Mitochondrial proteome of ES1 KO mouse brain

Mitochondrial fraction was obtained using the mitochondrial isolation kit (KC010100, BioChain Institute). Each tissue was finely chopped and homogenized 40 strokes in two volumes of mitochondrial isolation buffer, and then centrifuged at 600×g for 10 min. The supernatant was collected and centrifuged at 12,000×g for 15 min. The supernatant was used as cytosolic fraction containing lysosomes and microsomes. The mitochondrial fraction was obtained from the pellet and resuspended in mitochondrial isolation buffer. The suspended mitochondria were sonicated (TOMY, Ultrasonic disruptor UD-100, output level 30, 30 sec×2) and centrifuged at 105,000×g for 30 min. The supernatant was used as the mitochondrial soluble fractions, containing mitochondrial intermembrane space (IMS) and matrix (MAT). The pellet was resuspended in RIPA buffer and centrifuged at 20,000×g for 30 min. The supernatant was used as the mitochondrial membrane fractions, containing mitochondrial outer membrane (OM) and inner membrane (IM). All the procedures were performed at 4°C.
Mitochondrial and cytosolic fractions of WT and ES1-KO mouse brain were isolated as described 2.1 in 1% triton x-100 in 20 mM Tris-HCl. These samples were concentrated by SDS-PAGE and cut out from gels. The sectioned gels were finely chopped and washed with water. Then, the gel pieces were destained 50% methanol and vortexed in 25 mM ammonium bicarbonate containing 50% acetonitrile for 5 min. The supernatant was removed and the gel pieces were soaked 100% acetonitrile for 1 min. After removing the solution, the gel pieces were dried in vacuo, and rehydrated with a reducing solution (50 mM ammonium bicarbonate and 25 mM dithiothreitol), and incubated for 40 min at 56°C. The reducing buffer was removed and gel pieces were soaked in an alkylating solution (50 mM ammonium bicarbonate containing 55 mM iodoacetamide) for 30 min at room temperature in the dark. After removing the alkylation solution, the gel pieces were washed twice with water and vortexed in 25 mM ammonium bicarbonate containing 50% acetonitrile for 5 min, and then soaked 100% acetonitrile for 1 min. After removing the solution, the gel pieces were dried in vacuo and rehydrated with 50 mM ammonium bicarbonate containing 4 ng/μL sequencing grade trypsin and 0.01% Protease Max Surfactant. After incubating 10 min on ice, an equivalent volume of 50 mM ammonium bicarbonate containing 0.01% Protease Max Surfactant was added and incubated for 3 h at 37°C to digest the proteins in the gels with a protease. The equivalent volume of 1% trifluoroacetic acid was added and vortexed for 10 min for extraction of the tryptic fragments. After centrifuged at 12,000×g for 15 min, the peptide solutions were obtained from the supernatant.
Ten microliters of each peptide solution were diluted with 190 μL of 100 mM ammonium bicarbonate and filtered on with a 0.22 μm using the amicon ultrafiltration unit (Amicon Ultra, 3 kDa, Millipore, MA, USA). Concentrated samples were denatured with an equivalent volume of trifluoroethanol (25 μL) and reduced with 1 μL of 200 mM dithiothreitol (DTT). The samples were incubated at 90°C for 30 min and cooled to room temperature. Free cysteine residues were alkylated with 4 μL of 200 mM iodoacetamide for 60 min at room temperature in the dark and the remaining iodoacetamide was quenched by adding 1 μL of 200 mM DTT. The samples were then mixed with 300 μL of 100 mM ammonium bicarbonate. Fifteen microliters of the sample were diluted with 85 μL of 100 mM ammonium bicarbonate and incubated with 1 μg trypsin (TPCK treated, AB Sciex, Framingham, MA, USA) at 37 °C for 18 h. The samples were desalted with C18 ZipTip (Millipore, Bedford, MA, USA) and eluted with H2O/acetonitrile (5/5; v/v). The ZipTip eluates were dried in a vacuum centrifuge. Desalted samples were rehydrated in 0.1% formic acid (FA) and were analyzed by LC-MS using a nanoLC Eksigent 400 system (Eksigent, AB Sciex), coupled online to a TripleTOF6600 mass spectrometer (AB Sciex). Peptide separation was performed using liquid chromatography with a trap and elution configuration using a nano trap column (350 μm×0.5 mm, 3 μm, 120 Å, AB Sciex) and a nano ChromXP C18 reverse-phase column (75 μm×15 cm, 3 μm, 120 Å, AB Sciex) at 300 nL/min with a 90 min linear gradient of 8-30% acetonitrile in 0.1% FA, and then, with a 10 min linear gradient of 30% to 40% acetonitrile in 0.1% FA. The mass spectrometer was operated in information-dependent acquisition (IDA) mode, scanning full spectra (400–1500 m/z) for 250 ms, followed by up to 30 MS/MS scans (100–1800 m/z for 50 ms each), for a cycle time of 1.8 s. Candidate ions with a charge state between +2 and + 5 and counts above a minimum threshold of 125 counts per second were isolated for fragmentation, and one MS/MS spectrum was collected before adding those ions to the exclusion list for 12 s. The rolling collision energy was used with a collision energy spread of 15. The mass spectrometer was operated using the Analyst TF 1.7.1 software program (AB Sciex). For data-dependent acquisition (DDA, SWATH acquisition), the parameters were set as follows: 100 ms TOF MS scan, followed by 200 variable SWATH windows each at 50 ms accumulation time for m/z 400–1250. MS/MS SWATH scans were set at 5 Da window overlapping by 1 Da for m/z 400–1250 and varied on each side of the mass range. The total cycle time was 9.6 s. Rolling collision energy (CE) parameter script was used to automatically control the CE.
Acquired spectra were searched against the UniProt reviewed database using the Paragon algorithm embedded in the ProteinPilot 5.0.1 software program (AB Sciex), with the following search parameters: (i) sample type: identification, (ii) Cys alkylation: iodoacetamide, (iii) digestion: trypsin, (iv) instrument: TripleTOF 6600, (v) species: Mus musculus, (vi) ID focus: biological modifications, (vii) detected protein threshold: > 0.05 (10% confidence). The detected protein threshold was set to the minimum level to enhance the number of wrong answers to enable the curve fitting by an independent FDR analysis. This was carried out by the target-decoy approach provided with the ProteinPilot software program, which was used to assess the quality of the identifications. Positive identifications were considered when identified proteins and peptides reached a 1% local FDR. The resulting group file was loaded into Peakview (v2.2.0, AB Sciex) and peaks from SWATH runs were extracted with a peptide confidence threshold of 99% and a false discovery rate <1%. The SWATH files were then exported to the MarkerView software program (version 1.3.0.1; AB Sciex) and the peak areas of individual peptides were normalized to the sum of the peak areas of all detected peptides.

