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.

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 Development of Methodology to Investigate the Surface SMALPome of Mammalian Cells

2021 ◽  
Vol 8 ◽  
Author(s):  
Kerrie A. Morrison ◽  
Kate J. Heesom ◽  
Karen J. Edler ◽  
James Doutch ◽  
Gareth J. Price ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Plasma Membrane ◽  
Membrane Proteins ◽  
Mammalian Cells ◽  
Analytical Techniques ◽  
Protein Characterization ◽  
Surface Proteins ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Detergent Extraction

Extraction of membrane proteins from biological membranes has traditionally involved detergents. In the past decade, a new technique has been developed, which uses styrene maleic acid (SMA) copolymers to extract membrane proteins into nanodiscs without the requirement of detergents. SMA nanodiscs are compatible with analytical techniques, such as small-angle scattering, NMR spectroscopy, and DLS, and are therefore an attractive medium for membrane protein characterization. While mass spectrometry has also been reported as a technique compatible with copolymer extraction, most studies have focused on lipidomics, which involves solvent extraction of lipids from nanodiscs prior to mass-spectrometry analysis. In this study, mass spectrometry proteomics was used to investigate whether there are qualitative or quantitative differences in the mammalian plasma membrane proteins extracted with SMA compared to a detergent control. For this, cell surface proteins of 3T3L1 fibroblasts were biotinylated and extracted using either SMA or detergent. Following affinity pull-down of biotinylated proteins with NeutrAvidin beads, samples were analyzed by nanoLC-MS. Here, we report for the first time, a global proteomics protocol for detection of a mammalian cell “SMALPome”, membrane proteins incorporated into SMA nanodiscs. Removal of SMA from samples prior to processing of samples for mass spectrometry was a crucial step in the protocol. The reported surface SMALPome of 3T3L1 fibroblasts consists of 205 integral membrane proteins. It is apparent that the detergent extraction method used is, in general, quantitatively more efficient at extracting proteins from the plasma membrane than SMA extraction. However, samples prepared following detergent extraction contained a greater proportion of proteins that were considered to be “non-specific” than in samples prepared from SMA extracts. Tantalizingly, it was also observed that proteins detected uniquely or highly preferentially in pull-downs from SMA extracts were primarily multi-spanning membrane proteins. These observations hint at qualitative differences between SMA and detergent extraction that are worthy of further investigation.

scholarly journals Effect of Ocular Hypertension on D-β-Aspartic Acid-Containing Proteins in the Retinas of Rats

2019 ◽  
Vol 2019 ◽  
pp. 1-8 ◽  
Author(s):  
Takashi Kanamoto ◽  
Takashi Tachibana ◽  
Yasushi Kitaoka ◽  
Toshio Hisatomi ◽  
Yasuhiro Ikeda ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Ocular Hypertension ◽  
Aspartic Acid ◽  
Atp Synthase ◽  
Wistar Rats ◽  
Protein Band ◽  
Mass Spectrometry Analysis ◽  
Liquid Chromatography Mass Spectrometry ◽  
Spectrometry Analysis ◽  
Sds Page

Purpose. To investigate the effect of ocular hypertension-induced isomerization of aspartic acid in retinal proteins. Methods. Adult Wistar rats with ocular hypertension were used as an experimental model. D-β-aspartic acid-containing proteins were isolated by SDS-PAGE and western blot with an anti-D-β-aspartic acid antibody and identified by liquid chromatography-mass spectrometry analysis. The concentration of ATP was measured by ELISA. Results. D-β-aspartic acid was expressed in a protein band at around 44.5 kDa at much higher quantities in the retinas of rats with ocular hypertension than in those of normotensive rats. The 44.5 kDa protein band was mainly composed of α-enolase, S-arrestin, and ATP synthase subunits α and β, in both the ocular hypertensive and normotensive retinas. Moreover, increasing intraocular pressure was correlated with increasing ATP concentrations in the retinas of rats. Conclusion. Ocular hypertension affected the expression of proteins containing D-β-aspartic acid, including ATP synthase subunits, and up-regulation of ATP in the retinas of rats.

scholarly journals N-Terminomics Strategies for Protease Substrates Profiling

Molecules ◽  
2021 ◽  
Vol 26 (15) ◽  
pp. 4699
Author(s):  
Mubashir Mintoo ◽  
Amritangshu Chakravarty ◽  
Ronak Tilvawala
Keyword(s):  
Mass Spectrometry ◽  
Protease Activity ◽  
Viral Infections ◽  
Mass Spectrometry Analysis ◽  
Post Translational Modification ◽  
Spectrometry Analysis ◽  
Physiological Conditions ◽  
Inflammatory Conditions ◽  
Biological Mixtures

Proteases play a central role in various biochemical pathways catalyzing and regulating key biological events. Proteases catalyze an irreversible post-translational modification called proteolysis by hydrolyzing peptide bonds in proteins. Given the destructive potential of proteolysis, protease activity is tightly regulated. Dysregulation of protease activity has been reported in numerous disease conditions, including cancers, neurodegenerative diseases, inflammatory conditions, cardiovascular diseases, and viral infections. The proteolytic profile of a cell, tissue, or organ is governed by protease activation, activity, and substrate specificity. Thus, identifying protease substrates and proteolytic events under physiological conditions can provide crucial information about how the change in protease regulation can alter the cellular proteolytic landscape. In recent years, mass spectrometry-based techniques called N-terminomics have become instrumental in identifying protease substrates from complex biological mixtures. N-terminomics employs the labeling and enrichment of native and neo-N-termini peptides, generated upon proteolysis followed by mass spectrometry analysis allowing protease substrate profiling directly from biological samples. In this review, we provide a brief overview of N-terminomics techniques, focusing on their strengths, weaknesses, limitations, and providing specific examples where they were successfully employed to identify protease substrates in vivo and under physiological conditions. In addition, we explore the current trends in the protease field and the potential for future developments.

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