Direct profiling of multiple enzyme activities in human cell lysates by affinity chromatography/electrospray ionization mass spectrometry: Application to clinical enzymology

We describe a new method for enzyme analysis using affinity capture followe d by electrospray ionization mass spectrometry (ACESIMS) for the quantitati ve determination of the initial velocities of four heparin-modifying enzyme s. These enzymes, when defective in affected children, lead to the lysosoma l storage disease known as Sanfilippo syndrome. The method relies on substr ates and internal standards conjugated to the molecular handle biotin via's heavy isotope-encodable, mass-adjustable linker. Reaction velocities of th e Sanfilippo enzymes in a crude lysate prepared from as little as 2500 huma n skin fibroblasts can be determined. In addition, the ACESIMS method is wi dely applicable to the simultaneous analysis of multiple enzymes in a compl ex biological sample by a single analytical technique and will thus serve a s a useful tool in basic and clinical biomedical research.