Tandem Mass Spectrometry for the Direct Assay of Enzymes in Dried Blood Spots: Application to Newborn Screening for Krabbe Disease

Tandem mass spectrometry (MS/MS) for newborn screening allows detection of abnormal or excess metabolites in dried blood spots (1)(2)(3)(4)(5). Dried blood spots provide a source of active enzymes for the detection of Fabry (6), Hunter(7), Hurler (7), Pompe(8), Gaucher(9), Niemann–Pick (9), and Tay–Sachs(10) diseases. We investigated whether MS/MS can be used to directly assay enzymes in dried blood spots, in this case, galactocerebroside β-galactosidase (GALC; EC 3.2.1.46) for the detection of Krabbe disease. Development of a GALC assay is challenging because of the low GALC activity in cell lysates. It has been argued that a neonatal screening method for Krabbe disease is needed (11). This is the first report of an assay for Krabbe disease that uses dried blood spots and the first use of MS/MS for direct enzyme assay from newborn-screening cards.The GALC assay uses commercially available reagents and is based on the reaction shown in Fig. 1⇓ and the use of electrospray ionization (ESI) MS/MS. GALC converts β-Gal-C8-Cer to C8-Cer. A known amount of substrate analog with two extra methylene groups, C10-Cer, is added as internal standard. After collision-induced dissociation, C8-Cer and C10-Cer give rise to the same fragment ion ( m/z 264.3); thus C8-Cer and C10-Cer are distinguished by the Q1 quadrupole. The indicated fragmentation pathway is the major process, allowing for high-sensitivity detection of C8-Cer and C10-Cer.A simple, one-step, solid-phase extraction procedure was developed (see below) to remove the large amount of detergent before ESI-MS/MS. The product C8-Cer and internal standard C10-Cer have nearly identical ESI-MS/MS ionization efficiencies (see Fig. 1⇓ in the online Data Supplement that accompanies this …