Proteomic analysis of ubiqutiin binding proteins

2.1 Laboratory component 2.1.1 Materials used Coupling solution: • 100mM NaHCO3 • 0.5M NaCl • pH8.3 Blocking solution: • 1M ethanolamine • pH8 Column buffer • 10mM Tris • 150mM NaCl • 0.01% sodium azide • pH7.5 Sample buffer • 1% v/v Triton X100 detergent • 50mM Tris HCl 2.1.2 Conjugation of bait to sepharose beads 5ml of coupling solution was run through each sepharose bead column, containing 0.3ml of beads each. A 5ml solution of coupling buffer with an additional 0.6mg/ml ubiquitin was mixed with one column for a period of two hours, the control contained only coupling solution. Each column was then rinsed with blocking solution and stored in the blocking solution at -20oC. Upon thawing columns were given 3 cycles of 0.1M sodium acetate (pH4) / coupling buffer rinses to remove residual ethanolamide, the columns were stored in column buffer at 4oC and a small aliquot was taken to confirm ubiquitin binding by boiling followed by SDS PAGE analysis (standard protocols). 2.1.3 Tissue preparation and crude cell extract analysis. ~16g of porcine brain tissue was hand dissected, then processed in ~4g batches, each batch was placed in 20ml ice cold sample buffer with * protease inhibitor cocktail. The solutions were homogenised using both a an electric and dounce homogeniser, followed by centrifugation at 20,000rpm for 30 minutes at 4oC. The sample supernatants were then filtered through glass wool and a 10µl aliquot was taken for SDS PAGE analysis (12.5% acrylamide gels, by standard protocols). Sample solutions were stored at -80oC prior to Ub-sepharose analysis. 2.2 Affinity purification of UBPs In addition to the two prepared Ub and control columns, two extra columns were provided by demonstrators, one containing the quadruple ubiquitin mutant at the same protein:bead concentration (10mg/ml). Columns were run dry and rinsed with sample buffer to equilibrate the columns. 20ml of cell extract was then run through each column and the flow through recycled three times. Each column was then washed with sample buffer five times in order to remove unbound proteins, and then allowed to run dry. For the Ub-sepharose and a control, a 900µl of sample buffer was used to re-suspend the beads, and 200µl aliquots were taken (~50µl of beads in each), which were sent off for external LC MS.MS analysis.* 2.2 Data analysis 2.2.1 Mass spectroscopy of SDS PAGE bands Mass spectroscopy data were analysed using Mascot, a peptide mass fingerprint (PMF) analysis service available from ExPASy (Expert Protein Analysis System). Bands from between the 1-2kD range were inputted, with the following parameters; taxonomy-human, enzyme-trypsin, fixed modification-carboxymethyl, peptide tolerance 1Da and mass values – MH+. Returned results were then searched firstly for those with known Ub interactions, and then by known mass and checked against approximate mass estimated from the SDS PAGE gel. 2.2.2 Tandem mass spectroscopy By refining the mascot search a data set was formed containing all of the control hits, all hits containing unique peptide peaks (+RBR) and the top 300 results from the default algorithm which does not require unique peptides(-RBR). Control hits were removed from the dataset, in addition duplicates of +RBR found in the less stringent –RBR set. Likely contaminants containing the key words: ubiquitin, keratin, globin, DEAD, myosin and ribosomal were removed, in accordance with the non-specific binding partners identified by Trinkly-Mulcahy et al27 A batch NCBI conserved domain database (CDD) search of all protein hits, using default parameters, returned a further data set linking proteins to domain hits, with descriptions of each domain. These domains were then manually assigned to five categories, one domain was allowed to appear in multiple categories: nuclear, proteasomal, trafficking, signalling and “known ubiquitin binding”. A table was produced for each category to show the proteins assigned to that group and the entire dataset was uploaded to figshare.com to make it available for meta-analysis.

