Oxidative stress-induced mitochondrial protein degradation and peptide release, Released peptide 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:1. Flow diagram for the treatment and proteomic quantification and identification. Isolated mitochondria from potato tubers were treated under three different conditions (control or KCN). The mitochondria were pelleted by centrifugation and the supernatant fractionated on a spin-filter allowing only molecules &lt;10 kDa to pass through. The mitochondria were incubated for 1 min at the end of the incubation (immediately prior to pelletation) with the pore-forming peptide alamethicin, which is known to make the IMM permeable to smaller moleculesThe flow-through was up-concentrated by micro-column reverse phase chromatography before analysis by LC-MS and bioinformatic identification. The peptides were finally analyzed with LC-MS and identified using bioinformatics.Samples 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 iTraq data: https://doi.org/10.6084/m9.figshare.28607600.v1

本研究通过体外孵育的方式，探究了氧化应激条件下线粒体蛋白质的周转过程：将从马铃薯块茎（*Solanum tuberosum* L.）中分离的线粒体分别置于三种培养基中孵育：含有底物混合液与三磷酸腺苷（ATP）的培养基（对照组）、同培养基添加FCCP的体系（即活性氧（ROS）低生成的解偶联对照组），以及添加甲基紫精与氰化钾（KCN）以阻断电子传递并最大化ROS生成的氧化应激组。

孵育15分钟后，收集线粒体沉淀，经胰蛋白酶消化、同位素标记相对和绝对定量（iTRAQ）标记后混合所有样品，随后通过液相色谱-质谱联用法进行分析。本研究发现，经氧化应激处理后，共有69条胰蛋白酶肽段的相对含量相较于同一蛋白内的其他肽段出现下降，而FCCP处理组未出现该现象，提示这些肽段发生了氧化修饰。

发生修饰的蛋白涵盖多个功能类别，但呼吸复合体、三羧酸循环酶类及ROS清除酶类的占比显著偏高。从这些蛋白中释放出大量肽段，而在加入成孔肽段丙甲菌素（alamethicin）后，肽段释放量进一步提升，尤其在线粒体基质与线粒体内膜（IMM）组分中，这与线粒体内膜上大孔径孔道的形成相符。氧化应激处理组中，来自线粒体基质与IMM蛋白的肽段释放量为对照组的两倍，提示蛋白质周转过程加速。

随后，我们将呼吸链复合体组分释放的肽段序列与其亲本蛋白（已知线粒体内膜定位）的序列进行比对，由此确定了肽段释放的初始位点：线粒体基质、IMM、膜间间隙或胞质/培养基。结合切割位点的位置与序列特征，我们还可预测出哪些已知的ATP依赖型与非ATP依赖型线粒体蛋白酶参与了肽段的释放过程。

综上，体外孵育15分钟的分离线粒体可检测到蛋白质周转过程；而在严重氧化应激条件下，该周转过程会显著加速，释放出大量有望作为细胞核逆向信号的肽段。

实验流程详见workflow.png：
1. 处理与蛋白质组学定量鉴定流程图。将从马铃薯块茎中分离的线粒体分为三种不同条件处理：对照组、解偶联对照组与氧化应激组。通过离心收集线粒体沉淀，将上清液通过超滤离心管进行分级分离，仅允许分子量小于10 kDa的分子通过。在孵育结束前（即离心收集线粒体前1分钟），加入成孔肽段丙甲菌素，该试剂可破坏线粒体内膜的通透性，使小分子可自由透过。
超滤后的流出液经微柱反相色谱法浓缩富集后，通过液相色谱-质谱联用法（LC-MS）与生物信息学手段完成肽段的分析与鉴定。

将含有生物素标记肽段的样品重悬于5 μL 0.1%甲酸溶液中，上样至配置双柱的EASY nLC液相色谱系统（赛默飞世尔科技，Thermo Fisher Scientific）。预柱（内径100 μm，柱长2 cm）与分析柱（内径75 μm，柱长15 cm）均采用熔融石英管自行填充C18 ReproSil反相填料。以250 nL/min的流速进行洗脱，采用55分钟的梯度洗脱程序（流动相A：0.1%甲酸水溶液；流动相B：0.1%甲酸、90%乙腈水溶液），将B相从0%升至38%以分离肽段，随后用100% B相冲洗12分钟。

分析柱流出液分别连接至两种质谱仪：LTQ Orbitrap Velos Pro质谱仪（赛默飞世尔科技）用于释放肽段的分析，Q-Exactive Plus质谱仪（赛默飞世尔科技）用于消化后沉淀样品的分析。两台质谱仪均采用正离子模式与数据依赖性采集模式运行。

Velos质谱仪的主要参数设置如下：先以30000的分辨率采集350~1650 m/z范围的全扫描质谱图，随后选取电荷数大于+1、信号强度高于15000计数的前10个强度最高的前体离子，采用35%的归一化碰撞能量进行碰撞诱导解离（CID）碎裂；已选取的碎裂前体离子将被动态排除30秒。

Q-Exactive质谱仪的主要参数设置如下：先以70000的分辨率采集400~1200 m/z范围的全扫描质谱图，随后选取电荷数大于+1的前12个强度最高的前体离子，采用34%的归一化碰撞能量进行高能碰撞诱导解离（HCD）碎裂，最后以17500的分辨率在Orbitrap检测器中完成检测；已选取的碎裂前体离子将被动态排除20秒。

iTRAQ相关数据集：https://doi.org/10.6084/m9.figshare.28607600.v1