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 = 5.4 nM), dynamic range (nanomolar to micromolar), and linear response (average R(2) = 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.