Figure 1. Liquid Chromatography Multiple Reaction Monitoring Mass Spectrometry: Principle and Practice.

After reversed-phase HPLC separation, peptides are selected by their mass-to-charge ratio and dissociated by collisions with background gas before the fragment ions are mass selected to enable specific detection and quantification of individual peptides in complex mixtures (A). This technique was applied to the measurement of expression of apoptosis-regulating proteins in the Bcl-2 family to determine the mechanism for PTEN null melanoma cells' resistance to the BRAF V600E inhibitor, PLX4720 (inset). Both the heat map and the bar graph indicate that the major difference between the two cell lines was the upregulation of Bim, which caused apoptosis in PTEN positive cells. Regulation of the other Bcl-2 family proteins was similar regardless of PTEN status.