Targeted Approach for Proteomic Analysis of a Hidden Membrane Protein
Given the properties of plasma membrane proteins, namely, their hydrophobicity, low solubility, and high resistance to digestion and extraction, their identification by traditional mass spectrometry (MS) has been a challenging task. Hence, proteomic studies involving the transmembrane protein connexin43 (Cx43) are scarce. Additionally, studies demonstrating the presence of proteins embedded in the lipid bilayer of extracellular vesicles (EVs) are difficult to perform and require specific changes and fine adjustments in the experimental and technical procedure to allow their detection by MS. In this review, we provide a detailed description of the protocol we have used to detect Cx43 in EVs of human peripheral blood. This includes some of the modifications that we have introduced in order to improve the detection of Cx43 in EVs, including an optimization of vesicle isolation, Cx43 purification, MS acquisition data, and further analysis.
Key wordsSWATH-MS Proteomics Extracellular vesicles Cx43 Serum
This work was supported by the Portuguese Foundation for Science and Technology (FCT) grants, FCT-UID/NEU/04539/2013, PTDC/NEU-NMC/0205/2012, POCI-01-0145-FEDER-007440, and PTDC/NEU-SCC/7051/2014, by REDE/1506/REM/200 and by HealthyAging2020 CENTRO-01-0145-FEDER-000012-N2323. TMM was supported by PD/BD/106043/2015, SIA by SFRH/BD/81495/2011, and TRR by PD/BD/52294/2013.
- 13.Théry C, Amigorena S, Raposo G et al (2006) Isolation and characterization of exosomes from cell culture supernatants and biological fluids. In: Current protocols in cell biology. John Wiley & Sons, Inc., Hoboken, NJ, pp 3.22.1–3.22.29Google Scholar