Wang Kaijuan, Liu Ruichen, Li Li, Duan Xuejing, Zhao Xianglong, Cong Hongying, Gao Xiaojing, Yin Kunlun, Gao Zhangwei, Zhou Zhou
Center of Laboratory Medicine, Beijing, Key Laboratory for Molecular Diagnostics of Cardiovascular Diseases, State Key Laboratory of Cardiovascular Disease, National Center for Cardiovascular Diseases & Fuwai Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100037, China.
Shanghai AB Sciex Analytical Instrument Trading Co., Ltd., Shanghai, 201100, China.
Anal Chim Acta. 2025 Oct 22;1372:344453. doi: 10.1016/j.aca.2025.344453. Epub 2025 Jul 18.
The treatment and prognosis of cardiac amyloidosis (CA) depend heavily on the accurate identification of amyloid protein types. Histopathological methods are the most commonly used approach, but often produce inconclusive results. The application of mass spectrometry with laser microdissection mass spectrometry based on non-targeted proteomics in CA diagnosis is gradually being recognized, but it is expensive, time-consuming, and still in the early stages of scientific research applications. This study aims to establish a novel typing method based on targeted semi-quantitative proteomics to address the shortcomings of existing methods.
Formalin-fixed, paraffin-embedded (FFPE) myocardial tissue samples from 52 CA and 52 hypertrophic cardiomyopathy (HCM) patients were analyzed. A semi-quantitative typing method was developed using triple quadrupole mass spectrometry, with laser microdissection mass spectrometry (LMD-MS) serving as the reference standard. A total of 52 peptides were analyzed. Key amyloid-associated proteins (AAPs) -apolipoprotein A-IV (apo A-IV), apolipoprotein E (apo E), and serum amyloid P component (SAP) - showed high diagnostic accuracy, with AUC values of 0.964, 0.999, and 0.923, respectively. Transthyretin (TTR), immunoglobulin light chains- κ (IGL - κ), and IGL-λ were semi-quantified using normalized scores (NS) adjusted for microdissection and peptide peak areas. An NS cutoff of 0.066 distinguished transthyretin amyloidosis (ATTR), while NS-NS differentiated AL-κ (≤-0.04758) from AL-λ (>0.00999). The method successfully identified 15 ATTR, 10 AL-κ, 21 AL-λ, and mixed subtypes, aligning with LMD-MS results. By contrast, immunohistochemistry accurately typed only 14 cases, with most results inconclusive.
This targeted semi-quantitative mass spectrometry method has high consistency with non-targeted LMD-MS typing, with an accuracy higher than IHC (100 % vs. 30.8 %), while compensating for the shortcomings of non-targeted proteomics. It provides a practical method for CA typing in routine clinical laboratories and may help identify rare subtypes of amyloidosis in the future.
心脏淀粉样变性（CA）的治疗和预后在很大程度上取决于淀粉样蛋白类型的准确识别。组织病理学方法是最常用的方法，但往往产生不确定的结果。基于非靶向蛋白质组学的质谱联用激光显微切割质谱在CA诊断中的应用逐渐得到认可，但该方法昂贵、耗时，且仍处于科研应用的早期阶段。本研究旨在建立一种基于靶向半定量蛋白质组学的新型分型方法，以弥补现有方法的不足。
对52例CA患者和52例肥厚型心肌病（HCM）患者的福尔马林固定、石蜡包埋（FFPE）心肌组织样本进行了分析。采用三重四极杆质谱开发了一种半定量分型方法，以激光显微切割质谱（LMD-MS）作为参考标准。共分析了52种肽段。关键的淀粉样蛋白相关蛋白（AAP）——载脂蛋白A-IV（apo A-IV）、载脂蛋白E（apo E）和血清淀粉样蛋白P成分（SAP）——显示出较高的诊断准确性，曲线下面积（AUC）值分别为0.964、0.999和0.923。转甲状腺素蛋白（TTR）、免疫球蛋白轻链κ（IGL-κ）和IGL-λ通过针对显微切割和肽峰面积调整的标准化分数（NS）进行半定量。NS临界值为0.066可区分转甲状腺素蛋白淀粉样变性（ATTR），而NS-NS可区分AL-κ（≤-0.04758）与AL-λ（>0.00999）。该方法成功识别出15例ATTR、10例AL-κ、21例AL-λ和混合亚型，与LMD-MS结果一致。相比之下，免疫组化仅准确分型了14例，大多数结果不确定。
这种靶向半定量质谱方法与非靶向LMD-MS分型具有高度一致性，准确性高于免疫组化（100%对30.8%），同时弥补了非靶向蛋白质组学的不足。它为常规临床实验室的CA分型提供了一种实用方法，未来可能有助于识别淀粉样变性的罕见亚型。