本研究采用线粒体分离试剂盒（KC010100，BioChain研究所）获取线粒体组分（mitochondrial fraction）。将各组织充分切碎后，于两倍体积的线粒体分离缓冲液中匀浆40次，随后以600×g离心10 min。收集上清液并以12,000×g离心15 min，所得上清液即为包含溶酶体与微粒体的胞质组分（cytosolic fraction）。从沉淀中获取线粒体组分，并重悬于线粒体分离缓冲液中。将重悬的线粒体进行超声破碎（TOMY，超声破碎仪UD-100，输出强度30，30秒×2次），随后以105,000×g离心30 min。所得上清液即为线粒体可溶性组分（mitochondrial soluble fractions），包含线粒体膜间隙（mitochondrial intermembrane space, IMS）与线粒体基质（mitochondrial matrix, MAT）。将沉淀重悬于RIPA缓冲液中，以20,000×g离心30 min，所得上清液即为线粒体膜组分（mitochondrial membrane fractions），包含线粒体外膜（mitochondrial outer membrane, OM）与线粒体内膜（mitochondrial inner membrane, IM）。所有操作均于4℃下进行。

野生型（WT）与ES1基因敲除（ES1-KO）小鼠脑组织的线粒体及胞质组分，按照2.1节所述方法，于含1% Triton X-100的20 mM Tris-HCl缓冲液中进行分离。将上述样品经SDS-PAGE浓缩后从凝胶中切取目标条带。将切下的凝胶条带充分切碎，并用纯水洗涤。随后将凝胶碎片用50%甲醇脱色，再置于含50%乙腈的25 mM碳酸氢铵溶液中涡旋振荡5 min。弃去上清液，将凝胶碎片用100%乙腈浸泡1 min。移除溶液后，将凝胶碎片真空干燥，再用还原溶液（50 mM碳酸氢铵与25 mM二硫苏糖醇）重悬，于56℃孵育40 min。弃去还原缓冲液，将凝胶碎片于室温避光条件下，置于含55 mM碘乙酰胺的50 mM碳酸氢铵烷基化溶液中浸泡30 min。移除烷基化溶液后，用纯水洗涤凝胶碎片两次，再置于含50%乙腈的25 mM碳酸氢铵溶液中涡旋振荡5 min，随后用100%乙腈浸泡1 min。移除溶液后，将凝胶碎片真空干燥，再用含4 ng/μL测序级胰蛋白酶与0.01% Protease Max表面活性剂的50 mM碳酸氢铵溶液重悬。冰上孵育10 min后，加入等体积的含0.01% Protease Max表面活性剂的50 mM碳酸氢铵溶液，于37℃孵育3 h以酶解凝胶中的蛋白质。加入等体积的1%三氟乙酸，涡旋振荡10 min以提取胰酶酶解肽段。以12,000×g离心15 min后，上清液即为肽段溶液。

