samples. Subsequent steps for the presented projects are mainly focused on the implementation of the developed MRM methods with isotopically labelled standards, the validation of the obtained results by complementary techniques (ELISA) and the development of kits that can be used in clinical practice for diagnostics or follow-up of patients suffering from various diseases or in forensic investigations

Ensuring the resilience of wireless sensor networks to malicious data injections through measurements inspection

Malicious data injections pose a severe threat to the systems based on \emph{Wireless Sensor Networks} (WSNs) since they give the attacker control over the measurements, and on the system's status and response in turn. Malicious measurements are particularly threatening when used to spoof or mask events of interest, thus eliciting or preventing desirable responses. Spoofing and masking attacks are particularly difficult to detect since they depict plausible behaviours, especially if multiple sensors have been compromised and \emph{collude} to inject a coherent set of malicious measurements. Previous work has tackled the problem through \emph{measurements inspection}, which analyses the inter-measurements correlations induced by the physical phenomena. However, these techniques consider simplistic attacks and are not robust to collusion. Moreover, they assume highly predictable patterns in the measurements distribution, which are invalidated by the unpredictability of events. We design a set of techniques that effectively \emph{detect} malicious data injections in the presence of sophisticated collusion strategies, when one or more events manifest. Moreover, we build a methodology to \emph{characterise} the likely compromised sensors. We also design \emph{diagnosis} criteria that allow us to distinguish anomalies arising from malicious interference and faults. In contrast with previous work, we test the robustness of our methodology with automated and sophisticated attacks, where the attacker aims to evade detection. We conclude that our approach outperforms state-of-the-art approaches. Moreover, we estimate quantitatively the WSN degree of resilience and provide a methodology to give a WSN owner an assured degree of resilience by automatically designing the WSN deployment. To deal also with the extreme scenario where the attacker has compromised most of the WSN, we propose a combination with \emph{software attestation techniques}, which are more reliable when malicious data is originated by a compromised software, but also more expensive, and achieve an excellent trade-off between cost and resilience.Open Acces

The mutual interplay of gut microbiota, diet and human disease

The intestinal milieu harbours the gut microbiota, consisting of a complex community of bacteria, archaea, fungi, viruses, and protozoans that bring to the host organism an endowment of cells and genes more numerous than its own. In the last ten years, mounting evidence has highlighted the prominent influence of the gut mutualistic bacterial communities on human health. Microbial colonization occurs alongside with immune system development and plays a role in intestinal physiology. The community of the gut microbiota does not undergo significant fluctuations throughout adult life. However, bacterial infections, antibiotic treatment, lifestyle, surgery, and diet might profoundly affect it. Gut microbiota dysbiosis, defined as marked alterations in the amount and function of the intestinal microorganisms, is correlated with the aetiology of chronic non-communicable diseases, ranging from cardiovascular, neurologic, respiratory, and metabolic illnesses to cancer. In this review, we focus on the interplay among gut microbiota, diet, and host to provide a perspective on the role of microbiota and their unique metabolites in the pathogenesis and/or progression of various human disorders. We discuss interventions based on microbiome studies, i.e. faecal microbiota transplantation, probiotics, and prebiotics, to introduce the concept that correcting gut dysbiosis can ameliorate disease symptoms, thus offering a new approach toward disease treatment

Iterative schemes for surfactant transport in porous media

In this work, we consider the transport of a surfactant in variably saturated porous media. The water flow is modelled by the Richards equations and it is fully coupled with the transport equation for the surfactant. Three linearization techniques are discussed: the Newton method, the modified Picard, and the L-scheme. Based on these, monolithic and splitting schemes are proposed and their convergence is analyzed. The performance of these schemes is illustrated on five numerical examples. For these examples, the number of iterations and the condition numbers of the linear systems emerging in each iteration are presented.publishedVersio