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

摘要: Abstract Glycolysis is a 10-step metabolic pathway involved in producing cellular energy. Many tumors exhibit accelerated glycolytic rates, and enzymes that participate this are focal points of cancer research. Here, novel method for the measurement glycolysis reactants from vitro samples presented. Fast direct achieved by an automated system couples on-line solid phase extraction (SPE) with tandem mass spectrometry (MS/MS). The single analytical enables multiple to be measured concurrently, sustains cycle time 8 s, permits up 10,000 per day. Concentration–response curves were conducted using standards 10 intermediates, results demonstrate detection strategy has excellent sensitivity (average limit detection = 5.4 nM), dynamic range (nanomolar micromolar), linear response R 2  = 0.998). To test analysis on reactions, pyrophosphate-dependent phosphofructokinase (PPi–PFK) was used as model system. Data corroborate activation inhibition PPi–PFK presented, ways which SPE–MS/MS simplifies experimental design interpretation highlighted. In summary, measuring intermediates described here demonstrates unprecedented speed, performance, versatility.