Introduction: The membrane receptor P2X3 belongs to the P2X class of purinergic receptors which are gated by extracellular ATP. P2X3 is implicated in nociception and its role in the modulation of chronic pain in currently an active topic of study. In this poster we demonstrate the use of a fully integrated 2D LC-MS/MS system (Proteome X), in conjuction with ICAT, for the relative quantification of P2X3 and other membrane proteins in cell cultures grown under different conditions. The use of Xpress, an automated algorithm for the calculation of the d0/d8 ratio, is also demonstrated.
Methods: Membrane enriched fractions from differentially grown human embryonic kidney cells were processed through part of the ICAT labeling procedure in order to produce d0/d8-cysteine labeled tryptic peptides. The complex mixture of peptides (i.e. labeled and unlabeled) was separated using a new and fully-integrated 2D LC-MS/MS (Proteome X) via strong-cation exchange and reversed phase chromatography followed by ion trap detection. Protein identification was done by Sequest search of the human database. Realtive quantification of d0/d8 peptides was automated using the Xpress algorithm.
Preliminary Data: Preliminary studies using known ratios of d0/d8 labeled membrane preparations have yieled both the correct identification and quantification of P2X3. More than 500 other membrane proteins have also been indentified with high confidence in the same membrane preparations. We are currently in the process of preparing differential cultures for futher analysis.
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