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Targeted Approach for Proteomic Analysis of a Hidden Membrane Protein

  • Tania Martins-Marques
  • Sandra I. Anjo
  • Teresa Ribeiro-Rodrigues
  • Bruno Manadas
  • Henrique GiraoEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1619)

Abstract

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 words

SWATH-MS Proteomics Extracellular vesicles Cx43 Serum 

Notes

Acknowledgments

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.

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Tania Martins-Marques
    • 1
    • 2
  • Sandra I. Anjo
    • 2
    • 3
    • 4
  • Teresa Ribeiro-Rodrigues
    • 1
    • 2
  • Bruno Manadas
    • 2
    • 3
  • Henrique Girao
    • 1
    • 2
    Email author
  1. 1.Institute of Biomedical Imaging and Life Sciences (IBILI), Faculty of MedicineUniversity of CoimbraCoimbraPortugal
  2. 2.CNC.IBILIUniversity of CoimbraCoimbraPortugal
  3. 3.CNC—Center for Neuroscience and Cell BiologyUniversity of CoimbraCoimbraPortugal
  4. 4.Faculty of Sciences and TechnologyUniversity of CoimbraCoimbraPortugal

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