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Quantitative characterization of p-sites from the human T-lymphocyte phosphoproteome using isotopic labeling and mass spectrometry

AuthorsCasas, Vanessa ; Carrascal, Montserrat ; Gay, Marina ; Gelpí, Emili ; Abián, Joaquín
Issue Date2012
AbstractT-Iymphocytes are white blood cells that mediate cellular and humoral defense against foreign bodies or autoantigens. A key process for Iymphocyte activation is protein phosphorylation-dephosphorylation. Characterization of the phosphoprotein profiles of T-Iymphocytes in physiological conditions and the determination of these changes during cell activation is principal for the understanding of the processes invoJved in body defense and in autoimmune diseases.In this context we are interested in the characterization of phosphoproteins and p-sites in human primary T-cells and we are studyillg the qualltitative changes produced by differellt activators on the phosphoproteome of these cells. We established a strategy to increase the number and confidence of the identified sequences based on using different search engines, which was previously described in the study of the plasma phosphoproteome (Can-ascal et al , IPR 2010) . We used SEQUEST, Phenyx and OMSSA. These tools make use of different algorithms and scores for the identification of sequences in the database matching a mas s spectrum. Thus, identification of the same sequence by more than one search engine greatly increases confidence in the match. To select the best matches we aligned the SEQUEST identifications with an Xcon- > 2, the Phenyx identifications with z-score > 5 and the OMSSA identification with expectation value < 1 and we selected as con-ect those that were identified at Ieast with two search engines. With this approach we obtained a FDR < 0.7%. Peptide concentration differences were expressed using the natural logarithm of the A/C ratio (LAC). The LAC of the non phosphorytaled peptides were adjusted to a Gaussian distribution and the JI and swere caIculated. We selected as differential phosphopeptides those wiht a JI-2s< LAC < JI-2s . These studies have produced the first collection cun-ently available of primary T-Iymphocyte phosphorylation sites. The complete set of data obtained so far is stored in our LymPHOS database that is publicIy available at http://lvmphos.org (Ovelleiro et al. Proteomics 2009). We have implemented an automatic workflow for the annotation of the database that incIudes tools for MS data filtering , accurate phosphorylation site assignation and quantification. This data constitutes the only phosphorylation map available for human primary T-Iymphocytes. Several novellymphocyte specific p-sites are described and quantified. This information could be the basis for future studies on the role of phosphorylation in T-cell functions and the effect of pharmacological and immunological agonists and conditions in T-Iymphocyte activation.
DescriptionTrabajo presentado al 6th European Summer School in Proteomic Basics FEBS Advanced Lecture Course - High Performance Proteomics (Italia, Agosto 2012) y al 8th FENS Forum of Neuroscience (Barcelona, Julio 2012).
Appears in Collections:(IIBB) Comunicaciones congresos
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