A Novel Lithium Ion-Assisted TPP Approach for Enhanced Target Discovery

Traditional
thermal proteome profiling (TPP) suffers from limitations
in detecting membrane and weakly bound proteins, potentially overlooking
up to 20% of drug targets. To overcome this, we developed a lithium
ion-assisted TPP (Li-TPP) method that significantly enhances protein
thermal stability detection. Li-TPP successfully captured thermal
shifts in weakly bound proteins like lactate dehydrogenase A (LDHA)
and resolved changes within NMDA receptor complexes. Compared to traditional
TPP, Li-TPP identified 718 additional proteins (R2 >
0.8,
min_slope ≤ −0.06) and significantly increased thermal
displacement values (ΔTm ≥ 1.5 °C) in 368 coidentified
proteins. Importantly, Li-TPP identified known mania-associated targets
like GSK3β as well as novel regulatory proteins such as CRMP2.
Functional enrichment analysis revealed that Li-TPP-identified proteins
were significantly enriched in pathways critical to bipolar disorder,
including microtubule dynamics (MT3 complex), synaptic plasticity
(NMDA receptor assemblies), and energy metabolism (ATP-binding proteins).
This provides mechanistic insights into lithium’s therapeutic
action, highlighting its role in stabilizing neuronal energy homeostasis
and microtubule architecture. This study demonstrates Li-TPP’s
superior performance in detecting challenging protein targets and
provides a powerful new platform for elucidating drug mechanisms and
accelerating the development of multitarget therapeutics for bipolar
disorder.

传统热蛋白质组分析（thermal proteome profiling, TPP）在检测膜蛋白与弱结合蛋白时存在局限，可能会遗漏多达20%的药物靶点。为克服这一缺陷，本研究开发了锂离子辅助热蛋白质组分析（Li-TPP）方法，可显著提升蛋白质热稳定性检测效能。Li-TPP可成功捕获乳酸脱氢酶A（LDHA）这类弱结合蛋白的热位移变化，并解析N-甲基-D-天冬氨酸（NMDA）受体复合物内的动态变化。与传统热蛋白质组分析相比，Li-TPP额外鉴定出718个蛋白质（决定系数R²>0.8，最小斜率min_slope≤−0.06），并在368个共同鉴定到的蛋白质中显著提升了热位移值（熔点变化ΔTm≥1.5℃）。值得注意的是，Li-TPP不仅鉴定出了已知的躁狂相关靶点糖原合成激酶3β（GSK3β），还发现了新型调控蛋白如崩溃反应介质蛋白2（CRMP2）。功能富集分析结果显示，Li-TPP鉴定得到的蛋白质在双相情感障碍的关键通路中显著富集，包括微管动力学（MT3复合物）、突触可塑性（NMDA受体组装体）以及能量代谢（ATP结合蛋白）相关通路。该发现为锂盐的治疗作用提供了机制层面的深入见解，揭示了其在维持神经元能量稳态与微管结构稳定性中的关键作用。本研究证实Li-TPP在检测高挑战性蛋白质靶点方面性能更优，并为阐明药物作用机制、加速双相情感障碍多靶点治疗药物的开发提供了强有力的全新研究平台。