Profiling lipid-protein interactions using nonquenched fluorescent liposomal nanovesicles and proteome microarrays

Kuan Yi Lu, Sheng Ce Tao, Tzu Ching Yang, Yu Hsuan Ho, Chia Hsien Lee, Chen Ching Lin, Hsueh Fen Juan, Hsuan Cheng Huang, Chin Yu Yang, Ming Shuo Chen, Yu Yi Lin, Jin Ying Lu, Heng Zhu, Chien Sheng Chen

研究成果: Article同行評審

29 引文 斯高帕斯(Scopus)


Fluorescent liposomal nanovesicles (liposomes) are commonly used for lipid research and/or signal enhancement. However, the problem of self-quenching with conventional fluorescent liposomes limits their applications because these liposomes must be lysed to detect the fluorescent signals. Here, we developed a nonquenched fluorescent (NQF)1 liposome by optimizing the proportion of sulforhodamine B (SRB) encapsulant and lissamine rhodamine B-dipalmitoyl phosphatidylethanol (LRBDPPE) on a liposomal surface for signal amplification. Our study showed that 0.3% of LRB-DPPE with 200 μM of SRB provided the maximal fluorescent signal without the need to lyse the liposomes. We also observed that the NQF liposomes largely eliminated self-quenching effects and produced greatly enhanced signals than SRB-only liposomes by 5.3-fold. To show their application in proteomics research, we constructed NQF liposomes that contained phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) and profiled its protein interactome using a yeast proteome microarray. Our profiling led to the identification of 162 PI(3,5)P2-specific binding proteins (PI(3,5)P2-BPs). We not only recovered many proteins that possessed known PI(3,5)P2-binding domains, but we also found two unknown Pfam domains (Pfam-B-8509 and Pfam-B-10446) that were enriched in our dataset. The validation of many newly discovered PI(3,5)P2-BPs was performed using a bead-based affinity assay. Further bioinformatics analyses revealed that the functional roles of 22 PI(3,5)P2-BPs were similar to those associated with PI(3,5)P2, including vesicle-mediated transport, GTPase, cytoskeleton, and kinase. Among the 162 PI(3,5)P 2-BPs, we found a novel motif, HRDIKP[ES]NJLL that showed statistical significance. A docking simulation showed that PI(3,5)P2 interacted primarily with lysine or arginine side chains of the newly identified PI(3,5)P2-binding kinases. Our study showed that this new tool would greatly benefit profiling lipid-protein interactions in high-throughput studies.

頁(從 - 到)1177-1190
期刊Molecular and Cellular Proteomics
出版狀態Published - 2012 11月

All Science Journal Classification (ASJC) codes

  • 分析化學
  • 生物化學
  • 分子生物學


深入研究「Profiling lipid-protein interactions using nonquenched fluorescent liposomal nanovesicles and proteome microarrays」主題。共同形成了獨特的指紋。