Characterization and Proteomic Analysis of Endometrial Stromal Cell–Derived Small Extracellular Vesicles

2021 ◽  
Author(s):  
Chia-Yi Hsu ◽  
Tsung-Hua Hsieh ◽  
Hsiao-Yun Lin ◽  
Chi-Yu Lu ◽  
Hui-Wen Lo ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Extracellular Vesicles ◽  
Potential Role ◽  
Protein Composition ◽  
Mass Spectrometry Analysis ◽  
Endometrial Stromal Cells ◽  
Angiogenic Potential ◽  
Spectrometry Analysis ◽  
Novel Therapeutic Strategies

Abstract Context Small extracellular vesicles (sEVs) have emerged as modulators of the disease microenvironment, thereby supporting disease progression. However, the potential role of EVs and their content to the pathophysiology of endometriosis remain unclear. Objective This work aimed to investigate whether the EVs from eutopic (Eu) and ectopic (Ec) endometrial stromal cells (ESCs) differ with respect to protein composition and role in endometriosis. Methods Human Eu and Ec endometrium–derived ESCs were isolated from samples of the same patients (n = 3). sEVs were isolated from ESCs via ultracentrifugation; these sEVs were characterized by Western blotting, transmission electron microscopy, and nanoparticle tracking analysis and analyzed using mass spectrometry. The potential role of EcESCs-derived sEVs (EcESCs-sEVs) in endometriosis was explored by assaying their effects on cell viability/proliferation, migration, and angiogenesis. Results In total, 105 ESCs-sEV–associated proteins were identified from EcESCs-sEVs and EuESCs-sEVs by mass spectrometry analysis. The protein content differed between EcESCs-sEVs and EuESCs-sEVs, with annexin A2 (ANXA2) being the most prominent difference—present in EcESCs-sEVs but not EuESCs-sEVs. We also found that sEVs-ANXA2 regulates the motility, proliferation, and angiogenesis of ESCs via the extracellularly regulated kinase (ERK)/STAT3 pathway. Notably, treatment of ESCs with sEVs-ANXA2 resulted in increased proliferation and motility, suggesting that sEVs-ANXA2 may be involved in regulating endometriosis. Our data suggest that EcESCs-sEVs-ANXA2 regulates the motility and the angiogenic potential of ESCs, implying a role for sEVs-ANXA2 in the pathogenesis of endometriosis. Conclusion The study of sEVs-ANXA2 from Ec endometriotic cells uncovers a new mechanism of endometriosis progression and will inform the development of novel therapeutic strategies.

scholarly journals Treatment of Microglia with Anti-PrP Antibodies Induces Neuronal Allergenicity

2020 ◽  
Author(s):  
Utpal Kumar Adhikari ◽  
Elif Sakiz ◽  
Umma Habiba ◽  
Sachin Kumar ◽  
Meena Mikhael ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Molecular Mechanisms ◽  
Neuroblastoma Cell ◽  
Mass Spectrometry Analysis ◽  
Cross Linking ◽  
Antibody Treatment ◽  
Spectrometry Analysis ◽  
N2a Cells ◽  
Related Proteins

Abstract Background: Previous reports identified proteins associated with ‘apoptosis’ following cross-linking PrPC with motif-specific anti-PrP antibodies in vivo and in vitro. The molecular mechanisms underlying this IgG-mediated neurotoxicity and the role of the activated proteins in the apoptotic pathways leading to neuronal death has not been properly defined. Previous reports implicated a number of proteins, including apolipoprotein E, cytoplasmic phospholipase A2, prostaglandin and calpain with anti-PrP antibody-mediated ‘apoptosis’, however, these proteins are also known to play an important role in allergy. In this study, we investigated whether cross-linking PrPC with anti-PrP antibodies stimulates a neuronal allergenic response.Methods: Initially, we predicted the allergenicity of the epitope sequences associated with ‘neurotoxic’ anti-PrP antibodies using allergenicity prediction servers. We then investigated whether anti-PrP antibody treatment of neuronal (N2a) and microglia (N11) cell lines leads to a neuronal allergenic response.Results: We found that both tail- and globular-epitopes were allergenic. Specifically, binding regions that contain epitopes for ‘neurotoxic’ antibodies such as ICSM18 (146-159), ICSM35 (91-110), POM 1 (138-147), POM 2 (57-88) and POM 3 (95-100) lead to activation of allergenic related proteins. Following direct application of anti-PrPC antibodies on N2a cells, mass spectrometry analysis identified 4 neuronal allergenic-related proteins when compared with untreated cells. Furthermore, mass spectrometry analysis identified 8 neuronal allergenic-related proteins following cross-linking N11 cells with anti-PrPC antibodies prior to co-culture with N2a cells, when compared with untreated cells. Of importance, we showed that the allergenic effects triggered by the anti-PrP antibodies were more potent when antibody-treated microglia were co-cultured with the neuroblastoma cell line. Furthermore, in both direct and co-culture with antibody-treated microglia, we demonstrate that the allergenic proteome was part of the PrPC-interactome. Conclusions: This study showed for the first time that anti-PrP antibody binding to PrPC triggers a neuronal allergenic response (we termed ‘IgG-Mediated Neuronal Allergenic Toxicity’) and highlights the important role of microglia in triggering IgG-mediated neuronal allergenic toxicity. Moreover, this study provides an important impetus for including allergenic assessment of therapeutic antibodies for neurodegenerative to derive safe and targeted biotherapeutics.