2.1 实验组分
2.1.1 所用材料
偶联液（coupling solution）：
• 100mM 碳酸氢钠（NaHCO₃）
• 0.5M 氯化钠（NaCl）
• pH 8.3
封闭液（blocking solution）：
• 1M 乙醇胺（ethanolamine）
• pH 8
柱缓冲液（column buffer）：
• 10mM 三（羟甲基）氨基甲烷（Tris）
• 150mM 氯化钠（NaCl）
• 0.01% 叠氮化钠（sodium azide）
• pH 7.5
样品缓冲液（sample buffer）：
• 1% 体积分数的曲拉通X-100（Triton X-100）去污剂
• 50mM 三（羟甲基）氨基甲烷盐酸盐（Tris-HCl）

2.1.2 诱饵蛋白与琼脂糖珠（sepharose beads）的偶联
将5mL偶联液通过每根含0.3mL琼脂糖珠的层析柱。将5mL添加了0.6mg/mL泛素（ubiquitin）的偶联缓冲液与一根层析柱混合孵育2小时，对照组仅使用未添加泛素的偶联液。随后每根柱用封闭液冲洗，并置于封闭液中于-20℃保存。解冻后，对柱进行3次0.1M乙酸钠（pH 4）/偶联缓冲液冲洗循环以去除残留的乙醇胺，之后将柱置于柱缓冲液中于4℃保存；取少量分装样品，通过煮沸后进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE，标准操作流程）分析，以验证泛素结合效果。

2.1.3 组织制备与粗细胞提取物分析
取约16g猪脑组织进行手工解剖，随后以约4g为一批进行处理：每批样品置于20mL预冷的样品缓冲液中，并加入*蛋白酶抑制剂混合物（protease inhibitor cocktail）。使用电动匀浆器与杜恩斯匀浆器（dounce homogeniser）对样品进行匀浆，随后在4℃下以20000rpm离心30分钟。取样品上清液通过玻璃棉过滤，之后取10μL分装样品进行SDS-PAGE分析（采用12.5%丙烯酰胺凝胶，按标准操作流程进行）。在进行泛素-琼脂糖珠（Ub-sepharose）分析前，将样品溶液保存于-80℃。

2.2 泛素结合蛋白（UBPs）的亲和纯化
除两根已制备的泛素柱与对照柱外，实验演示人员还提供了额外两根层析柱：其中一根含有四倍泛素突变体，蛋白与微珠的浓度比与此前一致（10mg/mL）。将柱抽干后用样品缓冲液冲洗以平衡柱体。将20mL细胞提取物通过每根柱，收集流出液并循环过柱三次。随后每根柱用样品缓冲液冲洗五次以去除未结合的蛋白质，再将柱抽干。针对泛素-琼脂糖珠柱与对照柱，使用900μL样品缓冲液重悬微珠，取200μL分装样品（每管约含50μL微珠），送至外部机构进行液相色谱-串联质谱（LC-MS/MS）分析。

2.2.1 SDS-PAGE条带的质谱分析
质谱数据通过Mascot进行分析，该工具是ExPASy（Expert Protein Analysis System，专家蛋白质分析系统）提供的肽质量指纹图谱（PMF，peptide mass fingerprint）分析服务。将分子量介于1-2kD之间的条带导入分析，参数设置如下：物种分类-人类，酶解酶-胰蛋白酶，固定修饰-羧甲基化，肽段耐受误差1Da，质量离子类型-MH+。对返回的结果首先检索已知的泛素相互作用蛋白，随后根据已知分子量，并与SDS-PAGE凝胶估算的近似分子量进行比对验证。

2.2.2 串联质谱分析
通过优化Mascot检索流程，构建得到一组数据集，包含所有对照匹配结果、所有含独特肽峰的匹配结果（+RBR组），以及无需独特肽段的默认算法返回的前300条结果（-RBR组）。从数据集中移除对照匹配结果，同时移除在宽松阈值的-RBR组中出现重复的+RBR组条目。根据Trinkly-Mulcahy等人[27]鉴定的非特异性结合蛋白，移除包含泛素、角蛋白、珠蛋白、DEAD家族蛋白、肌球蛋白以及核糖体相关关键词的潜在污染物。使用默认参数对所有匹配蛋白进行批量NCBI保守结构域数据库（CDD，conserved domain database）检索，得到另一组将蛋白与结构域匹配结果关联的数据集，并附带各结构域的描述信息。随后手动将这些结构域划分为五大类别，允许一个结构域归入多个类别：细胞核相关、蛋白酶体相关、转运相关、信号通路相关以及"known ubiquitin binding"类别。为每个类别制作表格以展示归入该组的蛋白，且将完整数据集上传至figshare.com平台，以供后续元分析使用。