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Issue 4, 2013
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Combining enzymatic 18O-labeling and 2-D LC-MS/MS for a study of protein interactions in primary T cells

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Abstract

Affinity-MS experiments with primary cells require alternative proteomic approaches, as the widely used metabolic labeling method SILAC is impracticable. We now describe a novel application of a recently developed 2-D LC-MS/MS approach for identification of phosphorylation-dependent protein interactions in human primary T cells. Using this approach, we identified a set of Tyr 595-phosphorylated ADAP interaction partners which belong to the larger TCR proximal signaling complex. The results show that a combination of two-dimensional RP–RP LC-MS/MS and 18O-labeling is a powerful means for peptide-based affinity MS experiments.

Graphical abstract: Combining enzymatic 18O-labeling and 2-D LC-MS/MS for a study of protein interactions in primary T cells

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Supplementary files

Article information


Submitted
30 Oct 2012
Accepted
29 Dec 2012
First published
03 Jan 2013

Anal. Methods, 2013,5, 1058-1061
Article type
Technical Note

Combining enzymatic 18O-labeling and 2-D LC-MS/MS for a study of protein interactions in primary T cells

D. Lang, S. Anker, B. Kuropka and E. Krause, Anal. Methods, 2013, 5, 1058
DOI: 10.1039/C2AY26298C

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