scholarly journals Structural Architectural Features of Cyclodextrin Oligoesters Revealed by Fragmentation Mass Spectrometry Analysis

Molecules ◽  
2018 ◽  
Vol 23 (9) ◽  
pp. 2259 ◽  
Author(s):  
Cristian Peptu ◽  
Maksym Danchenko ◽  
Ľudovít Škultéty ◽  
Jaroslav Mosnáček
Keyword(s):  
Mass Spectrometry ◽  
Practical Importance ◽  
Gel Permeation Chromatography ◽  
Mass Spectrometry Analysis ◽  
Single Chain ◽  
Spectrometry Analysis ◽  
Architectural Features ◽  
Classical Characterization ◽  
Fragmentation Patterns

Cyclodextrins (CDs) were used in the present study for the ring-opening oligomerization (ROO) of l-lactide (LA) in order to synthesize biodegradable products with possible applications in pharmaceutical and medical fields. The practical importance of ROO reactions may reside in the possibility of synthesizing novel CD derivatives with high purity due to the dual role played by CDs, the role of the initiator through the hydroxylic groups, and the role of the catalyst by monomer inclusion in the CD cavity. The analyzed compounds were CDs modified with oligolactides obtained through ROO reactions of l-lactide in dimethylformamide. The resulting CD isomeric mixtures were investigated using classical characterization techniques such as gel permeation chromatography and nuclear magnetic resonance. Moreover, advanced mass spectrometry (MS) techniques were employed for the determination of the average number of monomer units attached to the cyclodextrin and the architecture of the derivatives (if the monomer units were attached as a single chain or as multiple chains). Thus, fragmentation studies effectuated on two different instruments (ESI Q-TOF and MALDI TOF) allowed us to correlate the size of the oligolactide chains attached to the CD with the observed fragmentation patterns.

Abstract WMP72: Peptide Composition of Stroke Causing Emboli Correlate With Serum Predictors of Atherosclerosis

Stroke ◽  
2017 ◽  
Vol 48 (suppl_1) ◽  
Author(s):  
Neal M Rao ◽  
Joseph Capri ◽  
Whitaker Cohn ◽  
Maram Y Abdaljaleel ◽  
Lucas Restrepo-Jimenez ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Acute Stroke ◽  
Small Sample Size ◽  
Protein Composition ◽  
Left Ventricular ◽  
Small Sample ◽  
Mass Spectrometry Analysis ◽  
Molecular Characteristics ◽  
Stroke Patients ◽  
Spectrometry Analysis

