Functional proteomics protocol for the identification of interaction partners in Tetrahymena thermophila

Syed Nabeel-Shah; Jyoti Garg; Pata-Eting Kougnassoukou Tchara; Ronald E Pearlman; Jean-Philippe Lambert; Jeffrey Fillingham

doi:10.1016/j.xpro.2021.100362

Functional proteomics protocol for the identification of interaction partners in Tetrahymena thermophila

STAR Protoc. 2021 Mar 4;2(1):100362. doi: 10.1016/j.xpro.2021.100362. eCollection 2021 Mar 19.

Authors

Syed Nabeel-Shah^{1

2

3}, Jyoti Garg^{1

4}, Pata-Eting Kougnassoukou Tchara^{5

6}, Ronald E Pearlman⁴, Jean-Philippe Lambert^{5

6}, Jeffrey Fillingham¹

Affiliations

¹ Department of Chemistry and Biology, Ryerson University, 350 Victoria Street, Toronto, ON M5B 2K3, Canada.
² Donnelly Centre, University of Toronto, Toronto, ON, M5S 3E1, Canada.
³ Department of Molecular Genetics, University of Toronto, Toronto, ON, M5S 1A8, Canada.
⁴ Department of Biology, York University, 4700 Keele Street, Toronto, ON, M3J 1P3, Canada.
⁵ Department of Molecular Medicine, Cancer Research Center, Big Data Research Center, Université Laval, Quebec City, QC, Canada.
⁶ CHU de Québec Research Center, CHUL, 2705 Laurier Boulevard, Quebec City, QC, G1V 4G2, Canada.

Abstract

We describe an optimized protocol for one-step affinity purification of FZZ-tagged proteins followed by mass spectrometry analysis for the identification of protein-protein interactions in the ciliate protozoan Tetrahymena thermophila. The FZZ epitope tag contains 2 protein A moieties (ZZ) and a 3xFLAG separated by a TEV cleavage site, which can also be employed in tandem affinity purification. This protocol is versatile and is suitable to use for other common epitope tags and can be adapted for other ciliates. For complete details on the use and execution of this protocol, please refer to Garg et al. (2019).

Keywords: Mass spectrometry; Model organisms; Molecular biology; Protein biochemistry; Protein expression and purification; Proteomics.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Proteomics*
Protozoan Proteins / metabolism*
Tetrahymena thermophila / metabolism*

Substances

Protozoan Proteins

Grants and funding

MOP13347 /CIHR/Canada