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

Figure 4. Multiple File Consensus Report (composite) . Results To monitor the effectiveness of ICAT derivatization of P2X3, Western blot analysis was done using sample preparations before and after ICAT labeling (Figure 3). The results shown in Figure 3 confirmed the accessibility of free thiols for labeling by ICAT. This result was also consistent with previous reports on the effects of labeling on the migration behavior in SDS-PAGE.

Using a new feature in BioWorks 3.0 called the Multiple File Consensus Report, a composite of the results output was created, shown in Figure 4. This composite shows the 4357 gene products identified in a single 2D LC-MS/MS experiment. Only highly significant MS/MS matches (Xcorr>2.0) were considered for positive identification. P2X3 was ranked as #537 on the list.

Figure 5 shows the MS/MS spectrum (bottom left) and calculated d0: d8 ratio (bottom right) of the ICAT-labeled peptide TGGVLGIKIGWVC*IPK using XPRESS. Note that in addition to the expected yand b-ions labeled with blue and red in the spectrum, the spectrum also shows two diagnostic ICAT ions at m/z 288 and 485.

There were a total of seven Cys-containing fragments in P2X3 (Table 1). At least two of them (T1, T10) were inaccessible for labeling due to their location inside trans-membrane regions. In fact, the only labeled peptides detected in our experiments came from four Cys residues in the large extracellular loop.

Three other non-Cys containing peptides were also identified with high confidence between residues 218-243 and 358-367. Figure 5. MS
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