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

using a ProteomeX Workstation (Thermo Finnigan, San Jose, CA). The system was fitted with a strong cation exchange column (SCX, 320 m ID 100 mm, DEV SCX, Thermo Hypersil- Keystone) and two C18 reversed phase columns (RP, 180 m x 100 mm, BioBasic C18, 5 m, Thermo Hypersil-Keystone), as shown in Figure 1. The salt steps used were 0, 25, 50, 100, 150, 200, 250 and 500 mM NH4Cl synchronized with seven 90-min RP gradients. RP solvents were 0.1% HCOOH in either water (A) or acetonitrile (B). Data Dependent mass spectral acquisition (MS/MS) was used for the most intense ion in the range of 300-1800 m/z (full-scan) with the following Dynamic Exclusion settings: repeat count, 1; repeat duration, 0.5 min; exclusion duration, 3.0 min. SCX and RP gradient programming, as well as MS settings, which can be set using the Multidimensional Protein ID method of the ProteomeX Workstation (Figure 2).

Data Analysis
Database searching was performed using the TurboSEQUEST program in the BioWorks 3.0 software suite. The database used was a subset (human) of the NR database available on 02/20/02 (ftp:// ftp. ncbi. nih. gov/blast/db/). No enzyme was specified for the search. Calculation of the d0:d8 ratios was performed automatically using the XPRESS feature of BioWorks 3.0. Only MS/MS spectra with Xcorr > 2.0 were considered for positive identification of a gene product. For the compilation of d0: d8 ratios, only those MS/MS spectra tagged by XPRESS, that also showed at least two of the ICAT diagnostic ions, were considered. Figure 3. Western blot of P2X3 expressed in HEK cells.


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