Endogenous protein interactomes resolved through immunoprecipitation-coupled quantitative proteomics in cell lines

Raman Kumar; Karthik S Kamath; Luke Carroll; Peter Hoffmann; Jozef Gecz; Lachlan A Jolly

doi:10.1016/j.xpro.2022.101693

Endogenous protein interactomes resolved through immunoprecipitation-coupled quantitative proteomics in cell lines

STAR Protoc. 2022 Dec 16;3(4):101693. doi: 10.1016/j.xpro.2022.101693. Epub 2022 Sep 18.

Authors

Raman Kumar¹, Karthik S Kamath², Luke Carroll³, Peter Hoffmann⁴, Jozef Gecz⁵, Lachlan A Jolly⁶

Affiliations

¹ Adelaide Medical School and the Robinson Research Institute, University of Adelaide, Adelaide, SA 5005, Australia. Electronic address: raman.sharma@adelaide.edu.au.
² Australian Proteome Analysis Facility (APAF), Macquarie University, North Ryde, NSW 2109, Australia. Electronic address: karthik.kamath@hdr.mq.edu.au.
³ Australian Proteome Analysis Facility (APAF), Macquarie University, North Ryde, NSW 2109, Australia.
⁴ Clinical and Health Sciences, University of South Australia, Adelaide, SA 5000, Australia.
⁵ Adelaide Medical School and the Robinson Research Institute, University of Adelaide, Adelaide, SA 5005, Australia; South Australian Health and Medical Research Institute, Adelaide, SA 5000, Australia.
⁶ Adelaide Medical School and the Robinson Research Institute, University of Adelaide, Adelaide, SA 5005, Australia. Electronic address: lachlan.jolly@adelaide.edu.au.

Abstract

Immunoprecipitation (IP) of endogenously expressed proteins is one of the most biologically relevant techniques to identify protein-protein interactions. We describe an adaptable IP protocol reliant on a specific antibody to the target protein. We detail a quantitative proteomics workflow for the unbiased identification of co-immunoprecipitating proteins, known collectively as an interactome. This includes protocols for the tryptic digestion, Tandem Mass Tag labeling and fractionation of peptides, and their identification and quantification using liquid chromatography-mass spectrometry including computational and statistical analysis. For complete details on the use and execution of this protocol, please refer to Johnson et al. (2020).

Keywords: Cell biology; Mass spectrometry; Protein expression and purification; Proteomics.

Publication types

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

MeSH terms

Cell Line
Immunoprecipitation
Mass Spectrometry / methods
Proteins*
Proteomics* / methods

Substances

Proteins