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Characterization of the phosphoproteome in LNCaP prostate cancer cells by in-gel isoelectric focusing and tandem mass spectrometry.

Reversible protein phosphorylation forms the basis of cell signaling networks. Aberrations in protein phosphorylation have been linked to human diseases including cancer. Phosphoproteomics has recently emerged as an approach that focuses on analysis of protein phosphorylation on a global scale. We have recently developed a new methodology, termed in-gel IEF LC-MS/MS, and we have adapted this methodology for phosphoproteome analysis. Here, we report on the application of in-gel IEF LC-MS/MS to the mapping of the phosphoproteome in the LNCaP human prostate cancer cell line. The analytical methodology used in the study included separation of the LNCaP proteins by in-gel isoelectric focusing (IEF), digestion of the proteins with trypsin, enrichment of the digests for phosphopeptides with Immobilized Metal Ion Affinity Chromatography (IMAC), analysis of the enriched digests by LC-MS/MS, and identification of the phosphorylated peptides/proteins through searches of a protein sequence database. With this analytical platform, we have characterized over 600 different phosphorylation sites in 296 phosphoproteins. This panel of the LNCaP phosphoproteins is 3-fold larger than the panel obtained in our previous work, which attests to the power of the chosen analytical methodology. The characterized phosphoproteins are functionally diverse and include a number of proteins relevant to cancer.