Department of Translational DMPK, Lundbeck, Ottiliavej 9, DK-2500 Valby, Copenhagen, Denmark.
ICON Bioanalytical Laboratories, Amerikaweg 18, 9407TK Assen.
J Am Soc Mass Spectrom. 2023 Mar 1;34(3):441-451. doi: 10.1021/jasms.2c00324. Epub 2023 Jan 31.
Hyperphosphorylated tau protein is well-known to be involved in the formation of neurofibrillary tangles and the progression of age-related neurodegenerative diseases (tauopathies), including Alzheimer's Disease (AD). Tau protein phosphorylated at serine-396 (pS396-tau) is often linked to disease progression, and we therefore developed an analytical method to measure pS396-tau in cerebrospinal fluid (CSF) in humans and animal models of AD. In the S396-region, multiple phosphorylation sites are present, causing structural complexity and sensitivity challenges for conventional bottom-up mass spectrometry approaches. Here, we present an indirect LC-MS/MS method for quantification of pS396-tau. We take advantage of the reproducible miscleavage caused by S396 being preceded by a lysine (K395) and the proteolytic enzyme trypsin not cleaving when the following amino acid is phosphorylated. Therefore, treatment with trypsin discriminates between the forms of tau with and without phosphorylation at S396 and pS396-tau can be quantified as the difference between total S396-tau and nonphosphorylated S396-tau. To qualify the method, it was successfully applied for quantification of pS396-tau in human CSF from healthy controls and patients with Mild Cognitive Impairment and AD. In addition, the method was applied for rTg4510 mice where a clear dose dependent decrease in pS396-tau was observed in CSF following intravenous administration of a monoclonal antibody (Lu AF87908, hC10.2) targeting the tau epitope containing pS396. Finally, a formal validation of the method was conducted. In conclusion, this sensitive LC-MS/MS-based method for measurement of pS396-tau in CSF allows for quantitative translational biomarker applications for tauopathies including investigations of potential drug induced effects.
高度磷酸化的 tau 蛋白被广泛认为参与了神经原纤维缠结的形成和与年龄相关的神经退行性疾病（tau 病）的进展，包括阿尔茨海默病（AD）。在丝氨酸-396 位点（pS396-tau）磷酸化的 tau 蛋白通常与疾病进展有关，因此我们开发了一种分析方法来测量 AD 患者和动物模型的脑脊液（CSF）中的 pS396-tau。在 S396 区域存在多个磷酸化位点，这给传统的自下而上的质谱方法带来了结构复杂性和灵敏度方面的挑战。在这里，我们提出了一种间接 LC-MS/MS 方法来定量 pS396-tau。我们利用 S396 之前的赖氨酸（K395）引起的可重复的错误切割，以及当后续氨基酸被磷酸化时胰蛋白酶不会切割的特性。因此，用胰蛋白酶处理可以区分 tau 中 S396 是否磷酸化的两种形式，而 pS396-tau 可以被定量为总 S396-tau 和非磷酸化 S396-tau 之间的差值。为了验证该方法，我们成功地将其应用于健康对照者、轻度认知障碍和 AD 患者的 CSF 中 pS396-tau 的定量。此外，该方法还应用于 rTg4510 小鼠，在静脉注射靶向含有 pS396 的 tau 表位的单克隆抗体（Lu AF87908，hC10.2）后，CSF 中的 pS396-tau 水平明显呈剂量依赖性下降。最后，我们对该方法进行了正式验证。总之，该方法灵敏、基于 LC-MS/MS，可用于 CSF 中 pS396-tau 的定量，可用于包括 tau 病在内的定量转化生物标志物研究，包括潜在药物诱导作用的研究。
