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Investigation of protein–protein interactions in living cells by chemical crosslinking and mass spectrometry

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Abstract

The identification of protein–protein interactions within their physiological environment is the key to understanding biological processes at the molecular level. However, the artificial nature of in vitro experiments, with their lack of other cellular components, may obstruct observations of specific cellular processes. In vivo analyses can provide information on the processes within a cell that might not be observed in vitro. Chemical crosslinking combined with mass spectrometric analysis of the covalently connected binding partners allows us to identify interacting proteins and to map their interface regions directly in the cell. In this paper, different in vivo crosslinking strategies for deriving information on protein–protein interactions in their physiological environment are described.

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Abbreviations

aaRS:

Aminoacyl-tRNA synthetase

FTICR:

Fourier transform ion cyclotron resonance

HBH:

Histidine–biotin–histidine

LC:

Liquid chromatography

MS:

Mass spectrometry

NHS:

N-hydroxysuccinimide

PAL:

Photoaffinity labeling

PIR:

Protein interaction reporter

QTAX:

Quantitative analysis of tandem-affinity purified crosslinked protein complexes

SILAC:

Stable isotope labeling using amino acids in cell culture

TAP:

Tandem-affinity purification

TRAP:

Targeted and releasable affinity probe

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Authors and Affiliations

  1. Department of Pharmaceutical Chemistry & Bioanalytics, Institute of Pharmacy, Martin-Luther-Universität Halle-Wittenberg, Wolfgang-Langenbeck-Straße 4, 06120, Halle (Saale), Germany

    Andrea Sinz

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  1. Andrea Sinz
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Correspondence to Andrea Sinz.

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Sinz, A. Investigation of protein–protein interactions in living cells by chemical crosslinking and mass spectrometry. Anal Bioanal Chem 397, 3433–3440 (2010). https://doi.org/10.1007/s00216-009-3405-5

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  • DOI: https://doi.org/10.1007/s00216-009-3405-5

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