Introduction: The specific protein composition of stroke-causing emboli is unknown. Because ischemic stroke has a varied etiology, it is possible that the composition of the thrombus from which an embolus originated will have distinctive molecular characteristics reflective of the underlying pathophysiology. In this study, we used mass spectrometry to evaluate the protein composition of retrieved emboli from patients with differing stroke etiologies and correlated the protein levels to serum predictors of atherosclerosis. Methods: Emboli from 20 consecutive acute stroke patients were retrieved by embolectomy during routine stroke care. Thrombus proteins were extracted, digested and multidimentional fractionation of peptides was performed. Fractionated peptides underwent nano-liquid chromatography with tandem mass spectrometry. Spectra were searched using Mascot software in which results with p<0.05 (95% confidence interval) were considered significant and indicating identity. The results were correlated to A1C, LDL, and ESR taken on admission. Results: Demographics: 55% had atrial fibrillation, 20% had significant proximal vessel atherosclerosis, 10% were cryptogenic, and three had other identified etiologies (left ventricular thrombus, dissection, endocarditis). Eighty-one common proteins (eg hemoglobin, fibrin, actin) were found in all 20 emboli. Serum LDL levels correlated with Septin-2 (r s =0.78, p=0.028), Phosphoglycerate Kinase 1 (r s =0.75, p=0.036), Integrin Alpha-M (r s =0.68, p=0.033) and Glucose-6-phosphate dehydrogenase (r s =0.63, p=0.05). Septin-7 levels inversely correlated to ESR (r s = -0.84, p=0.01). No significant protein correlations to A1C were found. Conclusion: To our knowledge, this study is the first mass spectrometry analysis of thrombi retrieved from acute stroke patients and the first to correlate the thrombus proteome to clinical features of the patient. Notably, we found proteins associated with inflammation (eg Integrin Alpha–M) in emboli from patients with high LDL. Although these findings are tempered by a small sample size, we provide preliminary support for the feasibility of utilizing proteomic analysis of emboli to discover proteins that may be used as markers for stroke etiology.

scholarly journals Analysis of the Anaplasma marginale Major Surface Protein 1 Complex Protein Composition by Tandem Mass Spectrometry

2006 ◽  
Vol 188 (13) ◽  
pp. 4983-4991 ◽  
Author(s):  
Henriette Macmillan ◽  
Kelly A. Brayton ◽  
Guy H. Palmer ◽  
Travis C. McGuire ◽  
Gerhard Munske ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Tandem Mass Spectrometry ◽  
Protein Composition ◽  
Surface Protein ◽  
Anaplasma Marginale ◽  
Mass Spectrometry Analysis ◽  
Tandem Mass ◽  
Spectrometry Analysis ◽  
Major Surface Protein ◽  
Major Surface

ABSTRACT The protective major surface protein 1 (MSP1) complex of Anaplasma marginale is a heteromer of MSP1a and MSP1b, encoded by a multigene family. The msp1β sequences were highly conserved throughout infection. However, liquid chromatography-tandem mass spectrometry analysis identified only a single MSP1b protein, MSP1b1, within the MSP1 complex.

scholarly journals Quantitative Proteomic Analysis of Biogenesis-Based Classification for Extracellular Vesicles

Proteomes ◽  
2020 ◽  
Vol 8 (4) ◽  
pp. 33
Author(s):  
Linwen Zhang ◽  
Jeremie Parot ◽  
Vincent A. Hackley ◽  
Illarion V. Turko
Keyword(s):  
Mass Spectrometry ◽  
Plasma Membrane ◽  
Extracellular Vesicles ◽  
Specific Protein ◽  
Mass Spectrometry Analysis ◽  
Protein Markers ◽  
Spectrometry Analysis ◽  
Endosomal Membrane ◽  
Size Limitation ◽  
Endosomal Membranes

Extracellular vesicles (EVs) are traditionally divided into two major groups: (i) large vesicles originating from plasma membrane and called microvesicles, and (ii) small vesicles originating from the endoplasmic membrane and called exosomes. However, it is increasingly clear that the actual composition of a particular EV preparation cannot be adequately described with these two simple terms and is much more complex. Since the cell membrane origin of EVs predetermines their biological functions, the understanding of EV biogenesis is important for accurate interpretation of observed results. In the present study, we propose to take advantage of selective expression of some proteins in plasma or endosomal membranes and to use these proteins as plasma membrane-specific or endosomal membrane-specific markers. We have demonstrated that a quantitative mass spectrometry analysis allows simultaneous measurement of plasma membrane-specific and endosomal membrane-specific proteins in microvesicles and exosomes obtained after differential ultracentrifugation. Before mass spectrometry analysis, we also used sonicated platelets as a model of mixed EVs and multidetector asymmetrical-flow field-flow fractionation as an analytical method to verify a possible cross contamination of obtained microvesicles and exosomes. Based on the quantitative appearance of membrane-specific protein markers in EV preparations from human plasma and from human ARPE-19 cell medium, we concluded that there is no actual size limitation and both microvesicles and exosomes can be represented by large and small vesicles.

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