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Vogt, A.* ; Fuerholzner, B.* ; Kinkl, N.* ; Boldt, K.* ; Ueffing, M.

Isotope Coded Protein Labeling Coupled Immunoprecipitation (ICPL-IP): A novel approach for quantitative protein complex analysis from native tissue.

Mol. Cell. Proteomics 12, 1395-1406 (2013)
Postprint Manuskript DOI PMC
Open Access Green
High confidence definition of protein interactions is an important objective towards biological systems understanding. Isotope labeling in combination with affinity-based isolation of protein complexes has increased in accuracy and reproducibility, yet, larger organisms - including humans - are hardly accessible to metabolic labeling and thus, a major limitation has been its restriction to small animals, cell lines and yeast. As composition as well as stoichiometries of protein complexes can significantly differ in primary tissues, there is a great demand for methods capable to combine the selectivity of affinity-based isolation as well as the accuracy and reproducibility of isotope-based labeling with its application towards analysis of protein interactions from intact tissue. Towards this goal, we combined isotope coded protein labeling (ICPL) with immunoprecipitation (IP) and quantitative mass spectrometry (MS). ICPL-IP allows sensitive and accurate analysis of protein interactions from primary tissue. We applied ICPL-IP to immuno-isolate protein complexes from bovine retinal tissue. Protein complexes of immunoprecipitated β-tubulin, a highly abundant protein with known interactors as well as the lowly expressed small GTPase RhoA were analyzed. The results of both analyses demonstrate sensitive and selective identification of known as well as new protein interactions by our method
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Publication type Article: Journal article
Document type Scientific Article
Corresponding Author
ISSN (print) / ISBN 1535-9476
e-ISSN 1535-9484
Journal Molecular and Cellular Proteomics
Quellenangaben Volume: 12, Issue: 5, Pages: 1395-1406 Article Number: , Supplement: ,
Publisher American Society for Biochemistry and Molecular Biology
Non-patent literature Publications
Reviewing status Peer reviewed
Institute(s) CF Metabolomics & Proteomics (CF-MPC)