Neuronal ceroid lipofuscinoses (NCLs) are a group of neurodegenerative diseases predominantly in childhood that are characterized by psychomotor deterioration, epilepsy, and early death of patients. The NCLs analyzed in the present study are caused by defects of the specific enzymes, CLN1 (palmitoyl protein thioesterase 1; PPT1), CLN2 (tripeptidyl peptidase 1; TPP1), and CLN10 (cathepsin D). Specific and sensitive diagnostic assays of NCLs were the main goal of this study. They are of increasing importance, particularly since enzyme replacement therapy (ERT) for NCL2 has recently become available for clinical treatment, and ERTs for further NCLs are under development. Here, we report specific and sensitive determinations for CLN1, CLN2, and CLN10 on dried blood spots by tandem mass spectrometry using multiple reaction monitoring mass spectrometry (MRM‐MS). Identical substrates suitable for (i) fluorimetric determination of single enzymes and (ii) for MRM‐MS determination of multiple enzymes were synthesized by chemical coupling of alkyl‐umbelliferone building blocks with the corresponding peptidyl‐substrate groups recognized by the target enzyme. Enzymatic determinations were performed both by fluorimetry and MRM‐MS in patients with NCL1, NCL2, and NCL10 and showed good agreement in single assays. Moreover, duplex and triplex determinations were successfully performed for NCL1, NCL2, and NCL10. Specific peptidyl‐(4‐alkyl‐umbelliferone) substrates were also synthesized for mass spectrometric determinations of different cathepsins (cathepsins‐D, ‐F, and ‐B), to provide a differentiation of proteolytic specificities.

中文翻译：

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使用用于伴随串联质谱法和荧光法的底物对干血斑中的神经元脂褐质糖进行酶学诊断

神经元蜡样脂褐质沉积症 (NCLs) 是一组主要发生在儿童期的神经退行性疾病，其特征是精神运动功能恶化、癫痫和患者过早死亡。本研究中分析的 NCL 是由特定酶 CLN1（棕榈酰蛋白硫酯酶 1；PPT1）、CLN2（三肽基肽酶 1；TPP1）和 CLN10（组织蛋白酶 D）的缺陷引起的。本研究的主要目标是对 NCL 进行特异性和灵敏的诊断分析。它们变得越来越重要，特别是因为 NCL2 的酶替代疗法 (ERT) 最近已可用于临床治疗，并且用于进一步 NCL 的 ERT 正在开发中。在这里，我们报告了 CLN1、CLN2、和 CLN10 通过串联质谱法使用多反应监测质谱法 (MRM-MS) 在干血斑上进行检测。通过烷基-伞形酮结构单元与目标酶识别的相应肽基-底物基团的化学偶联，合成了适用于（i）单一酶的荧光测定和（ii）多种酶的 MRM-MS 测定的相同底物。通过荧光法和 MRM-MS 对 NCL1、NCL2 和 NCL10 患者进行酶促测定，并在单次测定中显示出良好的一致性。此外，成功地对 NCL1、NCL2 和 NCL10 进行了双工和三工测定。还合成了特定的肽基-（4-烷基-伞形酮）底物，用于质谱测定不同组织蛋白酶（组织蛋白酶-D、-F 和-B），