Development of mass spectrometry-based identification and quantification methods for protein biomarkers in Alzheimer’s and Parkinson’s diseases

Ziel der Arbeit war es, Massenspektrometrie(MS)-basierte Methoden für die Quantifizierung der beiden für Morbus Alzheimer (AD) etablierten Biomarker (A$\beta$-Peptid und Tau-Protein) zu entwickeln und potentielle Biomarker für Morbus Parkinson (PD) zu identifizieren. Eine neue $\textit {direct-infusion}$ MS-basierte Methode ermöglichte nach A$\beta$-Anreicherung mittels Immunpräzipitation eine relative Quantifizierung der A$\beta$-Peptide. Tau-Isoformen wurden aus dem zerebralen Cortex von AD Patienten sowie entsprechender Kontrollen mit Hilfe von Western Blot und HPLC-MS identifiziert. Um die Etablierung eines optimierten Workflows für die Identifizierung von PD Biomarker zu ermöglichen, wurde eine robuste CSF Analyse mit zwei verschiedenen Methoden durchgeführt: ein "$\textit{machine learning}$" Ansatz und die Kombination zweier Quantifizierungssoftwares innerhalb eines $\textit{discovery sets}$. Mittels dieser zweiten Strategie konnten 13 potentielle Kandidaten identifiziert werden, die die PD Diagnose unterstützen könnten

Autoinflammatory Features in Gouty Arthritis

In the panorama of inflammatory arthritis, gout is the most common and studied disease. It is known that hyperuricemia and monosodium urate (MSU) crystal-induced inflammation provoke crystal deposits in joints. However, since hyperuricemia alone is not sufficient to develop gout, molecular-genetic contributions are necessary to better clinically frame the disease. Herein, we review the autoinflammatory features of gout, from clinical challenges and differential diagnosis, to the autoinflammatory mechanisms, providing also emerging therapeutic options available for targeting the main inflammatory pathways involved in gout pathogenesis. This has important implication as treating the autoinflammatory aspects and not only the dysmetabolic side of gout may provide an effective and safer alternative for patients even in the prevention of possible gouty attacks

A Pro-Inflammatory Signature Constitutively Activated in Monogenic Autoinflammatory Diseases

Autoinflammatory diseases (AIDs) are disorders characterised by recurrent inflammatory episodes in charge of different organs with no apparent involvement of autoantibodies or antigen-specific T lymphocytes. Few common clinical features have been identified among all monogenic AIDs (mAIDs), while the search for a common molecular pattern is still ongoing. The aim of this study was to increase knowledge on the inflammatory pathways in the development of mAIDs in order to identify possible predictive or diagnostic biomarkers for each disease and to develop future preventive and therapeutic strategies. Using protein array-based systems, we evaluated two signalling pathways known to be involved in inflammation and a wide range of inflammatory mediators (pro-inflammatory cytokines and chemokines) in a cohort of 23 patients affected by different mAIDs, as FMF, TRAPS, MKD, Blau syndrome (BS), and NLRP12D. Overall, we observed upregulation of multiple signalling pathway intermediates at protein levels in mAIDs patients’ PBMCs, compared with healthy controls, with significant differences also between patients. FMF, TRAPS, and BS presented also peculiar activations of inflammatory pathways that can distinguish them. MAPK pathway activation, however, seems to be a common feature. The serum level of cytokines and chemokines produced clear differences between patients with distinct diseases, which can help distinguish each autoinflammatory disease. The FMF cytokine production profile appears broader than that of TRAPS, which, in turn, has higher cytokine levels than BS. Our findings suggest an ongoing subclinical inflammation related to the abnormal and constitutive signalling pathways and define an elevated inflammatory cytokine signature. Moreover, the upregulation of Th17-related cytokines emphasises the important role for Th17 and/or Th17-like cells also in monogenic AIDs

Autoinflammatory Mechanisms in Crystal-Induced Arthritis

Crystal-induced arthritides have been classified as "type-1 autoinflammatory diseases" for their main features which resemble those of the monogenic autoinflammatory syndromes. They are in fact characterized by spontaneous onset, recurrence of the episodes, self-limitation and resolution, inflammasome activation with huge production of IL-1\u3b2 and a prevalent involvement of the innate immune system. The term "auto" refers also to the induction of IL-1\u3b2 gene expression, processing and secretion by IL-1\u3b2 itself. The concept of autoinflammation in crystal-induced arthritis has been finally reinforced by the efficacy of IL-1 blockade in treating acute and chronic state of this disease. The aim of this article is to review the autoinflammatory mechanisms in crystal-induced arthritis, considering both clinical and molecular aspects

Regulation of crystal induced inflammation: current understandings and clinical implications