取10 μL各肽段溶液，用190 μL 100 mM碳酸氢铵稀释，通过0.22 μm滤膜（采用Amicon Ultra超滤装置，3 kDa截留分子量，默克密理博，马萨诸塞州，美国）过滤。将浓缩后的样品用等体积三氟乙醇（25 μL）变性，再用1 μL 200 mM二硫苏糖醇（DTT）还原。将样品于90℃孵育30 min后冷却至室温。用4 μL 200 mM碘乙酰胺于室温避光条件下对游离半胱氨酸残基进行烷基化，随后加入1 μL 200 mM DTT以淬灭剩余的碘乙酰胺。将样品与300 μL 100 mM碳酸氢铵混合。取15 μL样品，用85 μL 100 mM碳酸氢铵稀释，加入1 μg经TPCK处理的胰蛋白酶（AB Sciex，弗雷明汉，马萨诸塞州，美国），于37℃孵育18 h。用C18 ZipTip（默克密理博，贝德福德，马萨诸塞州，美国）对样品进行脱盐，用H₂O/乙腈（5/5；v/v）洗脱。将ZipTip洗脱液置于真空离心浓缩仪中干燥。将脱盐后的样品重悬于0.1%甲酸（FA）中，采用nanoLC Eksigent 400系统（Eksigent，AB Sciex）连接TripleTOF 6600质谱仪（AB Sciex）进行LC-MS分析。采用捕集-洗脱液相色谱配置进行肽段分离：使用纳米捕集柱（350 μm×0.5 mm，3 μm，120 Å，AB Sciex）与纳米ChromXP C18反相柱（75 μm×15 cm，3 μm，120 Å，AB Sciex），流速为300 nL/min，采用90 min线性梯度（8%~30%乙腈于0.1% FA中），随后以10 min线性梯度（30%~40%乙腈于0.1% FA中）洗脱。质谱仪以信息依赖采集（information-dependent acquisition, IDA）模式运行：先以250 ms扫描全谱（400–1500 m/z），随后进行最多30次MS/MS扫描（每次50 ms，扫描范围100–1800 m/z），循环周期为1.8 s。将电荷态为+2至+5、计数高于每秒125个最低阈值的候选离子分离并进行碎裂，在将这些离子加入12 s的排除列表前采集一次MS/MS谱图。采用滚动碰撞能量，碰撞能量扩散值为15。质谱仪通过Analyst TF 1.7.1软件（AB Sciex）控制。对于数据依赖采集（data-dependent acquisition, DDA，SWATH采集），参数设置如下：100 ms的TOF MS扫描，随后对m/z 400–1250范围进行200个可变SWATH窗口采集，每个窗口累积时间为50 ms。MS/MS SWATH扫描窗口设置为5 Da，重叠1 Da，覆盖m/z 400–1250范围，且在质荷比范围两侧均有变化。总循环周期为9.6 s。采用滚动碰撞能量（CE）参数脚本自动控制碰撞能量。

采用嵌入于ProteinPilot 5.0.1软件（AB Sciex）中的Paragon算法，将采集到的谱图与UniProt已审核数据库进行检索，检索参数如下：(i) 样品类型：鉴定；(ii) 半胱氨酸烷基化试剂：碘乙酰胺；(iii) 酶解方式：胰蛋白酶；(iv) 仪器类型：TripleTOF 6600；(v) 物种：小家鼠（Mus musculus）；(vi) 鉴定聚焦：生物修饰；(vii) 检测蛋白阈值：>0.05（10%置信度）。将检测蛋白阈值设置为最低水平，以增加假阳性结果的数量，从而通过独立的错误发现率（false discovery rate, FDR）分析进行曲线拟合。该分析采用ProteinPilot软件自带的目标-诱饵（target-decoy）方法完成，用于评估鉴定结果的质量。当鉴定到的蛋白质与肽段的局部错误发现率（local FDR）达到1%时，即认定为阳性鉴定。将所得的分组文件导入Peakview（v2.2.0，AB Sciex），以99%的肽段置信度阈值与<1%的错误发现率提取SWATH运行数据中的峰。随后将SWATH文件导出至MarkerView软件（版本1.3.0.1；AB Sciex），将单个肽段的峰面积归一化至所有检测到的肽段的峰面积总和。