蛋白复合物的鉴定<em>大肠杆菌</em使用顺序肽亲和纯化技术相结合串联质谱法
Summary
亲和纯化标签蛋白结合质谱（APMS）是一个功能强大的蛋白质相互作用网络系统的映射方法和调查的生物过程的机理的基础。在这里，我们描述了一个优化的顺序肽亲和力（SPA）APMS程序开发的细菌<em>大肠杆菌</em>，可用于分离和表征到接近均匀性，甚至从每个细胞的拷贝数低的稳定的多蛋白复合物。
Abstract
由于大多数大分子组件，系统识别的蛋白-蛋白相互作用（PPI）和确定的亚基组成的多蛋白复合物，可以帮助了解基因的功能和增进了解的生物系统1，2介导的细胞过程。可以被映射的物理相互作用，与高的信心vialarge规模接近生理条件下，基于与质谱（APMS）亲和纯化的染色体标记的蛋白质组合的内源性蛋白复合物的分离和鉴定的。这种方法已成功地应用在进化上不同的生物，包括酵母，果蝇，蠕虫病毒，哺乳动物细胞和细菌1-6。特别是，我们已经产生了一个羧基末端的顺序肽亲和（SPA）亲和纯化的天然蛋白复合物从培养革兰阴性大肠杆菌双标记系统，使用GEnetically听话的主机实验室菌株全基因组调查的基本生物学和保守的过程中原核生物1，2，7，非常适合。我们的SPA标记系统是类似于酵母8，9最初开发的串联亲和纯化方法，由钙调蛋白结合肽（CBP）的后跟的高度特异性的烟草蚀刻病毒 （TEV）蛋白酶的切割位点和三个副本FLAG表位（3X FLAG），允许两个连续两轮亲和富集。盒扩增后，序列特异性的线性编码的SPA-tag和可选择标记的PCR产物是集成的，表示帧作为羧基末端融合在DY330背景下，瞬时表达被诱导高效率的异源λ噬菌体重组系统10。随后的双步纯化使用调钙蛋白和抗FLAG亲和珠使高度选择性甚至大型文化低丰度蛋白复合物和有效的恢复。串联质谱法，然后使用，以确定稳定地共同净化蛋白具有高灵敏度（低纳克检出限）。
在这里，我们将详细介绍一步一步的程序，我们通常使用的系统蛋白标记，分离纯化和质谱为基础的分析可溶性蛋白复合物E.大肠杆菌 ，可以按比例增加和潜在的针对其他细菌物种，包括某些服从重组工程的机会病原体。由此产生的物理相互作用通常可以发现有趣的组件和连接提出新的机械链接。 PPI数据整合与的备用分子协会提供的数据，如遗传（基因 – 基因）的相互作用和基因组上下文（GC）的预测可以方便的多蛋白复合物的的全球分子组织的澄清在BI目的论的途径。 E.产生的网络大肠杆菌可以使用的功能结构，其他微生物的同源基因产品的功能注释目前缺乏深入了解。
Protocol
Representative Results
Discussion
这里所描述的一个关键方面SPA基于APMS方法的的标记内的天然染色体背景下进行，从而确保维持正常的基因调节（ 即原生饵子保存的，因此，表达水平不扰动）和本机稳定相关蛋白质复合物中回收在近内源性水平。正负极性问题，也避免包括选择标记朝外启动。这SPA-标记的方法是有效的，足以净化组件的低丰度的配合物不久的均匀性，包括膜相关的组件，即使亚基表达下降到只有几个分子?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是从加拿大安大略基因组研究所，加拿大基因组，创新，创新的安大略省，加拿大卫生研究院研究补助金JG和AE红表示E.基金会的资金支持coli菌株DY330从Donald L.法院（美国国家癌症研究所，冯检基，MD）是一种礼物。
Materials
| Materials | Vendor | and Catalog Numbers | |
| I. Antibiotics | |||
| Kanamycin | Bioshop | #KAN201 | |
| Ampicillin | Bioshop | #AMP201 | |
| 2. Terrific-Broth medium | |||
| Bio-Tryptone | Bioshop | #TRP 402 | |
| Yeast extract | Bioshop | #YEX 555 | |
| Glycerol | Bioshop | #GLY 002 | |
| K2HPO4 | Bioshop | #PPM 302 | |
| KH2PO4 | Bioshop | #PPM 303 | |
| 3. Bacterial Strain and Plasmid | |||
| DY330 | Yu et al. (2000)10 | ||
| pJL148 | Zeghouf et al. (2004)7 | ||
| 4. PCR and Electrophoresis Reagents | |||
| Taq DNA polymerase | Fermentas | # EP0281 | |
| 10 X PCR buffer | Fermentas | # EP0281 | |
| 10 mM dNTPs | Fermentas | # EP0281 | |
| 25 mM MgCl2 | Fermentas | # EP0281 | |
| Agarose | Bioshop | # AGA002 | |
| Loading dye | NEB | #B7021S | |
| Ethidium bromide | Bioshop | # ETB444 | |
| 10X TBE buffer | Thermo Scientific | #28355 | |
| Tris Base | Bioshop | #TRS001 | |
| Boric acid | Bioshop | #BOR001 | |
| 0.5 M EDTA (pH 8.0) | Sigma | # E6768 | |
| DNA ladder | NEB | #N3232L | |
| 5. Plasmid isolation and Clean-up Kits | |||
| Plasmid Midi kit | Qiagen | #12143 | |
| QIAquick PCR purification kit | Qiagen | #28104 | |
| 6. PCR and Transformation Equipments | |||
| Thermal cycler | BioRad | iCycler | |
| Agarose gel electrophoresis | BioRad | ||
| Electroporator | Bio-Rad | GenePulser II | |
| 0.2 cm electroporation cuvette | Bio-Rad | ||
| 42 °C water bath shaker | Innova 3100 | ||
| Beckman Coulter TJ-25 centrifuge | Beckman Coulter | TS-5.1-500 | |
| 32 °C Shaker | New Brunswick Scientific, USA | ||
| 32 °C large Shaker | New Brunswick Scientific, USA | ||
| 32 °C plate incubator | Fisher Scientific | ||
| 7. Electrophoresis and Western blotting | |||
| Acrylamide monomer, N,N’- methylenebis-acrylamide | Bio-Rad | #161-0125 | |
| Ammonium persulfate | Bioshop | # AMP001 | |
| n-butanol | Sigma | # B7906 | |
