High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

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A Systematic Analysis of Eluted Fraction of Plasma Post Immunoaffinity Depletion: Implications in Biomarker Discovery

Plasma is the most easily accessible source for biomarker discovery in clinical proteomics. However, identifying potential biomarkers from plasma is a challenge given the large dynamic range of proteins. The potential biomarkers in plasma are generally present at very low abundance levels and hence identification of these low abundance proteins necessitates the depletion of highly abundant proteins. Sample pre-fractionation using immuno-depletion of high abundance proteins using multi-affinity removal system (MARS) has been a popular method to deplete multiple high abundance proteins. However, depletion of these abundant proteins can result in concomitant removal of low abundant proteins. Although there are some reports suggesting the removal of non-targeted proteins, the predominant view is that number of such proteins is small. In this study, we identified proteins that are removed along with the targeted high abundant proteins. Three plasma samples were depleted using each of the three MARS (Hu-6, Hu-14 and Proteoprep 20) cartridges. The affinity bound fractions were subjected to gelC-MS using an LTQ-Orbitrap instrument. Using four database search algorithms including MassWiz (developed in house), we selected the peptides identified at <1% FDR. Peptides identified by at least two algorithms were selected for protein identification. After this rigorous bioinformatics analysis, we identified 101 proteins with high confidence. Thus, we believe that for biomarker discovery and proper quantitation of proteins, it might be better to study both bound and depleted fractions from any MARS depleted plasma sample

Proteomic Analysis of Polypeptides Captured from Blood during Extracorporeal Albumin Dialysis in Patients with Cholestasis and Resistant Pruritus

Albumin dialysis using the molecular adsorbent recirculating system (MARS) is a new therapeutic approach for liver diseases. To gain insight into the mechanisms involved in albumin dialysis, we analyzed the peptides and proteins absorbed into the MARS strong anion exchange (SAX) cartridges as a result of the treatment of patients with cholestasis and resistant pruritus. Proteins extracted from the SAX MARS cartridges after patient treatment were digested with two enzymes. The resulting peptides were analyzed by multidimensional liquid chromatography coupled to tandem mass spectrometry. We identified over 1,500 peptide sequences corresponding to 144 proteins. In addition to the proteins that are present in control albumin-derived samples, this collection includes 60 proteins that were specific to samples obtained after patient treatment. Five of these proteins (neutrophil defensin 1 [HNP-1], secreted Ly-6/uPAR-related protein 1 [SLURP1], serum amyloid A, fibrinogen alpha chain and pancreatic prohormone) were confirmed to be removed by the dialysis procedure using targeted selected-reaction monitoring MS/MS. Furthermore, capture of HNP-1 and SLURP1 was also validated by Western blot. Interestingly, further analyses of SLURP1 in serum indicated that this protein was 3-fold higher in cholestatic patients than in controls. Proteins captured by MARS share certain structural and biological characteristics, and some of them have important biological functions. Therefore, their removal could be related either to therapeutic or possible adverse effects associated with albumin dialysis

Studying protein–protein affinity and immobilized ligand–protein affinity interactions using MS-based methods

This review discusses the most important current methods employing mass spectrometry (MS) analysis for the study of protein affinity interactions. The methods are discussed in depth with particular reference to MS-based approaches for analyzing protein–protein and protein–immobilized ligand interactions, analyzed either directly or indirectly. First, we introduce MS methods for the study of intact protein complexes in the gas phase. Next, pull-down methods for affinity-based analysis of protein–protein and protein–immobilized ligand interactions are discussed. Presently, this field of research is often called interactomics or interaction proteomics. A slightly different approach that will be discussed, chemical proteomics, allows one to analyze selectivity profiles of ligands for multiple drug targets and off-targets. Additionally, of particular interest is the use of surface plasmon resonance technologies coupled with MS for the study of protein interactions. The review addresses the principle of each of the methods with a focus on recent developments and the applicability to lead compound generation in drug discovery as well as the elucidation of protein interactions involved in cellular processes. The review focuses on the analysis of bioaffinity interactions of proteins with other proteins and with ligands, where the proteins are considered as the bioactives analyzed by MS

Training future generations to deliver evidence-based conservation and ecosystem management

Data availability statement: No data was used in this study.Peer review: The peer review history for this article is available at: https://publons.com/publon/10.1002/2688-8319.12032.Supporting Information: eso312032-sup-0001-SuppMat.docx (21.1 KB) available at: https://besjournals.onlinelibrary.wiley.com/action/downloadSupplement?doi=10.1002%2F2688-8319.12032&file=eso312032-sup-0001-SuppMat.docx. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.Copyright © 2021 The Authors. 1. To be effective, the next generation of conservation practitioners and managers need to be critical thinkers with a deep understanding of how to make evidence-based decisions and of the value of evidence synthesis. 2. If, as educators, we do not make these priorities a core part of what we teach, we are failing to prepare our students to make an effective contribution to conservation practice. 3. To help overcome this problem we have created open access online teaching materials in multiple languages that are stored in Applied Ecology Resources. So far, 117 educators from 23 countries have acknowledged the importance of this and are already teaching or about to teach skills in appraising or using evidence in conservation decision-making. This includes 145 undergraduate, postgraduate or professional development courses. 4. We call for wider teaching of the tools and skills that facilitate evidence-based conservation and also suggest that providing online teaching materials in multiple languages could be beneficial for improving global understanding of other subject areas. Making informed conservation and ecosystem management choices is based upon a sound understanding of the relevant evidence. There is an increasing wealth of conservation science available, and access to this is becoming easier. But, are conservation practitioners being trained to utilize this information? In conservation, decision-making is often based upon past experience or expert knowledge, as opposed to the full body of scientific literature (e.g., Pullin, Knight, Stone, & Charman, 2004; Rafidimanantsoa, Poudyal, Ramamonjisoa, & Jones, 2018). The failure to include scientific evidence in decision-making has the potential to reduce the effectiveness of management, or even lead to detrimental actions being undertaken (Walsh, Dicks, & Sutherland, 2015). Evidence-based conservation (EBC) seeks to avoid this by providing tools to facilitate and inform decision-making. To do this, scientific evidence is collated and critically appraised for its quality and relevance, and integrated with other knowledge, experience, values and costs (Sutherland, Pullin, Dolman, & Knight, 2004). Wider adoption of EBC requires conservation professionals to be trained in its principles and taught how to use it to inform conservation decision-making.MAVA Foundation; Arcadia Fund

2D DIGE analysis of serum after fractionation by proteominer™ beads

10.1007/978-1-61779-573-2_13Methods in Molecular Biology854181-19

Pluripotent stem cell heterogeneity and the evolving role of proteomic technologies in stem cell biology

Stem cells represent obvious choices for regenerative medicine and are invaluable for studies of human development and drug testing. The proteomic landscape of pluripotent stem cells (PSCs), in particular, is not yet clearly defined; consequently, this field of research would greatly benefit from concerted efforts designed to better characterize these cells. In this concise review, we provide an overview of stem cell potency, highlight the types and practical implications of heterogeneity in PSCs and provide a detailed analysis of the current view of the pluripotent proteome in a unique resource for this rapidly evolving field. Our goal in this review is to provide specific insights into the current status of the known proteome of both mouse and human PSCs. This has been accomplished by integrating published data into a unified PSC proteome to facilitate the identification of proteins, which may be informative for the stem cell state as well as to reveal areas where our current view is limited. These analyses provide insight into the challenges faced in the proteomic analysis of PSCs and reveal one area - the cell surface subproteome - that would especially benefit from enhanced research efforts. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.link_to_subscribed_fulltex