Oxidative stress-induced mitochondrial protein degradation and peptide release, iTRAQ data

The turnover of mitochondrial proteins under oxidative stress conditions was studied in vitro by incubating mitochondria isolated from potato tubers (<i>Solanum tuberosum</i> L.) in a medium containing a substrate cocktail and ATP (control), in the same medium plus FCCP (uncoupled control with low production of reactive oxygen species (ROS), or in the medium plus methyl viologen and KCN to block electron transport and maximize ROS production (oxidative stress). After 15 min incubation, the mitochondria were pelleted, digested with trypsin, iTRAQ-labelled and the samples pooled. The pooled sample was analyzed by liquid chromatography-mass spectrometry. We found that 69 tryptic peptides decreased in amount relative to other peptides in the same protein after the oxidative stress treatment, but not after FCCP treatment. This indicates that the peptides had been modified, probably by oxidation. The modified proteins represented a wide range of proteins, but the respiratory complexes, the tricarboxylic acid cycle enzymes and ROS-detoxifying enzymes were overrepresented. Many peptides were released from these POM and many more so in the presence of the pore-forming peptide alamethicin especially from the matrix and the inner mitochondrial membrane (IMM) consistent with the formation of large pores in the IMM. Twice as many peptides appeared from matrix and IMM proteins during the oxidative stress treatment pointing at an accelerated protein turnover. We then matched the sequence of the released peptides from respiratory chain complex components with that of their parent proteins (having a known IMM orientation), and this allowed us to identify the initial site of peptide release – matrix, IMM, intermembrane space, or cytosol/medium. The location of the cleavage site and its sequence signature also allowed us to predict which of the known ATP-dependent and ATP-independent mitochondrial proteases were likely responsible for the release. We conclude that isolated mitochondria respiring in vitro over 15 min have a detectable protein turnover. This protein turnover is greatly accelerated during severe oxidative stress leading to the release of many peptides that are potential retrograde signals to the nucleus.Workflow is depicted in workflow.png:Isolated mitochondria from potato tubers were treated under three different conditions (control (114 itraq channel), FCCP (115 itraq channel) or MV + KCN (117 itraq channel)). The mitochondrial pellet was digested using trypsin and the resulting peptides iTRAQ labelled before the different treatments were pooled and the peptides separated by HILIC. The peptides were finally analyzed with LC-MS:LC-MS/MSSamples containing biotinylated peptides were resuspended in 5 µl 0.1% formic acid and loaded on an EASY nLC system (Thermo Fisher Scientific) set up with two columns. Both the pre-column (100 μm inner diameter, 2 cm long) and the analytical column (75 μm inner diameter, 15 cm long) were in-house packed with C18 ReproSil reverse-phase material in fused silica. The flow of 250 nl/min was delivered and the peptides were separated using a 55 min gradient from 0-38% B buffer before washing with 100% B for 12 min (A buffer: 0.1% formic acid, B buffer: 0.1% formic acid, 90% acetonitrile). The flow from the analytical column was either coupled to an LTQ Orbitrap Velos Pro mass spectrometer (Thermo Fisher Scientific) for the analysis of released peptides or Q-Exactive Plus (Thermo Fisher Scientific) for the analysis of the digested pellets. The instruments were operated in positive ion mode with data-dependent acquisition. The Velos main settings were as follows: A full ion scan (from 350–1650 m/z) was acquired at resolution of 30,000 before the 10 most intense precursor ions with charge states larger than +1 and intensity above 15000 counts were selected for collision-induced disassociation (CID) fragmentation using a normalized collision energy of 35 %. Former target ions selected for fragmentation were dynamically excluded for 30 s. The Q-Exactive was operated with the following main settings: A full ion scan (from 400–1200 m/z) was acquired at resolution of 70,000 before the 12 most intense precursor ions with charge states larger than +1 were selected for higher-energy C-trap dissociation (HCD) fragmentation using a normalized collision energy of 34 % prior to detection in the Orbitrap with a resolution of 17,500. Former target ions selected for fragmentation were dynamically excluded for 20 s.For released peptide data: 10.6084/m9.figshare.28607927