INTRODUCTION: Accumulation of abnormal crystals in the body, derived from endogenous or exogenous materials can drive a wide spectrum of inflammatory disease states. It is well established that intra-articular deposition of monosodium urate (MSU) and calcium pyrophoshate (CPP) crystals contribute to joint destruction through pro-inflammatory processes.AREAS COVERED: This review will focus on current understanding and recent novelty about the mechanisms and the clinical implications of the inflammation induced by MSU and CPP crystals.EXPERT OPINION: Advances in molecular biology reveal that at the base of the inflammatory cascade, stimulated by MSU or CPP crystals, there are many complex cellular mechanisms mainly involving the NLRP3 inflammasome, the hallmark of autoinflammatory syndromes. The extensive studies carried out through in vitro and in vivo models along with a better clinical definition of the disease has led to an optimized use of existing drugs and the introduction of novel therapeutic strategies. In particular, the identification of IL-1 as the most important target in gout and pseudogout has made it possible to expand the pharmacological indications of anti-IL-1 biological drugs, opening new therapeutic perspectives for patients

Metabolic profiling of body fluids and multivariate data analysis

Metabolome analyses of body fluids are challenging due pre-analytical variations, such as pre-processing delay and temperature, and constant dynamical changes of biochemical processes within the samples. Therefore, proper sample handling starting from the time of collection up to the analysis is crucial to obtain high quality samples and reproducible results. A metabolomics analysis is divided into 4 main steps: 1) Sample collection, 2) Metabolite extraction, 3) Data acquisition and 4) Data analysis. Here, we describe a protocol for gas chromatography coupled to mass spectrometry (GC–MS) based metabolic analysis for biological matrices, especially body fluids. This protocol can be applied on blood serum/plasma, saliva and cerebrospinal fluid (CSF) samples of humans and other vertebrates. It covers sample collection, sample pre-processing, metabolite extraction, GC–MS measurement and guidelines for the subsequent data analysis. Advantages of this protocol include: • Robust and reproducible metabolomics results, taking into account pre-analytical variations that may occur during the sampling process • Small sample volume required • Rapid and cost-effective processing of biological samples • Logistic regression based determination of biomarker signatures for in-depth data analysi

Direct infusion-SIM as fast and robust method for absolute protein quantification in complex samples

Relative and absolute quantification of proteins in biological and clinical samples are common approaches in proteomics. Until now, targeted protein quantification is mainly performed using a combination of HPLC-based peptide separation and selected reaction monitoring on triple quadrupole mass spectrometers. Here, we show for the first time the potential of absolute quantification using a direct infusion strategy combined with single ion monitoring (SIM) on a Q Exactive mass spectrometer. By using complex membrane fractions of Escherichia coli, we absolutely quantified the recombinant expressed heterologous human cytochrome P450 monooxygenase 3A4 (CYP3A4) comparing direct infusion-SIM with conventional HPLC-SIM. Direct-infusion SIM revealed only 14.7% (±4.1 (s.e.m.)) deviation on average, compared to HPLC-SIM and a decreased processing and analysis time of 4.5 min (that could be further decreased to 30 s) for a single sample in contrast to 65 min by the LC–MS method. Summarized, our simplified workflow using direct infusion-SIM provides a fast and robust method for quantification of proteins in complex protein mixtures

Protein variability in cerebrospinal fluid and its possible implications for neurological protein biomarker research.

Cerebrospinal fluid is investigated in biomarker studies for various neurological disorders of the central nervous system due to its proximity to the brain. Currently, only a limited number of biomarkers have been validated in independent studies. The high variability in the protein composition and protein abundance of cerebrospinal fluid between as well as within individuals might be an important reason for this phenomenon. To evaluate this possibility, we investigated the inter- and intraindividual variability in the cerebrospinal fluid proteome globally, with a specific focus on disease biomarkers described in the literature. Cerebrospinal fluid from a longitudinal study group including 12 healthy control subjects was analyzed by label-free quantification (LFQ) via LC-MS/MS. Data were quantified via MaxQuant. Then, the intra- and interindividual variability and the reference change value were calculated for every protein. We identified and quantified 791 proteins, and 216 of these proteins were abundant in all samples and were selected for further analysis. For these proteins, we found an interindividual coefficient of variation of up to 101.5% and an intraindividual coefficient of variation of up to 29.3%. Remarkably, these values were comparably high for both proteins that were published as disease biomarkers and other proteins. Our results support the hypothesis that natural variability greatly impacts cerebrospinal fluid protein biomarkers because high variability can lead to unreliable results. Thus, we suggest controlling the variability of each protein to distinguish between good and bad biomarker candidates, e.g., by utilizing reference change values to improve the process of evaluating potential biomarkers in future studies