Fig 1.
Protein standard absolute quantification for measurement of cellular Ras abundance.
Isotopically-labelled Ras proteins are spiked into cell lysates at a known concentration, before fractionation, proteolysis and quantification of pre-defined proteotypic peptides using selected reaction monitoring to allow calculation of Ras isoform abundance.
Fig 2.
Isotopically-labelled Ras standards and proteotypic peptides used in this study.
His-tagged Ras protein standards are isolated to high purity (A) and exhibit >99% incorporation of isotope label (B). (C) Ras peptides used for quantification. Peptides in black were utilised for quantification, while those in grey were identified but were unreliably detected/ undetectable at endogenous levels in SW48 cells. All peptides are tryptic, bar FYTLVREI that is elastase derived. Sites of common Ras oncogenic mutations at codons 12 and 13 are underlined. (D) A linear relationship is observed (R2 > 0.97) between the abundance of the Ras peptides injected onto the mass spectrometer and the integrated intensity of transitions. Full-length, light and isotope-labelled Ras proteins were mixed at ratios from 0.2 to 20, before being subject to SDS-PAGE and in-gel tryptic digestion. The peptide mixtures were subject to SRM analysis and data analysed using Skyline.
Fig 3.
Selected reaction monitoring of proteotypic Ras peptides in SW48 isogenic cell lines.
(A) Individual transitions of endogenous (Lys0 Arg0) and heavy (Lys8 Arg10) peptides describing total wild type, HRAS, KRAS4B and NRAS abundance in the Parental (homozygous KRASWT) SW48 cell line. (B) Integrated transitions (normalised to heavy signal) of the proteotypic Ras peptides in Parental (PAR) and heterozygous KRAS G12D knock-in (KRASG12D) SW48 cells. Presented data representative of 3 biological replicates.
Fig 4.
Cellular Ras abundance in isogenic colorectal cell lines.
(A) Ras proteins are highly abundant but their levels vary across a panel of isogenic SW48 cells harbouring different oncogenic mutations in KRAS. Horizontal lines indicate the wild-type cell lines to aid comparison. (B) Quantitation of isoform-specific peptides is corroborated by peptides shared between isoforms. Percentage agreement was calculated as ((isoform-specific peptide + isoform-specific peptide) / shared peptide) × 100. Shared peptides were TGEGFLCVFAINNTK (HRAS and KRAS), SYGIPFIETSAK (KRAS and NRAS) and LVVVGAGGVGK (all wild-type isoforms). n ≥ 20 for H+K and N+K, n = 3 for Pan as only applicable to the wild-type Ras SW48 cell line. Bars represent mean ± SD. No significant difference (p>0.01 paired T-test) is observed between the total estimated molecules per cell of HRAS peptide + KRAS isoform-specific peptidse versus the HRAS/KRAS shared peptide, NRAS + KRAS isoform specific peptides versus KRAS/NRAS shared peptide and H+K+N isoform specific peptides versus the wild type Pan-Ras peptide. (C) KRAS represents >50% of total cellular Ras in the wild-type Ras SW48 cell line. For each graph in each panel, bars represent mean ± SD of ≥3 biological replicates. (D) Number of calculated mutant KRAS molecules changes between cells harbouring different KRAS mutations. Mutant KRAS levels were calculated by subtracting number of wild-type RAS molecules from the sum of H+K(B)+N isoform specific peptides.