Determination of specific glycobiomarkers in alpha-mannosidosis patients by NMR and mass spectrometry

Alpha-mannosidosis is a progressive lysosomal storage disorder, firstly described in 1967[1]. It is caused by a partial or complete deficiency of alpha-mannosidose, enzyme playing an essential role in oligosaccharide and glycoprotein degradation. Mannose-rich oligosaccharides are accumulated in lysosomes of all tissues, resulting in impaired cellular function and apoptosis [2]. Due to the variability of clinical manifestation and multisystematic involvement, its definitive diagnostics may be challenging. MALDI TOF/TOF mass spectrometric analyses of permethylated oligosaccharides from urine of alpha-mannosidosis patients revealed characteristic set of specific mannose-based glycobiomarkers, compiled from Man₂GlcNAc₁ (m/z 722.3) up to Man₈GlcNAc₁ (m/z 1947.0). In ¹H NMR spectra, characteristic signals of mannose units, originated from mannose in various linkages and locations in these oligosaccharides, as well as α,βGlcNAc from reducing ends were identified. Despite of non-quantitative nature of MALDI TOF/TOF analysis, ¹H NMR was used for an overall quantification of mannose oligosaccharides. It is based on the integration of the signal, representing the core 1,3,6-linked βMan unit bound to the reducing end GlcNAc unit. Significantly increased intensities of 1,3-linked αMan signals in ¹H NMR spectra confirmed results from mass spectrometry, suggesting Man₂GlcNAc₁ as the most abundant structure found in urine of alpha-mannosidosis patients. Presented methods were developed to become the basis for a precise monitoring of therapy efficacy in the future.