利用肽配体库分析蛋清中低丰度蛋白质组

利用肽配体库技术(CPLL)研究了蛋清中的低丰度蛋白质组。考察了盐浓度和洗脱顺序差异对CPLL富集效率的影响。结果表明:盐浓度越高,蛋清蛋白质溶液的离子相互作用越强。利用洗脱溶液解离差异,采用先HOS(有机水溶液)后尿素CHAPS(3-[3-(胆酰胺丙基)二甲氨基]-1-丙磺酸内盐)的洗脱顺序,溶菌酶能够得到高效分离。保持蛋清的原p H 8.8,25 mmol/L KH2PO4,150 mmol/L Na Cl,尿素CHAPS洗脱,是CPLL提供高效富集的前提。通过分析CPLL富集前后的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDSPAGE)条带差异,采用线性离子阱(LTQ)质谱在蛋清中检出45种低丰度蛋白质,其中两种未见报道。对蛋清中低丰度蛋白质亚细胞的定位分析发现,蛋清蛋白质的分泌蛋白居多。研究结果表明,CPLL技术能够实现蛋清中低丰度蛋白质的高效富集,分析深度达到亚细胞水平。

从组合化学肽库中筛选亲和配基

A new method for the rapid and efficient screening of affinity ligands to biological targets is reported. The fusion peptide of influenza virus A was used as the model target and immobilized on the PGMA beads. Antisense peptide YRSKQA of fusion peptide was chosen as the lead compound. The special positional scanning peptide libraries were designed based on YRSKQA and synthesized by utilizing solid phase peptide synthesis manually. The libraries were YRSKQX, YRSKXA, YRSXQA, YRXKQA, YXSKQA and XRSKQA, where X represented 18 L-amino acids(except for Cys and Trp). Each library was screened by affinity chromatography. The eluates from the fusion peptide affinity column were collected and analyzed by RP-HPLC and MS, respectively, in order to determine the kind of X at each position. After the preferred residues of six positions were decided, the two preferred peptide sequences, GRGKHK and TRGKHK, were obtained. The dissociation constants of GRGKHK, TRGKHK and YRSKQA, were 3.35×10 -6, 5.24×10 -6 and 1.15×10 -5 mol·L -1, respectively. The preferred peptides showed the higher affinity binding to immobilized fusion peptide than the lead peptide.

组合肽配体库技术富集蛋白研究进展

低丰度蛋白是生物体中重要的活性物质,参与新陈代谢、转录和翻译等多种生理及病理过程,因此对低丰度蛋白的研究具有重要意义。组合肽配体库技术(CPLL)是富集低丰度蛋白的一种有效方法,通过CPLL富集作用可以实现低丰度蛋白的检测与鉴定。该文总结了CPLL应用于不同领域中(包括人体、动物源、植物源等蛋白研究方面)低丰度蛋白样品的富集研究,可为CPLL的进一步应用提供参考。