Characterization of Protein Complexes Using Chemical Cross-Linking Coupled Electrospray Mass Spectrometry

Timothy D. Cummins, Gopal P. Sapkota (Lead / Corresponding author)

Research output: Chapter in Book/Report/Conference proceedingChapter (peer-reviewed)

1 Citation (Scopus)

Abstract

Identification and characterization of large protein complexes is a mainstay of biochemical toolboxes. Utilization of cross-linking chemicals can facilitate the capture and identification of transient or weak interactions of a transient nature (Huang and Kim, PloS One 8:e61430, 2013; Gao et al., J Vis Exp doi: 10.3791/51387, 2014). Here we describe a detailed methodology for a cell culture-based proteomic approach. We describe the generation of cells stably expressing green fluorescent protein (GFP)-tagged proteins under the tetracycline-inducible promoter and subsequent proteomic analysis of GFP-interacting proteins. We include a list of proteins that were identified as interactors of GFP.

Original languageEnglish
Title of host publicationTissue Proteomics
Subtitle of host publicationMethods and Protocols
EditorsM. Sarwal, T. Sigel
Place of PublicationNew York
PublisherHumana Press
Pages43-61
Number of pages19
Volume1788
ISBN (Electronic)9781493978540
ISBN (Print)9781493978526
DOIs
Publication statusPublished - 1 Jan 2018

Publication series

NameMethods in Molecular Biology
Volume1788
ISSN (Print)1064-3745

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Keywords

  • GFP affinity purification
  • Inducible expression
  • Interactome
  • Mass spectrometry
  • Protein complex
  • Protein–protein interaction

Cite this

Cummins, T. D., & Sapkota, G. P. (2018). Characterization of Protein Complexes Using Chemical Cross-Linking Coupled Electrospray Mass Spectrometry. In M. Sarwal, & T. Sigel (Eds.), Tissue Proteomics: Methods and Protocols (Vol. 1788, pp. 43-61). (Methods in Molecular Biology; Vol. 1788). Humana Press. https://doi.org/10.1007/7651_2017_85