Data-Independent Acquisition Proteomics and N‑Terminomics Methods Reveal Alterations in Mitochondrial Function and Metabolism in Ischemic-Reperfused Hearts

Myocardial ischemia-reperfusion (IR) (stunning) injury
triggers
changes in the proteome and degradome of the heart. Here, we utilize
quantitative proteomics and comprehensive degradomics to investigate
the molecular mechanisms of IR injury in isolated rat hearts. The
control group underwent aerobic perfusion, while the IR injury group
underwent 20 min of ischemia and 30 min of reperfusion to induce a
stunning injury. As MMP-2 activation has been shown to contribute
to myocardial injury, hearts also underwent IR injury with ARP-100,
an MMP-2-preferring inhibitor, to dissect the contribution of MMP-2
to IR injury. Using data-independent acquisition (DIA) and mass spectroscopy,
we quantified 4468 proteins in ventricular extracts, whereby 447 proteins
showed significant alterations among the three groups. We then used
subtiligase-mediated N-terminomic labeling to identify more than a
hundred specific cleavage sites. Among these protease substrates,
15 were identified following IR injury. We identified alterations
in numerous proteins involved in mitochondrial function and metabolism
following IR injury. Our findings provide valuable insights into the
biochemical mechanisms of myocardial IR injury, suggesting alterations
in reactive oxygen/nitrogen species handling and generation, fatty
acid metabolism, mitochondrial function and metabolism, and cardiomyocyte
contraction.

心肌缺血再灌注（IR，顿抑）损伤可引发心脏蛋白质组（proteome）与降解组（degradome）的改变。本研究利用定量蛋白质组学与全面降解组学技术，探究离体大鼠心脏IR损伤的分子机制。对照组仅接受有氧灌注，IR损伤组则施以20分钟缺血及30分钟再灌注以诱导顿抑损伤。鉴于已有研究证实基质金属蛋白酶2（MMP-2）的激活参与心肌损伤过程，本研究同时设置了给予MMP-2偏好性抑制剂ARP-100的IR损伤干预组，以解析MMP-2在IR损伤中的具体作用。
本研究采用数据非依赖采集（DIA）结合质谱技术，对心室提取物中的4468种蛋白质进行定量分析，结果显示三组间共有447种蛋白质存在显著表达差异。随后通过枯草杆菌蛋白酶介导的N端组学标记技术，鉴定出百余个特异性蛋白酶切割位点，在这些蛋白酶底物中，有15个的切割事件与IR损伤相关。
本研究发现，IR损伤后大量参与线粒体功能与代谢过程的蛋白质表达发生显著改变。本研究结果为阐明心肌IR损伤的生化机制提供了关键见解，提示活性氧/氮物种的代谢调控与生成、脂肪酸代谢、线粒体功能与代谢以及心肌细胞收缩相关通路均发生了显著异常。