MS RAW DATA CTPS.xlsx

<b>Tandem affinity purification (TAP) and Liquid chromatography - Mass spectrometry (LC-MS) Analyses</b> We used a Cts1-flag-3HA strain for our TAP and LC-MS analyses. For the TAP assay we used the FLAG-HA tandem affinity purification kit manufactured by Sigma (TP0010), and performed the protocol according to the manufacturer’s specifications. Briefly, Cts1-flag-3HA cells were cultured in rich media (YE4S) until they reached exponential phase. 50x10⁷ cells were collected and the cell lysate was extracted, as described in the co-IP protocol. The cell lysate was then incubated with anti-FLAG M2 resin rotating overnight at 4⁰C, ensuring efficient binding. The supernatant was then removed carefully and the resin was washed with RIPA buffer (Sigma, R0278) containing protease inhibitors (Sigma, P8215), in order to remove any unbound protein. The first elution of the protein complex bound on the resin was done by using 3XFLAG peptide, and in a following step the eluate was bound to anti-HA resin slurry. In the second elution of the protein complex, the anti-HA slurry was washed with TBS (50mM Tris-Cl, ph=7.5; 150mM NaCl), to remove any unbound protein. The final elution was done by using TBS with 100mM ammonium bicarbonate, and the sample was subsequently digested with trypsin overnight. Peptides were then separated and analyzed on an Easy-nLC 1000 system coupled to a Q Exactive HF (both - Thermo Scientific). About 2 µg of peptides were separated in an home-made column (75 µm x 15 cm) packed with C18 AQ (5 µm, 300Å, Michrom BioResources, Auburn, CA, USA) at a flow rate of 300 nL/min. Mobile phase A (0.1% formic acid in 2% ACN) and mobile phase B (0.1% formic acid in 98% ACN) were used to establish a 60 min gradient comprised of 2 min of 5% B, 40 min of 5-30% B, 6 min of 30-45% B, 2 min of 45-90% B and 10 min of 90% B. Peptides were then ionized by electrospray at 2.3 kV. A full MS spectrum (300-1800 m/z range) was acquired at a resolution of 120,000 at m/z 200 and a maximum ion accumulation time of 20 ms. Dynamic exclusion was set to 30 s. Resolution for HCD MS/MS spectra was set to 30,000 at m/z 200. The AGC setting of MS and MS2 were set at 3E6 and 1E5, respectively. The 20 most intense ions above a 1.3E4 counts threshold were selected for fragmentation by HCD with a maximum ion accumulation time of 80 ms. Isolation width of 1.6 m/z units was used for MS2. Single and unassigned charged ions were excluded from MS/MS. For HCD, normalized collision energy was set to 30%. The underfill ratio was defined as 1%. The raw data were processed and searched with MaxQuant 1.5.4.1 with MS tolerance of 4.5 ppm, and MS/MS tolerance of 20 ppm. The Uniprot Schizosaccharomyces pombe proteome database and the database for proteomics contaminants from MaxQuant were used for database searches. Reversed database searches were used to evaluate false discovery rate (FDR) of peptide and protein identifications. Two missed cleavage sites of trypsin were allowed. Oxidation (M), Acetyl (Protein N-term), deamidation (NQ) and GGE (K) were set as variable modifications. The FDR of both peptide identification and protein identification is set to be 1%. The option of “Second peptides”, “Match between runs” and “Dependent peptides” was enabled. Label-free quantification was used to quantify the difference of protein abundances between different samples. Each sheet on the xls file corresponds to a different trial.<br>

串联亲和纯化（Tandem affinity purification, TAP）与液相色谱-质谱联用（Liquid chromatography - Mass spectrometry, LC-MS）分析 本研究采用Cts1-flag-3HA菌株开展TAP与LC-MS分析。针对TAP实验，我们使用了Sigma公司生产的FLAG-HA串联亲和纯化试剂盒（货号TP0010），并严格按照厂商说明书的操作流程进行实验。简言之，将携带Cts1-flag-3HA的细胞在丰富培养基YE4S中培养至指数生长期，收集50×10^7个细胞，参照免疫共沉淀（co-immunoprecipitation, co-IP）实验方案提取细胞裂解液。随后将细胞裂解液与抗FLAG M2亲和树脂在4℃下旋转孵育过夜，以确保蛋白充分结合。小心移除上清液后，使用含蛋白酶抑制剂（Sigma货号P8215）的RIPA缓冲液（Sigma货号R0278）洗涤亲和树脂，以去除未结合的杂蛋白。第一步洗脱结合于树脂上的蛋白复合物采用3×FLAG肽段，后续将洗脱液与抗HA树脂悬液孵育以结合靶蛋白复合物。第二步洗脱前，先用Tris缓冲盐溶液（TBS，50mM Tris-Cl，pH=7.5；150mM NaCl）洗涤抗HA树脂悬液，以去除未结合的蛋白。最终洗脱采用含100mM碳酸氢铵的TBS缓冲液，随后将样品用胰蛋白酶过夜酶解。取约2μg肽段，经自制色谱柱（75μm×15cm，填充C18 AQ填料，粒径5μm，孔径300Å，购自Michrom BioResources，美国加利福尼亚州奥本市）分离，流速设置为300nL/min，采用Easy-nLC 1000液相色谱系统与Q Exactive HF质谱仪（均为赛默飞世尔科技Thermo Scientific产品）联用进行分析。采用流动相A（含0.1%甲酸的2%乙腈（ACN）水溶液）与流动相B（含0.1%甲酸的98%乙腈溶液）构建60分钟梯度洗脱程序：2分钟内维持5% B，40分钟内从5% B线性升至30% B，6分钟内从30% B线性升至45% B，2分钟内从45% B线性升至90% B，最后10分钟维持90% B。肽段经2.3kV电喷雾电离。全扫描质谱（MS）采集范围为300-1800 m/z，在m/z 200处分辨率为120000，最大离子积累时间为20ms。动态排除时长设置为30秒。高能碰撞解离（HCD）二级质谱（MS/MS）在m/z 200处分辨率设置为30000。MS与MS2的自动增益控制（AGC）目标值分别设为3×10^6与1×10^5。选取强度高于1.3×10^4计数阈值的前20个强度最高的离子进行HCD碎裂，最大离子积累时间为80ms。二级质谱分离窗口宽度设为1.6 m/z。单电荷离子与未指定电荷的离子被排除在MS/MS分析之外。HCD的归一化碰撞能量设置为30%。未填满比例定义为1%。原始数据使用MaxQuant 1.5.4.1软件进行处理与数据库检索，母离子质量容忍度设为4.5ppm，二级质谱质量容忍度设为20ppm。数据库检索采用Uniprot粟酒裂殖酵母（Schizosaccharomyces pombe）蛋白质组数据库，以及MaxQuant自带的蛋白质组学常见污染物数据库。采用反向数据库检索评估肽段与蛋白质鉴定的假发现率（false discovery rate, FDR）。允许胰蛋白酶最多2个错切位点。氧化（甲硫氨酸，M）、乙酰化（蛋白质N端）、脱酰胺（天冬酰胺/谷氨酰胺，NQ）与GGE修饰（赖氨酸，K）被设置为可变修饰。肽段鉴定与蛋白质鉴定的假发现率均设为1%。启用“Second peptides”、“Match between runs”与“Dependent peptides”选项。采用无标记定量方法对不同样本间的蛋白丰度差异进行定量。Excel表格文件中的每个工作表对应一次独立重复实验。