Immunological and mass spectrometry based methods for the sensitive and accurate determination of selected interleukins and growth factors, including IL-6, IL-8 and CXCL3

Bei der Anwendung von Standard Methoden für die Identifizierung und Quantifizierung von Proteinen, wie z.B. Western Blot (WB) Analyse oder ELISA, kann es auf Grund von unerwünschten Matrixeffekten zu verfälschten Ergebnissen kommen. Diese Effekte können die Robustheit, Sensitivität, Spezifität und Verlässlichkeit der Ergebnisse beeinträchtigen. Wir haben einen systematischen Vergleich von verschiedenen Methoden, welcher auf die Verwendung von Antikörpern und auf Massenspektrometrie basierenden Methoden durchgeführt, um die Vor- und Nachteile der jeweiligen Methoden und deren analytischen Grenzen festzustellen. Durch das Ausnützen der Vorteile beider Methoden, z.B. die hohe Sensitivität der Antikörper basierenden Methoden und die hohe Robustheit und Verlässlichkeit der MS-basierende Methoden, konnten bei deren Kombination die besten Ergebnisse erzielt. Diese Vorteile kommen besonders bei der Analyse von Proben mit komplexer Matrix zum Tragen.The use of standard methods for the identification or quantification of proteins using Western blot (WB) analysis or ELISA may lead to unexpected results with respect to sensitivity, specificity, reliability and robustness, because of matrix effects. We have performed a systematic comparison of different antibody-based and mass spectrometry-based methods in order to assess their advantages and disadvantages, as well as specific limits of each of them. By the combination of the advantages of both types of methods, high sensitivity of antibody-based methods and high reliability and robustness of mass spectrometry-based methods, the best results were obtained particularly when dealing with samples containing complex matrices

Translational rodent models for research on parasitic protozoa – a review of confounders and possibilities

Rodents, in particular Mus musculus, have a long and invaluable history as models for human diseases in biomedical research, although their translational value has been challenged in a number of cases. We provide some examples in which rodents have been suboptimal as models for human biology and discuss confounders which influence experiments and may explain some of the misleading results. Infections of rodents with protozoan parasites are no exception in requiring close consideration upon model choice. We focus on the significant differences between inbred, outbred and wild animals, and the importance of factors such as microbiota, which are gaining attention as crucial variables in infection experiments. Frequently, mouse or rat models are chosen for convenience, e.g., availability in the institution rather than on an unbiased evaluation of whether they provide the answer to a given question. Apart from a general discussion on translational success or failure, we provide examples where infections with single-celled parasites in a chosen lab rodent gave contradictory or misleading results, and when possible discuss the reason for this. We present emerging alternatives to traditional rodent models, such as humanized mice and organoid primary cell cultures. So-called recombinant inbred strains such as the Collaborative Cross collection are also a potential solution for certain challenges. In addition, we emphasize the advantages of using wild rodents for certain immunological, ecological, and/or behavioral questions. The experimental challenges (e.g., availability of species-specific reagents) that come with the use of such non-model systems are also discussed. Our intention is to foster critical judgment of both traditional and newly available translational rodent models for research on parasitic protozoa that can complement the existing mouse and rat models

Cerebrospinal Fluid Proteomic Characterization in Major Depressive Disorder

Major depressive disorder (MDD) is a highly prevalent and increasing disease, characterized by low mood, anhedonia, sadness, and guilt. Despite its heterogeneity, neither a biomarker nor a biosignature has been introduced to clinical practice, which would ensure greater diagnostic and prognostic accuracy. The review of the available literature reveals an increase in the number of studies being conducted to better understand the pathophysiologic mechanisms of MDD, as well as the urgent need to develop diagnostic biomarkers. Several molecules are discussed as promising indicators of depressive conditions, that are involved in different processes such as inflammation, structural and transportation molecules, and synaptic transmission. However, to confirm their diagnostic and prognostic value, additional large-scale studies and the application of cutting-edge analytical techniques are required

