Improved Matrix-assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometric Method for Identification of Amino Acid O-Glycosides in Patients with α-N-Acetylgalactosaminidase Deficiency

Abstract

Thin-layer chromatography (TLC) has been widely applied to the diagnosis of lysosomal storage diseases by analyzing abnormally excreted oligosaccharides in urine. However, TLC does not necessarily identify the excreted compounds accurately. Labeling the oligosaccharides with a tag, such as 8-aminonaphthalene-1,3,6-trisulfonic acid, allows easy identification of the excreted compounds by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) (1). However, this labeling is based on reductive amination of the reductive end of the oligosaccharides with the labeling compound, so abnormal amino acid O-glycosides with no reducing end cannot be identified. Indeed, patients with Kanzaki/Schindler disease or aspartylglucosaminuria, who mainly excrete abnormal amino acid O-glycosides, can be identified only with difficulty by this method. In the present study, we successfully identified abnormal amino acid O-glycosides in urine from Kanzaki/Schindler disease [α- N -acetylgalactosaminidase (α-NAGA) deficiency] patients (2), using partition chromatography and MALDI-TOF MS. The method involves three steps: purification on a solid-phase extraction column filled with cellulose;removal of sialic acids; and mass spectrometric analysis. This simple procedure reduces both the amount of urine and analysis time and precisely identifies excreted abnormal substances. For MS, 5- to 15-μL urine samples (equivalent to 5 μg of creatinine) were mixed with 250 pmol of raffinose as an internal standard. Urinary creatinine was measured by colorimetric analysis (3). The samples were then mixed with an ethanol–1-butanol solution (1:4 by volume) and applied to a column filled with …