Changes in the Proteome of Platelets from Patients with Critical Progression of COVID-19.

Platelets, the smallest cells in human blood, known for their role in primary hemostasis, are also able to interact with pathogens and play a crucial role in the immune response. In severe coronavirus disease 2019 (COVID-19) cases, platelets become overactivated, resulting in the release of granules, exacerbating inflammation and contributing to the cytokine storm. This study aims to further elucidate the role of platelets in COVID-19 progression and to identify predictive biomarkers for disease outcomes. A comparative proteome analysis of highly purified platelets from critically diseased COVID-19 patients with different outcomes (survivors and non-survivors) and age- and sex-matched controls was performed. Platelets from critically diseased COVID-19 patients exhibited significant changes in the levels of proteins associated with protein folding. In addition, a number of proteins with isomerase activity were found to be more highly abundant in patient samples, apparently exerting an influence on platelet activity via the non-genomic properties of the glucocorticoid receptor (GR) and the nuclear factor κ-light-chain-enhancer of activated B cells (NFκB). Moreover, carbonic anhydrase 1 (CA-1) was found to be a candidate biomarker in platelets, showing a significant increase in COVID-19 patients.

Quality Control-A Stepchild in Quantitative Proteomics: A Case Study for the Human CSF Proteome.

Proteomic studies using mass spectrometry (MS)-based quantification are a main approach to the discovery of new biomarkers. However, a number of analytical conditions in front and during MS data acquisition can affect the accuracy of the obtained outcome. Therefore, comprehensive quality assessment of the acquired data plays a central role in quantitative proteomics, though, due to the immense complexity of MS data, it is often neglected. Here, we address practically the quality assessment of quantitative MS data, describing key steps for the evaluation, including the levels of raw data, identification and quantification. With this, four independent datasets from cerebrospinal fluid, an important biofluid for neurodegenerative disease biomarker studies, were assessed, demonstrating that sample processing-based differences are already reflected at all three levels but with varying impacts on the quality of the quantitative data. Specifically, we provide guidance to critically interpret the quality of MS data for quantitative proteomics. Moreover, we provide the free and open source quality control tool MaCProQC, enabling systematic, rapid and uncomplicated data comparison of raw data, identification and feature detection levels through defined quality metrics and a step-by-step quality control workflow.

Protein Expression of AEBP1, MCM4, and FABP4 Differentiate Osteogenic, Adipogenic, and Mesenchymal Stromal Stem Cells.

Mesenchymal stem cells (MSCs) gain an increasing focus in the field of regenerative medicine due to their differentiation abilities into chondrocytes, adipocytes, and osteoblastic cells. However, it is apparent that the transformation processes are extremely complex and cause cellular heterogeneity. The study aimed to characterize differences between MSCs and cells after adipogenic (AD) or osteoblastic (OB) differentiation at the proteome level. Comparative proteomic profiling was performed using tandem mass spectrometry in data-independent acquisition mode. Proteins were quantified by deep neural networks in library-free mode and correlated to the Molecular Signature Database (MSigDB) hallmark gene set collections for functional annotation. We analyzed 4108 proteins across all samples, which revealed a distinct clustering between MSCs and cell differentiation states. Protein expression profiling identified activation of the Peroxisome proliferator-activated receptors (PPARs) signaling pathway after AD. In addition, two distinct protein marker panels could be defined for osteoblastic and adipocytic cell lineages. Hereby, overexpression of AEBP1 and MCM4 for OB as well as of FABP4 for AD was detected as the most promising molecular markers. Combination of deep neural network and machine-learning algorithms with data-independent mass spectrometry distinguish MSCs and cell lineages after adipogenic or osteoblastic differentiation. We identified specific proteins as the molecular basis for bone formation, which could be used for regenerative medicine in the future.

Correction: Gemoll et al. Protein Profiling of Serum Extracellular Vesicles Reveals Qualitative and Quantitative Differences after Differential Ultracentrifugation and ExoQuick™ Isolation. J. Clin. Med. 2020, 9, 1429.

The authors wish to make the following corrections to this paper [...].

Quantitative Mass Spectrometry-Based Proteomics: An Overview.

In recent decades, mass spectrometry has moved more than ever before into the front line of protein-centered research. After being established at the qualitative level, the more challenging question of quantification of proteins and peptides using mass spectrometry has become a focus for further development. In this chapter, we discuss and review actual strategies and problems of the methods for the quantitative analysis of peptides, proteins, and finally proteomes by mass spectrometry. The common themes, the differences, and the potential pitfalls of the main approaches are presented in order to provide a survey of the emerging field of quantitative, mass spectrometry-based proteomics.

Protein Profiling of Serum Extracellular Vesicles Reveals Qualitative and Quantitative Differences After Differential Ultracentrifugation and ExoQuickTM Isolation.

Solid tumor biopsies are the current standard for precision medicine. However, the procedure is invasive and not always feasible. In contrast, liquid biopsies, such as serum enriched for extracellular vesicles (EVs) represent a non-invasive source of cancer biomarkers. In this study, we compared two EV isolation methods in the context of the protein biomarker detection in inflammatory bowel disease (IBD) and colorectal cancer (CRC). Using serum samples of a healthy cohort as well as CRC and IBD patients, EVs were isolated by ultracentrifugation and ExoQuickTM in parallel. EV associated protein profiles were compared by multiplex-fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) and subsequent identification by mass spectrometry. Validation of gelsolin (GSN) was performed using fluorescence-quantitative western blot. 2D-DIGE resolved 936 protein spots in all serum-enriched EVs isolated by ultracentrifugation or ExoQuickTM. Hereof, 93 spots were differently expressed between isolation approaches. Higher levels of GSN in EVs obtained with ExoQuickTM compared to ultracentrifugation were confirmed by western blot (p = 0.0006). Although patient groups were distinguishable after both EV isolation approaches, sample preparation strongly influences EVs' protein profile and thus impacts on inter-study reproducibility, biomarker identification and validation. The results stress the need for strict SOPs in EV research before clinical implementation can be reached.

Protective effect of placenta extracts against nitrite-induced oxidative stress in human erythrocytes.

Aqueous-saline human placenta extract (HPE) is known to possess antioxidant activity due to the high concentration of bioactive substances. This fact allows its application in clinical practice in order to treat oxidation-induced diseases. Extract antioxidant activity is mainly conditioned by proteins. Freezing of extracts has been shown to lead to their antioxidant activity increasing due to protein conformation changes.Different biological models are widely used in order to evaluate efficacy of novel antioxidants and mechanisms of their action. One such model appears to be erythrocytes under nitrite-induced oxidative stress. Nitrite is known to be able to penetrate erythrocyte membrane and to oxidize hemoglobin. In order to investigate whether HPE is able to decrease nitrite-induced oxidative injuries and to evaluate the protein contribution to this process, spectrophotometric and electron spin resonance (ESR) assays were used.Experimental data have revealed that antioxidant activity of extracts and of some of their fractions correlates with methemoglobin concentration lowering. Preliminary erythrocyte incubation with an extract fraction of 12 kDa allows preservation of the structural-dynamic cytosol state the closest to the control.