Biochemical Genetics Laboratory, Greenwood Genetic Center, Greenwood, SC.
Department of Clinical Genetics, Greenwood Genetic Center, Charleston Office, North Charleston, SC.
Clin Chem. 2018 Dec;64(12):1772-1779. doi: 10.1373/clinchem.2018.289645. Epub 2018 Sep 10.
The glycoproteinoses are a subgroup of lysosomal storage diseases (LSDs) resulting from impaired degradation of N-linked oligosaccharide side chains of glycoproteins, which are commonly screened by detecting the accumulated free oligosaccharides (FOSs) in urine via thin layer chromatography (TLC). The traditional TLC method suffers from limited analytical sensitivity and specificity and lacks quantification capability. Therefore, we developed an analytically sensitive and relatively specific assay using ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for urinary FOS analysis and validated its use for urine screening of glycoproteinoses and other LSDs.
Urine volumes equivalent to 30 μg of creatinine were derivatized with butyl-4-aminobenzoate and then purified through a solid-phase extraction cartridge. A 7-min UPLC-MS/MS analysis was performed on a triple quadrupole mass spectrometer using an amide column for separation of derivatized FOS. Urine samples from >100 unaffected controls and 37 patients with various LSDs were studied.
Relative quantification was conducted on 7 selected FOSs using a single internal standard, which allowed the identification of patients with 1 of 8 different LSDs: aspartylglucosaminuria, α-fucosidosis, α-mannosidosis, β-mannosidosis, β-galactosidase deficiency, Sandhoff disease, sialidosis, and galactosialidosis. Patients treated with hematopoietic stem cell transplant show decreased FOS responses compared with untreated patients.
This UPLC-MS/MS assay offers a valuable tool for screening of glycoproteinoses and other LSDs, with potential use for future treatment monitoring.
糖蛋白贮积症是溶酶体贮积症（LSDs）的一个亚组，其起因是 N-连接寡糖侧链的糖蛋白降解受损，这些糖蛋白通常通过薄层色谱法（TLC）检测尿液中累积的游离寡糖（FOSs）进行筛选。传统的 TLC 方法存在分析灵敏度和特异性有限且缺乏定量能力的问题。因此，我们开发了一种使用超高效液相色谱-串联质谱法（UPLC-MS/MS）进行尿液 FOS 分析的灵敏且相对特异的分析方法，并验证了其用于糖蛋白贮积症和其他 LSDs 的尿液筛查。
用丁基-4-氨基苯甲酸对相当于 30 μg 肌酐的尿样进行衍生化，然后通过固相萃取小柱进行纯化。在三重四极杆质谱仪上进行 7 分钟的 UPLC-MS/MS 分析，使用酰胺柱分离衍生化的 FOS。研究了来自>100 名未受影响的对照者和 37 名患有各种 LSDs 的患者的尿样。
使用单个内标对 7 种选定的 FOS 进行相对定量，这允许鉴定患有 8 种不同 LSDs 之一的患者：天冬氨酰葡糖胺尿症、α-岩藻糖苷酶缺乏症、α-甘露糖苷症、β-甘露糖苷症、β-半乳糖苷酶缺乏症、桑格福德病、唾液酸贮积症和半乳糖脑苷脂贮积症。与未接受治疗的患者相比，接受造血干细胞移植治疗的患者 FOS 反应降低。
这种 UPLC-MS/MS 测定法为糖蛋白贮积症和其他 LSDs 的筛查提供了有价值的工具，具有用于未来治疗监测的潜力。
