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

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

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

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

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

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

Multi-omics Analysis of Serum Samples Demonstrates Reprogramming of Organ Functions Via Systemic Calcium Mobilization and Platelet Activation in Metastatic Melanoma

Pathophysiologies of cancer-associated syndromes such as cachexia are poorly understood and no routine biomarkers have been established, yet. Using shotgun proteomics, known marker molecules including PMEL, CRP, SAA, and CSPG4 were found deregulated in patients with metastatic melanoma. Targeted analysis of 58 selected proteins with multiple reaction monitoring was applied for independent data verification. In three patients, two of which suffered from cachexia, a tissue damage signature was determined, consisting of nine proteins, PLTP, CD14, TIMP1, S10A8, S10A9, GP1BA, PTPRJ, CD44, and C4A, as well as increased levels of glycine and asparagine, and decreased levels of polyunsaturated phosphatidylcholine concentrations, as determined by targeted metabolomics. Remarkably, these molecules are known to be involved in key processes of cancer cachexia. Based on these results, we propose a model how metastatic melanoma may lead to reprogramming of organ functions via formation of platelet activating factors from long-chain polyunsaturated phosphatidylcholines under oxidative conditions and via systemic induction of intracellular calcium mobilization. Calcium mobilization in platelets was demonstrated to alter levels of several of these marker molecules. Additionally, platelets from melanoma patients proved to be in a rather exhausted state, and platelet-derived eicosanoids implicated in tumor growth were found massively increased in blood from three melanoma patients. Platelets were thus identified as important source of serum protein and lipid alterations in late stage melanoma patients. As a result, the proposed model describes the crosstalk between lipolysis of fat tissue and muscle wasting mediated by oxidative stress, resulting in the metabolic deregulations characteristic for cachexia

Coffee consumption modulates inflammatory processes in an individual fashion

Anti-inflammatory effects of coffee consumption have been reported to be caused by caffeine and adenosine receptor signaling. However, contradictory effects have been observed. Many kinds of chronic diseases are linked to inflammation; therefore a profound understanding of potential effects of coffee consumption is desirable. We performed ex vivo experiments with eight individuals investigating peripheral blood mononuclear cells isolated from venous blood before and after coffee consumption, as well as in vitro experiments applying caffeine on isolated cells. After in vitro inflammatory stimulation of the cells, released cytokines, chemokines and eicosanoids were determined and quantified using targeted mass spectrometric methods. Remarkably, the release of inflammation mediators IL6, IL8, GROA, CXCL2, CXCL5 as well as PGA2, PGD2, PGE2, LTC4, LTE4 and 15S-HETE was significantly affected after coffee consumption. While in several individuals coffee consumption or caffeine treatment caused significant down-regulation of most inflammation mediators, in other healthy individuals exactly the opposite effects were observed. Ruling out age, sex, coffee consumption habits, the metabolic kinetics of caffeine in blood and the individual amount of regulatory T-cells or CD39 expression as predictive parameters, we demonstrated here that coffee consumption may have significant pro- or anti-inflammatory effects in an individual fashion