Measurement of glycolysis reactants by high-throughput solid phase extraction with tandem mass spectrometry: Characterization of pyrophosphate-dependent phosphofructokinase as a case study.

Glycolysis is a 10-step metabolic pathway involved in producing cellular energy. Many tumors exhibit accelerated glycolytic rates, and enzymes that participate in this pathway are focal points of cancer research. Here, a novel method for the measurement of glycolysis reactants from in vitro samples is presented. Fast and direct measurement is achieved by an automated system that couples on-line solid phase extraction (SPE) with tandem mass spectrometry (MS/MS). The single analytical method enables multiple reactants to be measured concurrently, sustains a cycle time of 8s, and permits the measurement of up to 10,000 samples per day. Concentration–response curves were conducted using standards for 10 metabolic intermediates, and the results demonstrate that the detection strategy has excellent sensitivity (average limit of detection=5.4nM), dynamic range (nanomolar to micromolar), and linear response (average R2=0.998). To test the analysis method on reactions, pyrophosphate-dependent phosphofructokinase (PPi–PFK) was used as a model system. Data that corroborate the activation and inhibition of PPi–PFK are presented, and the ways in which SPE–MS/MS simplifies experimental design and interpretation are highlighted. In summary, the method for measuring metabolic intermediates described here demonstrates unprecedented speed, performance, and versatility.