采用亲和去垢小柱净化，建立了水稻叶片蛋白质组的纳升液相色谱-串联质谱分析方法。水稻叶片蛋白质分别采用酚提取法结合十二烷基硫酸钠（sodium dodecyl sulfate，SDS）裂解，裂解液经亲和去垢小柱净化，酶解肽段用纳升液相色谱-线性离子阱/静电场轨道阱组合式高分辨质谱（nanoLC-LTQ / Orbitrap MS）分析，相关数据库检索鉴定。比较了超滤辅助样品制备法（FASP 法）、亲和去垢小柱法和丙酮沉淀法对 SDS 去除效率及对蛋白质鉴定结果的影响。结果表明：3种方法均有较好的 SDS 去除效果（去除效率均大于95％）；尽管3种方法鉴定的蛋白质种类具有一定的互补性，但以亲和去垢小柱法鉴定的蛋白质数目最多，为563种，远多于 FASP 法和丙酮沉淀法的196和306种；此外，亲和去垢小柱法适合于各种相对分子质量和不同 pI 值蛋白质的净化，而 FASP 法和丙酮沉淀法中不同相对分子质量和 pI 值蛋白质均有类似程度的损失。采用本文建立的方法，一次进样分析可鉴定出水稻叶片蛋白质多达588种；肽段匹配数≥2的296个蛋白质的生物学功能主要分为结合活性、酶活性、转移运输活性和结构组成等。该蛋白质分析方法可为开展水稻蛋白质组学研究提供技术参考。
A purification method was established for the analysis of proteomes in rice leaves based on a novel detergent removal spin column(DRSC). The proteins were extracted by phe-nol protein extraction method followed by sodium dodecyl sulfate( SDS)lysis. The lysate was purified by the detergent removal spin column and the enzymolytic peptides were detected by the nanoflow liquid chromatography-hybrid linear trap quadrupole orbitrap mass spectrometry (nanoLC-LTQ / Orbitrap). In terms of SDS removal efficiencies and protein identification,the method of DRSC was compared with those of filter aided sample preparation(FASP)and ace-tone precipitation. As a result,there were good efficiencies( > 95% )of SDS removal for the three methods. With the DRSC purification strategy,563 proteins were identified from rice leav-es,while only 196 and 306 proteins were identified by FASP and acetone precipitation proce-dures respectively,in spite of certain complementarities among these identified proteins by the three methods. DRSC is suitable for proteins with various relative molecular masses and pI values. However,there were similar losses of proteins with different relative molecular masses and pI values with the other two methods. Using the established method,588 proteins were identified by once injection analysis. According to the molecular functions,296 proteins with at least two identified peptides can be classified into eight categories with binding activity,enzyme activity,transporter activity,inhibitor activity,structural constitute,catalytic activity,other and unknown functions. The method provides technical reference for conducting rice proteomes.
参考文献
| [1] | Second T P;Blethrow J D;Schwartz J C.[J].Anal Chem,200981:7757. |
| [2] | Hsieh E J;Hoopmann M R;MacLean B.[J].J Proteome Res,20109:1138. |
| [3] | 于海洋;晏嘉泽;郭明.[J].色谱,201331(04):362. |
| [4] | Yates J R.[J].J Am Chem Soc,2013135(05):1629. |
| [5] | Jorrin-Novo J V;Komatsu S;Weckwerth W.Methods in Molecular Biology[M].New York:Humana Press,2014 |
| [6] | 李蓓;宁露云;张俊卫.[J].湖北农业科学,201352(32):5403. |
| [7] | Kim S T;Kim S G;Agrawal G K.[J].Proteomics,201414:593. |
| [8] | Masuda T;Tomita M;Ishihama Y.[J].J Proteome Res,20087:731. |
| [9] | Bereman M S;Egertson J D;MacCoss M J.[J].Proteomics,201111:2931. |
| [10] | Winter D;Steen H.[J].Proteomics,201111:4726. |
| [11] | Tanca A;Biosa G;Pagnozzi D.[J].Proteomics,201313:2597. |
| [12] | Zhang N;Chen R;Young N.[J].Proteomics,20077:484. |
| [13] | Lin Y;Liu H;Liu Z.[J].J Chromatogr B,2012901:18. |
| [14] | Botelho D;Wall M J;Vieira D B.[J].J Proteome Res,20109:2863. |
| [15] | Antharavally B S;Mallia K A;Rosenblatt M M.[J].Anal Biochem,2011416:39. |
| [16] | Hengel S M;Floyd E;Baker E S.[J].Proteomics,201212:3138. |
| [17] | Sheoran I S;Ross A R S;Olson D J H.[J].Plant Sci,2009176:99. |
| [18] | Wisˊ niewski J R;Zougman A;Nagaraj N.[J].Nat Meth-ods,20096:359. |
| [19] | Manza L L;Stamer S L;Ham A J L.[J].Proteomics,20055:1742. |
| [20] | Wisˊ niewski J R;Ostasiewicz P;Mann M.[J].J Proteome Res,201110:3040. |
| [21] | Wisˊ niewski J R;Zielinska D F;Mann M.[J].Anal Biochem,2011410:307. |
| [22] | Fic E;Kedracka-Krok S;Jankowska U.[J].Electropho-resis,201031:3573. |
| [23] | Muthurajan R;Shobbar Z S;Jagadish S V K.[J].Mol Biotechnol,201148:173. |
| [24] | Zhao C F;Wang J Q;Cao M L.[J].Proteomics,20055:961. |
| [25] | 魏开华;应天翼;胡良平.蛋白质组学实验技术精编[M].北京:化学工业出版社,2010 |
| [26] | Islam N;Lonsdale M;Upadhyaya N M.[J].Proteomics,20044:1903. |
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