Isolation protocols and mitochondrial content for plasma extracellular vesicles

Quantitative Analysis of Complement Membrane Attack Complex Proteins Associated with Extracellular Vesicles

“…We targeted the accurate measurement of protein stoichiometry of complement proteins involved in MAC formation, namely, C5b, C6, C7, C8, and C9 in the 20K-SEC-Hep and 106K-SEC-Hep EV preparations. We used a well-established MRM assay [ 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 ] to quantify these proteins. Nevertheless, we would like to emphasize key aspects of this technique and details of the EV isolation protocol that contribute to the accuracy and confidence of the measurements.…”

“…To further characterize the final preparations of EVs, we have measured five proteins (TSG101, flotillin-1, EHD4, moesin, and integrin beta-1) that are typical for any EV preparation ( Table 2 ). We also measured six plasma membrane-specific proteins and two endosomal membrane-specific proteins that are expected to appear mostly in 20K EVs and 106K EVs, respectively [ 23 , 24 , 25 , 26 ]. As expected, plasma membrane-specific proteins were found enriched in 20K-SEC-Hep EVs, while endosomal membrane-specific proteins were found enriched in 106K-SEC-Hep EVs.…”

“…EVs were isolated from 200 mL of pooled normal male human plasma K2EDTA (BioreclamationIVT, Westbury, NY, USA), as described previously [ 25 ]. The lot was pooled from 10 donors.…”

“…This approach allows for the simultaneous quantification of proteins of interest in a single liquid chromatography-mass spectrometry run. To ensure a high level of EV purity, a three-step isolation protocol was used [ 25 ]. The efficiency of this protocol was confirmed through the quantification of several EV proteins in parallel with the quantification of abundant plasma proteins, such as albumin and alpha-2-macroglobulin [ 25 ].…”

“…To ensure a high level of EV purity, a three-step isolation protocol was used [ 25 ]. The efficiency of this protocol was confirmed through the quantification of several EV proteins in parallel with the quantification of abundant plasma proteins, such as albumin and alpha-2-macroglobulin [ 25 ]. In the present research, the protocol was used to quantify 13 EV proteins side-by-side with MAC proteins.…”