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Quantification of the Purinergic Receptor P2X3 using ICAT and ,,, Orthogonal 2D LC-MS/MS with an Ion Trap Mass Spectrometer

ortant analytical challenge. As expected, cysteine thiols that occur in highly hydrophobic environments, such as transmembrane protein domains, are less accessible for labeling. In instances like this, the success of the analysis hinges on the ability to efficiently label free thiols (i.e., on cysteines localized in extra-cellular loops), and to resolve and measure the labeled peptides in the midst of a very complex matrix.

We are reporting on the quantification of the membrane receptor P2X3 expressed in HEK cells using ICAT and 2D LC-MS/MS. The P2X family of purino-receptors constitutes a class of ligand-gated ion channels where ATP is the ligand. P2X3 occurs in small diameter sensory neurons and is implicated in the modulation of chronic pain. Goal In this report, we describe the analysis and quantification of the membrane receptor P2X3 using a technique that shows:
  1. The ease-of-use of the ProteomeX Workstation for multi-dimensional separation of complex mixtures.
  2. The ability of TurboSEQUEST software to identify ICAT-labeled proteins.
  3. The power of XPRESS to provide quantitative information for the proteins of interest.
Methods Preparation of Cell Membrane Fractions
Cell membranes used in this experiment were prepared from approximately 110 9 HEK cells, grown to ca. 90% confluency. Cells were harvested in cold PBS, pelleted, re-suspended to 2 mL in PBS and frozen at 80C. The cell suspension was thawed, diluted to 5 mL in cold PBS, and sonicated for 30 seconds followed by ultracentrifugation at 100,000 xg for 1 hou
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