Figure 5. Label-free, gel-free, and antibody-free quantitation of Ras from complex mixture targeting exclusively proteoform-specific tryptic peptides detected in 3D-cultured and 2D-cultured NCI-H23 cells.

(A) Extracted ion chromatograms of the KRas4B^WT proteoform-specific tryptic peptide QGVDDAFYTLVR identified in control (i.e., 2D cultured NCI-H23 cells) and the sample (i.e., 3D-cultured NCI-H23 cells). (B) Extracted ion chromatograms of the KRas4B^G12C allele-specific N-terminal mutant tryptic peptide LVVVGACGVGK identified in control (i.e., 2D cultured NCI-H23 cells) and the sample (i.e., 3D-cultured NCI-H23 cells). (C) Extracted ion chromatogram of total Ras^WT specific N-terminal LVVVGAVGVGK peptide identified in control (i.e., 2D cultured NCI-H23 cells) and the sample (i.e., 3D-cultured NCI-H23). (D) 3D and 2D quantitative channels readings for the total Ras^WT specific N-terminal LVVVGAVGVGK peptide, the KRas4B^WT proteoform-specific tryptic peptide QGVDDAFYTLVR and the KRas4B^G12C allele-specific N-terminal mutant tryptic peptide LVVVGACGVGK, respectively. (E) Calculated 3D/2D ratios for total Ras^WT, KRas4B^WT, and KRas4B^G12C proteoforms detected in membrane fraction of NCI-H23 cells grown in 3D and 2D culture, respectively.