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

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

Research output: Contribution to journal › Article › peer-review

2 Citations (Scopus)

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).

Original language	English
Article number	101693
Journal	STAR Protocols
Volume	3
Issue number	4
DOIs	https://doi.org/10.1016/j.xpro.2022.101693
Publication status	Published or Issued - 16 Dec 2022
Externally published	Yes

Keywords

Cell biology
Mass spectrometry
Protein expression and purification
Proteomics

ASJC Scopus subject areas

General Neuroscience
General Biochemistry,Genetics and Molecular Biology
General Immunology and Microbiology

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10.1016/j.xpro.2022.101693

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title = "Endogenous protein interactomes resolved through immunoprecipitation-coupled quantitative proteomics in cell lines",

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).",

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AU - Kumar, Raman

AU - Kamath, Karthik S.

AU - Carroll, Luke

AU - Hoffmann, Peter

AU - Gecz, Jozef

AU - Jolly, Lachlan A.

PY - 2022/12/16

Y1 - 2022/12/16

N2 - 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).

AB - 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).

KW - Cell biology

KW - Mass spectrometry

KW - Protein expression and purification

KW - Proteomics

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DO - 10.1016/j.xpro.2022.101693

M3 - Article

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JO - STAR Protocols

JF - STAR Protocols

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ER -

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

Abstract

Keywords

ASJC Scopus subject areas

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