COVID-19 TESTING: ROCHE DIAGNOSTICS ROBUST PORTFOLIO

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to a new social order worldwide. The COVID-19 pandemic outbreak has affected many countries causing hundred thousand of deaths all around the world. It did not exercise its influence only on the citizens\u27 health status, but also had a tremendous impact on the economy and financial status of many countries. Nevertheless, all its clinical manifestations are still being elucidated. Together with its spread, proper protective measures, early and accurate detection of the COVID-19 became the most crucial and challenging step. Therefore, facing this global situation and need, Roche Diagnostics responded by launching the most innovative and accurate diagnostic assays towards fast, robust, and accurate detection of SARS-CoV-2. Here we present eight distinct diagnostic approaches covering molecular and serological testing for the evaluation of patient\u27s health condition, infectiousness and their exposure to the virus. All these tests have demonstrated high accuracy and precision with over 99.5% sensitivity and specificity as they were validated throughout a large number of samples

Peptidomic Approaches and Observations in Neurodegenerative Diseases

Mass spectrometry (MS), with its immense technological developments over the last two decades, has emerged as an unavoidable technique in analyzing biomolecules such as proteins and peptides. Its multiplexing capability and explorative approach make it a valuable tool for analyzing complex clinical samples concerning biomarker research and investigating pathophysiological mechanisms. Peptides regulate various biological processes, and several of them play a critical role in many disease-related pathological conditions. One important example in neurodegenerative diseases is the accumulation of amyloid-beta peptides (Aβ) in the brain of Alzheimer’s disease (AD) patients. When investigating brain function and brain-related pathologies, such as neurodegenerative diseases, cerebrospinal fluid (CSF) represents the most suitable sample because of its direct contact with the brain. In this review, we evaluate publications applying peptidomics analysis to CSF samples, focusing on neurodegenerative diseases. We describe the methodology of peptidomics analysis and give an overview of the achievements of CSF peptidomics over the years. Finally, publications reporting peptides regulated in AD are discussed

Quantification of Cytokines secreted by primary human cells using multiple reaction monitoring: evaluation of analytical parameters

Determination of secreted proteins provides highly valuable information about cell functions. While the typical methods for the determination of biologically relevant but low-abundant molecular species still relies on the use of specific antibodies, mass spectrometry-based methods are now gaining sufficient sensitivity to cope with such challenges as well. In the current study we have identified several cytokines and chemokines which were induced in primary human umbilical vein endothelial cells upon inflammatory activation. Based on the high-resolution mass spectrometry data obtained with a Q Exactive orbitrap, we built an MRM method to quantify the most relevant molecules selected from the screening experiment. All experimental data are available via ProteomeXchange, PXD002211/12, and Panorama, www.panoramaweb.org. Using nano-flow Chip-HPLC coupled to a 6490 triple-quadrupole MS for MRM analyses we achieved calibration curves covering a linear range of four orders of magnitude and detection limits in the low attomol per microliter concentration range. Carryover was consistently less than 0.005%, the accuracy was between 80% and 120%, and the median coefficient of variation for LC/MS was only 2.2%. When including the variance of quantification introduced by cell culture and digestion, the coefficient of variation was less than 20% for most peptides. With appropriate marker molecules we monitored typical variations introduced by cell culture caused by differences in cell numbers, proliferative states and cell death. As a result, here, we present a robust and efficient MRM-based assay for the accurate and sensitive determination of cytokines and chemokines representative for functional cell states and including comprehensive quality controls

Neutrophil Extracellular Trap Formation Correlates with Favorable Overall Survival in High Grade Ovarian Cancer

It is still a question of debate whether neutrophils, often found in the tumor microenvironment, mediate tumor-promoting or rather tumor-inhibiting activities. The present study focuses on the involvement of neutrophils in high grade serous ovarian cancer (HGSOC). Macroscopic features classify two types of peritoneal tumor spread in HGSOC. Widespread and millet sized lesions characterize the miliary type, while non-miliary metastases are larger and associated with better prognosis. Multi-omics and FACS data were generated from ascites samples. Integrated data analysis demonstrates a significant increase of neutrophil extracellular trap (NET)-associated molecules in non-miliary ascites samples. A co-association network analysis performed with the ascites data further revealed a striking correlation between NETosis-associated metabolites and several eicosanoids. The congruence of data generated from primary neutrophils with ascites analyses indicates the predominance of NADPH oxidase 2 (NOX)-independent NETosis. NETosis is associated with protein S100A8/A9 release. An increase of the S100A8/CRP abundance ratio was found to correlate with favorable survival of HGSOC patients. The analysis of additional five independent proteome studies with regard to S100A8/CRP ratios confirmed this observation. In conclusion, NET formation seems to relate with better cancer patient outcome

