HyTest Ltd., Turku, Finland;
Department of Biochemistry, School of Biology, Moscow State University, Moscow, Russia.
Clin Chem. 2017 Jun;63(6):1094-1100. doi: 10.1373/clinchem.2016.266635. Epub 2017 Apr 20.
Cardiac troponin T (cTnT) is an acknowledged biomarker of acute myocardial infarction (AMI) that is known to be prone to proteolytic degradation in serum. Such degradation is usually explained by the action of μ-calpain, although there could be other candidates for that role. In the current study, we explored the hypothesis that thrombin-mediated cTnT cleavage occurs as a result of the serum sample preparation.
cTnT degradation was studied by using immunoblotting and mass spectrometry (MS) analysis.
The comparison of cTnT isolated from AMI heparin plasma and serum samples showed that cTnT in the plasma samples was mainly present as the full-sized molecule (approximately 35 kDa), while in serum samples it was present as a 29-kDa fragment. The incubation of recombinant cTnT, or native ternary cardiac troponin complex with thrombin or in normal human serum (NHS), resulted in the formation of a 29-kDa product that was similar to that detected in AMI serum samples. No cTnT degradation was observed when thrombin or NHS was pretreated with hirudin, a specific inhibitor of thrombin, or during incubation of troponin in normal heparin plasma. When the products of thrombin-mediated cTnT proteolysis were analyzed by MS, 2 fragments consisting of amino acid residues (aar) 2-68 and 69-288 were identified, which suggests that thrombin cleaves cTnT between R68 and S69.
The results of this study suggest that the 29-kDa fragment of cTnT in AMI serum samples mainly appears due to the cleavage by thrombin during serum sample preparation.
心肌肌钙蛋白 T（cTnT）是公认的急性心肌梗死（AMI）生物标志物，已知其在血清中易发生蛋白水解降解。这种降解通常归因于μ-钙蛋白酶的作用，尽管可能有其他候选物也具有这种作用。在本研究中，我们探讨了这样一个假设，即凝血酶介导的 cTnT 裂解是由于血清样本制备引起的。
使用免疫印迹和质谱（MS）分析研究 cTnT 的降解情况。
比较 AMI 肝素血浆和血清样本中分离的 cTnT 发现，血浆样本中的 cTnT 主要以全长分子（约 35 kDa）形式存在，而在血清样本中则以 29-kDa 片段形式存在。重组 cTnT 或天然三价心肌肌钙蛋白复合物与凝血酶或正常人血清（NHS）孵育，会形成与在 AMI 血清样本中检测到的相似的 29-kDa 产物。当用凝血酶特异性抑制剂水蛭素预处理凝血酶或在正常肝素血浆中孵育肌钙蛋白时，未观察到 cTnT 降解。通过 MS 分析凝血酶介导的 cTnT 蛋白水解产物，鉴定出由氨基酸残基（aar）2-68 和 69-288 组成的 2 个片段，提示凝血酶在 R68 和 S69 之间裂解 cTnT。
本研究结果表明，AMI 血清样本中的 cTnT 29-kDa 片段主要是由于血清样本制备过程中凝血酶的切割而出现的。
