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

/MS spectrum and calculated d0: d8 ratio of P2X3 peptide.

Table 1. P2X3 ICATlabeled peptides and their corresponding d0:d8 ratios. Figure 6 shows a hydropathy plot for the P2X3 receptor protein. The y-axis plots the hydrophilicity/hydrophobicity of the protein for each of its amino acid residues along the x-axis. Each of the Cys-containing peptides listed in Table 1 are plotted along with their locations within the cell. Most of the labeled peptides are located within the extra-cellular loop, while two are found in trans-membrane regions. Figure 6. Hydropathy plot of P2X3 showing the location of Cys-containing peptides. Conclusions
  1. These experiments serve as a preliminary validation of the ProteomeX technology before attempting quantification of P2X3 in vitro and in vivo.
  2. Off-line IEX and affinity clean-up of samples prior to LC/ MS analysis was not required. Non-specific sample losses were minimized. Both ICAT- labeled and unlabeled peptides were seen.
  3. In the future, we plan to quantify the levels of P2X3 in systems treated with RiboTAG (Neuromics Inc., Minneapolis, MN) antisense oligonucleotides in gene function studies.
References

  1. Yates, J. R. et al. Nature Biotech. 17, 676-682 (1999)
  2. Aebersold, R. et al. Anal. Biochem. 297, 25-31 (2001)
  3. Aebersold, R. et al. Molecular Cellular Proteomics 1, 19-20 (2002)'"/>


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