Mass spectrometric biosensing: Quantitation of multiplex enzymes using single mass probe and fluorous affinity chip

This work presents a concept of "mass spectrometric biosensing" by using a chip to recognize the targets and mass spectrometry to detect the signals switched by the recognition. The chip is prepared on an ITO slide with a hydrophobic fluorous-tag monolayer to self-assemble the mixture of mass probe and fluorous-tagged cysteine as a spacer through fluorous affinity interaction. The presence of spacer provides suitable conditions for recognition reactions on the chip. By designing a single mass probe as the peptide substrates of corresponding target enzymes, a novel quantitative strategy based on the ratio of signal intensities of different species on the chip is developed for MALDI-MS assay of multiplex enzyme activities. Using caspase-3 and protein kinase A as targets, the reactions with designed mass probe produce three mass shifts to act as two "fingerprint" patterns for obtaining the dual enzyme activities. The proposed biosensing method shows the detectable ranges from 0.05 to 50 μU μL^-1 and 0.4-40 μU μL^-1 with correlation coefficients of 0.990 and 0.989 for PKA and caspase-3, respectively. The biosensing application has been demonstrated by monitoring these enzymes in cell lysates upon anti-cancer drug treatment, indicating the prospect of the novel biosensing protocol in biomedical study.