本研究以体外实验手段，探究了氧化应激条件下线粒体蛋白的周转情况。实验将从马铃薯块茎（*Solanum tuberosum* L.）中分离得到的线粒体，分别置于三种培养基中孵育：第一种为含底物混合液与三磷酸腺苷（ATP）的基础培养基（对照组）；第二种为添加羰基氰化物-4-(三氟甲氧基)苯腙（FCCP）的同型培养基（解偶联对照组，活性氧（reactive oxygen species, ROS）生成量较低）；第三种为添加甲基紫精与氰化钾（KCN）的培养基，该体系可阻断电子传递并最大化ROS生成，用以模拟氧化应激环境。孵育15分钟后，对线粒体进行离心沉淀，经胰蛋白酶消化、同量异位素相对和绝对定量标记（iTRAQ）后混合样本，随后采用液相色谱-质谱联用法（liquid chromatography-mass spectrometry, LC-MS）对混合样本进行分析。

我们发现，相较于同一蛋白内的其他肽段，氧化应激处理组中有69条胰蛋白酶肽段的丰度出现下降，而FCCP处理组未出现该现象，这表明这些肽段发生了氧化修饰。被修饰的蛋白涵盖范围广泛，但呼吸复合物、三羧酸循环酶类以及ROS解毒酶类的占比显著偏高。

诸多肽段从这些蛋白中释放，且在加入成孔肽段丙甲菌素（alamethicin）时释放量显著增多，尤其从线粒体基质与线粒体内膜（inner mitochondrial membrane, IMM）中释放的肽段更多，这与IMM中形成大孔的现象相符。氧化应激处理组中，从基质与IMM蛋白中释放的肽段数量为对照组的两倍，提示蛋白周转速率加快。

我们将呼吸链复合物组分释放的肽段序列与其亲本蛋白（已知IMM定位）进行匹配，由此确定了肽段释放的初始位点：线粒体基质、IMM、膜间腔或胞质/培养基。通过切割位点的位置及其序列特征，还可预测哪些已知的ATP依赖型与ATP非依赖型线粒体蛋白酶可能参与了肽段的释放。

本研究得出结论：体外培养的分离线粒体在15分钟内可检测到蛋白周转现象，而严重氧化应激会大幅加速该蛋白周转过程，导致大量肽段释放，这些肽段可能是向细胞核传递信号的潜在逆行信号分子。

实验流程详见workflow.png：从马铃薯块茎中分离的线粒体经三种不同条件处理：对照组（对应114号iTRAQ通道）、FCCP处理组（对应115号iTRAQ通道）、MV+KCN处理组（对应117号iTRAQ通道）。收集线粒体沉淀后用胰蛋白酶消化，所得肽段经iTRAQ标记，随后混合不同处理组的样本，通过亲水作用色谱（hydrophilic interaction liquid chromatography, HILIC）分离肽段。最终采用LC-MS进行分析：

含生物素标记肽段的样本重悬于5 μl 0.1%甲酸溶液中，上样至配置双柱的EASY nLC系统（赛默飞世尔科技（Thermo Fisher Scientific））。预柱（内径100 μm，长2 cm）与分析柱（内径75 μm，长15 cm）均采用熔融石英管自行填充C18 ReproSil反相材料。以250 nl/min的流速输送流动相，采用55分钟梯度洗脱（流动相A：0.1%甲酸；流动相B：0.1%甲酸、90%乙腈），梯度范围为0-38% B，随后用100% B冲洗12分钟。

分析柱流出液可连接至LTQ Orbitrap Velos Pro质谱仪（赛默飞世尔科技（Thermo Fisher Scientific））用于释放肽段的分析，或连接至Q-Exactive Plus质谱仪（赛默飞世尔科技（Thermo Fisher Scientific））用于消化沉淀的分析。两台仪器均采用正离子模式与数据依赖性采集模式运行。

Velos质谱的主要参数设置如下：先以30000的分辨率采集350–1650 m/z范围的全扫描离子谱图，随后选取电荷态大于+1、强度高于15000计数的10个强度最高的前体离子，采用归一化碰撞能量35%进行碰撞诱导解离（collision-induced dissociation, CID）碎裂。此前选中的碎裂目标离子将被动态排除30秒。

Q-Exactive质谱的主要参数设置如下：先以70000的分辨率采集400–1200 m/z范围的全扫描离子谱图，随后选取电荷态大于+1的12个强度最高的前体离子，采用归一化碰撞能量34%进行高能碰撞诱导解离（high-energy C-trap dissociation, HCD），之后以17500的分辨率在Orbitrap中进行检测。此前选中的碎裂目标离子将被动态排除20秒。

释放肽段数据集的DOI为：10.6084/m9.figshare.28607927