Figure 1. Experimental overview.
Samples for TMT analysis were prepared as illustrated above. PaSC were harvested following 12 hr serum starvation and subsequent 12 hr treatment with either nicotine or α-bungarotoxin. Cells were harvested and lysed so that proteins could be extracted and prepared for TMT-based mass spectrometry analysis. The additional step of TiO2 enrichment was performed for phosphopeptide analysis. Control samples were labeled with 126, 127N, and 127C; nicotine-treated samples were labeled with 128N, 128C, and 129N, while α-bungarotoxin-treated samples were labeled with 129C, 130N, and 130C.