Research Project: Memeli Hücre Yüzeyi Proteinlerinin Geniş Kapsamlı Proteomik Analizi için Yeni Metot Geliştirilmesi
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Contributors
Funders
ID
TB.00298
Authors
Yunt, Zeynep Sabahat
Teaching Faculty
Publications
Proximal biotinylation-based combinatory approach for isolating integral plasma membrane proteins
(American Chemical Society, 2020)
Department of Molecular Biology and Genetics
No
Akdağ, Mehmet
Kamacıoğlu, Altuğ
Qureshi, Mohammad Haroon
Comprehensive profiling of the cell-surface proteome has been challenging due to the lack of tools for an effective and reproducible way to isolate plasma membrane proteins from mammalian cells. Here we employ a proximity-dependent biotinylation approach to label and isolate plasma membrane proteins without an extra in vitro labeling step, which we call Plasma Membrane-BiolD. The lipid-modified BirA* enzyme (MyrPalm BirA*) was targeted to the inner leaflet of the plasma membrane, where it effectively biotinylated plasma membrane proteins. Biotinylated proteins were then affinity-purified and analyzed by mass spectrometry. Our analysis demonstrates that combining conventional sucrose density gradient centrifugation and Plasma Membrane-BioID is ideal to overcome the inherent limitations of the identification of integral membrane proteins, and it yields highly pure plasma components for downstream proteomic analysis.