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Phosphorylation Analysis of Primary Human T Lymphocytes Using Sequential IMAC and Titanium Oxide Enrichment

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journal contribution
posted on 05.12.2008, 00:00 by Montserrat Carrascal, David Ovelleiro, Vanessa Casas, Marina Gay, Joaquin Abian
T lymphocytes mediate cellular and humoral defense against foreign bodies or autoantigens. An understanding of T-cell information processing furthers studies of the immunological response. We describe a large-scale phosphorylation analysis of primary T cells using a multidimensional separation strategy, involving preparative SDS-PAGE for prefractionation, in-gel digestion and sequential phosphopeptide enrichment using IMAC and TiO2. A total of 281 phosphorylation sites (197 of high confidence, Ascore > 15), mapping to 204 human gene sequences, were identified by LC−MSn analysis in an LTQ linear ion trap. Subsequently, we created the LymPHOS database (http://lymphos.org), which links mass spectrometric peptide information to phosphorylation sites and phosphoprotein sequences.

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