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

Quality control - a stepchild in quantitative proteomics

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 $\it MaCProQC$, 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

Translation of Cryobiological Techniques to Socially Economically Deprived Populations—Part 1: Cryogenic Preservation Strategies

Use of cold for preservation of biological materials, avoidance of food spoilage and to manage a variety of medical conditions has been known for centuries. The cryobiological science justified these applications in the 1960s increasing their use in expanding global activities. However, the engineering and technological aspects associated with cryobiology can be expensive and this raises questions about the abilities of resource-restricted low and middle income countries (LMICs) to benefit from the advances. This review was undertaken to understand where or how access to cryobiological advances currently exist and the constraints on their usage. The subject areas investigated were based on themes which commonly appear in the journal Cryobiology. This led in the final analysis for separating the review into two parts, with the first part dealing with cold applied for biopreservation of living cells and tissues in science, health care and agriculture, and the second part dealing with cold destruction of tissues in medicine. The limitations of the approaches used are recognized, but as a first attempt to address these topics surrounding access to cryobiology in LMICs, the review should pave the way for future more subject-specific assessments of the true global uptake of the benefits of cryobiology.Fil: Buriak, Iryna. National Academy of Sciences of Ukraine; UcraniaFil: Fleck, Roland. Kings College London; Reino UnidoFil: Goltsev, Anatoliy. National Academy of Sciences of Ukraine; UcraniaFil: Shevchenko, Nadiya. National Academy of Sciences of Ukraine; UcraniaFil: Petrushko, Maryna. National Academy of Sciences of Ukraine; UcraniaFil: Yurchuk, Taisiia. National Academy of Sciences of Ukraine; UcraniaFil: Puhovkin, Anton. National Academy of Sciences of Ukraine; UcraniaFil: Rozanova, Svitlana. National Academy of Sciences of Ukraine; UcraniaFil: Guibert, Edgardo Elvio. Universidad Nacional de Rosario. Secretaria de Ciencia y Técnica. Centro Binacional de Investigación en Criobiología Clínica y Aplicada; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; ArgentinaFil: Robert, María Celeste. Universidad Nacional de Rosario. Secretaria de Ciencia y Técnica. Centro Binacional de Investigación en Criobiología Clínica y Aplicada; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; ArgentinaFil: Juan de Paz, Leonardo. Universidad Nacional de Rosario. Secretaria de Ciencia y Técnica. Centro Binacional de Investigación en Criobiología Clínica y Aplicada; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; ArgentinaFil: Powell Palm, Matthew J.. University of California at Berkeley; Estados UnidosFil: Fuller, Barry. University College London; Estados Unido