For analysis of CD3ζ phosphorylation in PSCA-8tBBZ samples, total CD3ζ was immunoprecipitated using a mouse monoclonal anti-CD3ζ antibody (F3, Santa Cruz Biotechnology) and Protein G beads (Millipore) following the protocol provided by the Santa Cruz Biotechnology. Immunoprecipitates were then separated by SDS-PAGE. A gel slice ranging from 21 to 25 kDa was excised and digested, as described above for CAR interactome studies. LC-MS/MS was performed as described above; to maximize the ability to detect specific peptides, parallel reaction monitoring (or targeted MS/MS) was programmed for the expected charge states of phosphopeptides that had been previously observed in our data or reported in the literature. Sequest and Mascot database searches were used to identify phosphorylated peptides that were manually verified by inspection of mass measurement accuracy and fragment ion matching in the tandem mass spectra. MS/MS spectrum of peptide GHDGLYQGLSTATK: pTyr142 was located by b5 and b6 ions because the mass/charge ratio (m/z) of the b6 ion indicates the inclusion of the phosphorylation (Sequest XCorr sc score, 2.62; ∆CN score, 0.71). MS/MS spectrum of peptide MAEAYSEIGMK: pTyr123 was located by y6 and y7 ions because the m/z of y7 ion indicates the presence of the phosphorylation (Sequest XCorr score, 2.60; ∆CN score, 0.74).

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