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Experimental overview of the SPS-MS3 strategy. Four cell types (PaSC—in duplicate, HEK293, HeLa, and SH-SY5Y) were propagated and designated cultures were mock or nicotine treated. Proteins were extracted and then digested with Lys-C and trypsin. The resulting peptides were labeled with TMT, pooled, and fractionated via basic pH reversed-phase high performance liquid chromatography (BPRP-HPLC) prior to MS3 analysis on an Orbitrap Fusion mass spectrometer.