| TEMED | Bioshop | #TEM001 | |
| Whatman No. 1 filter paper | Fischer Scientific | #09-806A | |
| Mini protean 3 cell | Bio-Rad | #165-3301 | |
| iBlot gel transfer device | Invitrogen | #IB1001 | |
| Nitrocellulose membranes | Bio-Rad | #162-0115 | |
| Monoclonal Anti-Flag M2 antibody | Sigma | #F3165 | |
| Horseradish peroxidase | Amersham | #NA931V | |
| Pre-stained protein molecular weight standards | Bio-Rad | #161-0363 | |
| Chemiluminescence reagent | PIERCE | #1856136 | |
| Autoradiography film | Clonex Corp | #CLEC810 | |
| Quick Draw blotting paper | Sigma | #P7796 | |
| C2 platform rocking shaker | New Brunswick Scientific, USA | ||
| 8. Sonication Equipment and Reagents | |||
| Sonicator | Branson Ultrasonic | #23395 | |
| NaCl | Bioshop | #SOD001 | |
| Protease inhibitors | Roche | #800-363-5887 | |
| 0.5 mM TCEP-HCl | Thermo Scientific | #20490 | |
| 9. Affinity Purification Reagents and Equipment | |||
| 0.8 x 4 cm Bio-Rad polypropylene column | Bio-Rad | #732-6008 | |
| Benzonase nuclease | Novagen | #70746 | |
| Anti-FLAG M2 agarose beads | Sigma | #A2220 | |
| Calmodulin-sepharose beads | GE Healthcare | #17-0529-01 | |
| TEV protease | Invitrogen | #12575-015 | |
| Triton X-100 | Sigma | #T9284 | |
| CaCl2 | Sigma | #C2661 | |
| EGTA | Sigma | #E3889 | |
| LabQuake Shaker | Thermolyne | #59558 | |
| 10. Silver Staining Reagents | |||
| Methanol | Bioshop | #MET302 | |
| Acetic acid | Bioshop | t#ACE222 | |
| Sodium-thiosulfate | Sigma | #S-7143 | |
| Silver nitrate | Fischer Scientific | #S181-100 | |
| Formaldehyde | Bioshop | #FOR201 | |
| Sodium carbonate | Bioshop | #SOC512 | |
| 11. Reagents and Equipment for Protein Identification | |||
| Trypsin Gold, Mass Spectrometry Grade | Promega | # V5280 | |
| 50 mM NH4HCO3 | Bioshop | #AMC555 | |
| 1 mM CaCl2 | Bioshop | #CCL302 | |
| Acetonitrile | Sigma | #A998-4 | |
| Formic acid | Sigma | #F0507 | |
| HPLC grade water | Sigma | #95304 | |
| Iodoacetamide | Sigma | #16125 | |
| Millipore Zip-Tip | Millipore | # ZTC18M960 | |
| ~10 cm of 3 μm Luna-C18 resin | Phenomenex | ||
| Proxeon nano HPLC pump | Thermo Fisher Scientific | ||
| LTQ Orbitrap Velos mass spectrometer | Thermo Fisher Scientific | ||
| 12. Labware | |||
| 4 liter conical flasks | VWR | #89000-372 | |
| 50 ml polypropylene falcon tubes | Any Vendor | ||
| 1.5 ml micro-centrifuge tubes | Any Vendor | ||
| 250 ml conical flaks | VWR | #29140-045 | |
| 15 ml sterile culture tubes | Thermo Scientific | #366052 | |
| Cryogenic vials | VWR | #479-3221 | |
| -80 °C freezer | Fisher Scientific | #13-990-14 | |
| Speed vacuum system | Thermo Scientific | ||
|
Buffers and Solutions 1. 1 liter Terrific Broth (TB) media 11 g Bio-Tryptone 2. Potassium Salt Stock Solution 1.5 M K2HPO4 3. Sonication Buffer 20 mM Tris-HCl (pH 7.9) 4. AFC buffer 30 mM Tris-HCl (pH 7.9) 5. TEV cleavage buffer 30 mM Tris-HCl (pH 7.9) 6. Calmodulin binding buffer 30 mM Tris-HCl (pH 7.9) 7. Calmodulin wash buffer 30 mM Tris-HCl pH 7.9 8. Calmodulin elution buffer 30 mM Tris-HCl (pH 7.9) 9. Developing solution (1L) 37% Formaldehyde 10. Digestion buffer 50 mM NH4HCO3 11. Wetting and Equilibration solution 70% acetonitrile (ACN) in 0.1% formic acid 12. Washing solution 100% H2O in 0.1% formic acid |
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