Gel Electrophoretic and Mass Spectrometry-based methodologies in the quality study of Biopharmaceuticals

Modern analytical methodologies play an essential role regarding the quality evaluation of complex components, such as biologicals, where their implementation turned out to be crucial to assure their quality, safety, and efficacy. Glycoprotein hormones possess a highly complex structure and the study of their quality requires advanced analytical methodologies. Therefore, the aim of this study was the evaluation of suitable analytical methods for the quality study of such complex compounds. In order to satisfy the aim, 1D-SDS-PAGE and 2D gel electrophoresis in conjunction with mass spectrometry-based analyses were implemented. Gel electrophoretic techniques revealed the pI, Mr and glycoforms pattern, while MS analyses shed light upon the identity, structural integrity and glycosylation extent. The results demonstrated high complexity and extreme heterogeneity, typical for glycoproteins. Mass spectrometry provided information regarding structural identity parameters and glycosylation model. This methodology proved to be capable for the determination of purity and structural integrity of such complex compounds, although further investigations are required to fully understand glycosylation pattern since it has a significant effect on the pharmacokinetics and their biological activity

NECTIN4 (PVRL4) as Putative Therapeutic Target for a Specific Subtype of High Grade Serous Ovarian Cancer—An Integrative Multi-Omics Approach

In high grade serous ovarian cancer patients with peritoneal involvement and unfavorable outcome would benefit from targeted therapies. The aim of this study was to find a druggable target against peritoneal metastasis. We constructed a planar—scale free small world—co-association gene expression network and searched for clusters with hub-genes associated to peritoneal spread. Protein expression and impact was validated via immunohistochemistry and correlations of deregulated pathways with comprehensive omics data were used for biological interpretation. A cluster up-regulated in miliary tumors with NECTIN4 as hub-gene was identified and impact on survival validated. High Nectin 4 protein expression was associated with unfavorable survival and (i) reduced expression of HLA genes (mainly MHC I); (ii) with reduced expression of genes from chromosome 22q11/12; (iii) higher BCAM in ascites and in a high-scoring expression cluster; (iv) higher Kallikrein gene and protein expressions; and (v) substantial immunologic differences; locally and systemically; e.g., reduced CD14 positive cells and reduction of different natural killer cell populations. Each three cell lines with high (miliary) or low NECTIN4 expression (non-miliary) were identified. An anti-Nectin 4 antibody with a linked antineoplastic drug−already under clinical investigation−could be a candidate for a targeted therapy in patients with extensive peritoneal involvement

Comprehensive Assessment of Proteins Regulated by Dexamethasone Reveals Novel Effects in Primary Human Peripheral Blood Mononuclear Cells

Inflammation is a physiological process involved in many diseases. Monitoring proteins involved in regulatory effects may help to improve our understanding of inflammation. We have analyzed proteome alterations induced in peripheral blood mononuclear cells (PBMCs) upon inflammatory activation in great detail using high-resolution mass spectrometry. Moreover, the activated cells were treated with dexamethasone to investigate their response to this antiphlogistic drug. From a total of 6886 identified proteins, 469 proteins were significantly regulated upon inflammatory activation. Data are available via ProteomeXchange with identifiers PXD001415–23. Most of these proteins were counter-regulated by dexamethasone, with some exceptions concerning members of the interferon-induced protein family. To confirm some of these results, we performed targeted MRM analyses of selected peptides. The inflammation-induced upregulation of proteins such as IL-1β, IL-6, CXCL2, and GROα was confirmed, however, with strong quantitative interindividual differences. Furthermore, the inability of dexamethasone to downregulate inflammation-induced proteins such as PTX3 and TSG6 was clearly demonstrated. In conclusion, the relation of cell function as well as drug-induced modulation thereof was successfully mapped to proteomes, suggesting targeted analysis as a novel and powerful drug evaluation method. Although most consequences of dexamethasone were found to be compatible with the expected mode of action, some unexpected but significant observations may be related to adverse effects