Direct Profiling of Multiple Enzyme Activities in Human Cell Lysates by Affinity Chromatography/Electrospray Ionization Mass Spectrometry:  Application to Clinical Enzymology

Abstract

We describe a new method for enzyme analysis using affinity capture followed by electrospray ionization mass spectrometry (ACESIMS) for the quantitative determination of the initial velocities of four heparin-modifying enzymes. These enzymes, when defective in affected children, lead to the lysosomal storage disease known as Sanfilippo syndrome. The method relies on substrates and internal standards conjugated to the molecular handle biotin via a heavy isotope-encodable, mass-adjustable linker. Reaction velocities of the Sanfilippo enzymes in a crude lysate prepared from as little as 2500 human skin fibroblasts can be determined. In addition, the ACESIMS method is widely applicable to the simultaneous analysis of multiple enzymes in a complex biological sample by a single analytical technique and will thus serve as a useful tool in basic and clinical biomedical research.

Direct Multiplex Assay of Lysosomal Enzymes in Dried Blood Spots for Newborn Screening

“…Major applications include (a) quantification of metabolites in dried blood spots (DBS) 6 by tandem mass spectrometry (1 ); (b) identification and quantification of protein biomarkers in serum by surfaceenhanced laser desorption/ionization mass spectrometry (2 ); (c) analysis of PCR-amplified DNA by MassArray to identify mutated genes (3 ); and (d) quantification of enzyme activities in cell lysates by affinity capture/ elution electrospray ionization mass spectrometry (4 ). Among these applications, only the implementation of tandem mass spectrometry for the detection of inborn errors of metabolism in DBS has become routine clinical practice (5,6 ).…”

Simultaneous Kinetic Characterization of Multiple Protein Forms by Top Down Mass Spectrometry

“…The recent proteomics revolution has brought the development of powerful new techniques for characterizing the sequence and posttranslational modifications of individual proteins that are isolated from biological systems. The advantages of mass spectrometry (MS) for kinetic studies of small molecules are well known [1][2][3][4], but the application of MS to larger biomolecule kinetics has been limited to a single modification of a single protein without isotopic resolution [5,6]. …”

A novel mass spectrometric assay for the cerebroside sulfate activator protein (saposin B) and arylsulfatase A

“…Mass spectrometry provides a valuable tool for the analysis of lipids and lipid-metabolizing enzymes (13). Previous work using mass spectrometry for the analysis of lipid-metabolizing enzymes has involved the use of matrix-assisted laser desorption ionization (MALDI) and electrospray ionization mass spectrometry (ESI) (14,15). In the present case, the possibility of following sulfatide depletion by ESI has been tested by taking advantage of the strong signals that these lipids yield in the negative ion mode (16,17); more importantly, the possibility of following cerebroside formation has been tested by taking advantage of the strong signals that these lipids yield as their lithiated adducts in the positive ion mode (18).…”

“…The assays have been used to reveal hitherto obscure but potentially important details of the sulfatide specificity for binding by CSAct and the saposin specificity for the ASA-catalyzed reaction. (behenic), and C 24 :1 15 (nervonic) were synthesized as described (19). Hydroxylated sulfatides with fatty acyl chain (R moiety; Fig.…”