MMCA heatmaps based on datasets from astrocytomas, meningiomas, oligodendrogliomas and metastases.

<p>The heatmaps are normalized and filtered using a p-value cut-off of 0.001 coupled to a bootstrap confidence interval of level 0.999. Metabolites are arranged according to the main biochemical modules.</p

Metabolic interactions between brain tumour cells and host cells.

<p>The lactate from tumour cell is transported to glial cells, where it is used a fuel to produce ATP. ATP and PCr are connected via the creatine-kinase reaction. PCr, phosphocreatine; Cr, creatine; TCA, tricarboxylic acid cycle.</p

Average HRMAS ¹H NMR spectra from the five tumour types.

<p>From bottom and upwards: glioblastomas (black, n = 132), astrocytomas (blue, n = 101), meningiomas (red, n = 75), oligodendrogliomas (brown, n = 37) and metastases (pink, n = 33).</p

Correlation coefficients of metabolite pairs across the five tumour types.

<p>Correlation coefficients of metabolite pairs across the five tumour types.</p

MR images and corresponding H&E sections from five types of brain tumours.

<p>The five tumour types illustrated are a glioblastoma (GBM), an astrocytoma (AST), a meningioma (MN), an oligodendroglioma (ODG) and a metastasis (MET) from a mammary carcinoma.</p

Diagrams illustrating biochemical mechanisms that could give rise to the observed correlations between metabolites.

<p>(A) Glycolytic products. The enzymes driving relevant reactions are: 1, lactate dehydrogenase; 2, alanine aminotransferase; 3, glutamine synthetase; 4, glutaminase. (B) Glutaminolysis pathway to bypass PKM2 inhibition. Active enzymes are: 1, glutaminase; 2, aspartate aminotransferase; 3, malate dehydrogenase; 4, malate decarboxylase. (C) Correlations involving the creatine pool. Dashed boxes indicate pools of metabolites: tCr, total creatines (creatine + phosphocreatine); tCho, total cholines (choline + phosphocholine + glycerophosphocholine). The enzyme creatine kinase is marked by the number 1. (D) Correlations involving the choline pool. Dashed boxes indicate pools of metabolites: tCr, total creatines (creatine + phosphocreatine); tCho, total cholines (choline + phosphocholine + glycerophosphocholine); tGlut, glutamate + glutamine pool; PtdCho, phosphatidylcholine. The enzymes marked are: 1, choline kinase; 2, CTP:phosphocholine cytidylyltransferase and choline phosphotransferase; 3, phospholipase Al; phospholipase A2, and lysophospholipase; 4, GPC:choline phosphodiesterase. Metabolites that were not detected by the ¹H HR-MAS NMR are shown in grey. Positive correlations are shown in red while negative correlations are in blue.</p

Correlation heatmap and selected metabolite distribution summaries based on glioblastoma datasets.

<p>The correlation heatmap shown is normalized and filtered using a p-value cut-off of 0.001 coupled to a bootstrap confidence interval of level 0.999. Metabolites are arranged according to the main biochemical modules. The highlighted squares in the heatmap show the correlations between lactate (Lac) and alanine (Ala), glutamate (Glu) and glutamine (Gln), as well as between tGlut (Glu+Gln) and lipid signals at 1.3ppm (Lip 13a+Lip13b).</p

Cellular Ras abundance in isogenic colorectal cell lines.

<p>(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). <i>n</i> ≥ 20 for H+K and N+K, <i>n</i> = 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.</p

Protein standard absolute quantification for measurement of cellular Ras abundance.

<p>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.</p

Selected reaction monitoring of proteotypic Ras peptides in SW48 isogenic cell lines.

<p>(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 KRAS^WT) SW48 cell line. (B) Integrated transitions (normalised to heavy signal) of the proteotypic Ras peptides in Parental (PAR) and heterozygous KRAS G12D knock-in (KRAS^G12D) SW48 cells. Presented data representative of 3 biological replicates.</p