Abstract
Affinity enrichment techniques in combination with quantitative proteomics enable the unbiased identification of protein–protein interaction, and thus the delineation of protein complexes and interaction networks. Here, we describe an immunoaffinity enrichment approach that employs covalently immobilized antibodies for the identification of protein–protein interactions of endogenously expressed proteins under near-to-physiological conditions. Specifically enriched proteins are identified using shotgun mass spectrometry and isobaric mass tag-based relative quantification.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Bantscheff M, Hopf C, Savitski MM et al (2011) Chemoproteomics profiling of HDAC inhibitors reveals selective targeting of HDAC complexes. Nat Biotechnol 29:255–265
Dawson MA, Prinjha RK, Dittmann A et al (2011) Inhibition of BET recruitment to chromatin as an effective treatment for MLL-fusion leukaemia. Nature 478:529–533
Kocher T, Superti-Furga G (2007) Mass spectrometry-based functional proteomics: from molecular machines to protein networks. Nat Methods 4:807–815
Gavin A-C, Aloy P, Grandi P et al (2006) Proteome survey reveals modularity of the yeast cell machinery. Nature 440:631–636
Krogan NJ, Cagney G, Yu H et al (2006) Global landscape of protein complexes in the yeast Saccharomyces cerevisiae. Nature 440:637–643
Song J, Hao Y, Du Z et al (2012) Identifying novel protein complexes in cancer cells using epitope-tagging of endogenous human genes and affinity-purification mass spectrometry. J Proteome Res 11:5630–5641
Malovannaya A, Lanz RB, Jung SY et al (2011) Analysis of the human endogenous coregulator complexome. Cell 145:787–799
Malovannaya A, Li Y, Bulynko Y et al (2010) Streamlined analysis schema for high-throughput identification of endogenous protein complexes. Proc Natl Acad Sci U S A 107:2431–2436
Mellacheruvu D, Wright Z, Couzens AL et al (2013) The CRAPome: a contaminant repository for affinity purification-mass spectrometry data. Nat Methods 10:730–736
Bantscheff M, Lemeer S, Savitski M et al (2012) Quantitative mass spectrometry in proteomics: critical review update from 2007 to the present. Anal Bioanal Chem 404:939–965
Selbach M, Mann M (2006) Protein interaction screening by quantitative immunoprecipitation combined with knockdown (QUICK). Nat Methods 3:981–983
Kruse U, Pallasch CP, Bantscheff M et al (2011) Chemoproteomics-based kinome profiling and target deconvolution of clinical multi-kinase inhibitors in primary chronic lymphocytic leukemia cells. Leukemia 25:89–100
Brehme M, Hantschel O, Colinge J et al (2009) Charting the molecular network of the drug target Bcr-Abl. Proc Natl Acad Sci U S A 106:7414–7419
Werner T, Becher I, Sweetman G et al (2012) High-resolution enabled TMT 8-plexing. Anal Chem 84:7188–7194
Olsen JV, de Godoy LMF, Li G et al (2005) Parts per million mass accuracy on an orbitrap mass spectrometer via lock mass injection into a C-trap. Mol Cell Proteomics 4:2010–2021
Eng J, McCormack A, Yates J (1994) An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J Am Soc Mass Spectrom 5:976–989
Perkins DN, Pappin DJC, Creasy DM et al (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20:3551–3567
Jr C, Neuhauser N, Michalski A et al (2011) Andromeda: a peptide search engine integrated into the MaxQuant environment. J Proteome Res 10:1794–1805
Acknowledgments
We would like to thank Frank Weisbrodt for help with the figures.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media New York
About this protocol
Cite this protocol
Dittmann, A., Ghidelli-Disse, S., Hopf, C., Bantscheff, M. (2014). Mapping Protein Complexes Using Covalently Linked Antibodies and Isobaric Mass Tags. In: Martins-de-Souza, D. (eds) Shotgun Proteomics. Methods in Molecular Biology, vol 1156. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0685-7_19
Download citation
DOI: https://doi.org/10.1007/978-1-4939-0685-7_19
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-0684-0
Online ISBN: 978-1-4939-0685-7
eBook Packages: Springer Protocols