We have developed a next-generation proteomics approach—in vitro proteome–assisted multiple reaction monitoring for protein absolute quantification (iMPAQT)—that allows genome-wide absolute quantification of the human proteome and is reliant on the production of ～18,000 recombinant proteins. We applied iMPAQT to delineate the metabolic landscape of human diploid fibroblasts. Oncogenic transformation of these cells gave rise to relatively small but global changes in metabolic pathways that account for aerobic glycolysis (Warburg effect) and increased rates of macromolecule synthesis. Modulation of metabolic enzyme expression revealed an unexpected functional interaction between glycolysis and the pentose phosphate pathway that facilitates nucleic acid synthesis. Furthermore, integration of proteomic and metabolomic data allowed construction of a mathematical model for identification of key enzymes responsible for the metabolic shift in cancer. We found that substantial remodeling in glutamine metabolism, which we call the "second" Warburg effect, is essential for malignant phenotypes of cancer cells. Our results thus provide a global view of metabolic restructuring in cancer that underlies adaptation to a rapid growth state.