多肽功能化亲和微球的制备与线粒体高选择性分离分析  

Preparation of peptide-functionalized affinity materials for the highly specific capture and analysis of mitochondria

作　　者：陈建 徐坤 高涵 赵睿[1,2] 黄嫣嫣 CHEN Jian;XU Kun;GAO Han;ZHAO Rui;HUANG Yanyan(Beijing National Laboratory for Molecular Sciences,CAS Key Laboratory of Analytical Chemistry for Living Biosystems,Institute of Chemistry,Chinese Academy of Sciences,Beijing 100190,China;University of Chinese Academy of Sciences,Beijing 100049,China)

机构地区：[1]北京分子科学国家研究中心,中国科学院化学研究所,中国科学院活体分析化学重点实验室,北京100190 [2]中国科学院大学,北京100049

出　　处：《色谱》2024年第6期555-563,共9页Chinese Journal of Chromatography

基　　金：国家自然科学基金项目(22122411,22374152,22174145).

摘　　要：线粒体作为细胞物质和能量代谢的主要细胞器,在维持细胞生理稳态中发挥关键作用,进而与诸多疾病的发生发展密切相关。从高度复杂的细胞组分中特异性分离分析线粒体,对于其功能解析、分子机制研究和化学干预具有重要意义,但仍存在困难与挑战。本研究以靶向多肽为识别元件,开展了线粒体亲和分离新材料的设计、合成和分析应用研究。以与线粒体膜具有特异性相互作用的线粒体穿透肽(mitochondrial penetrating peptide,MPP)为识别元件,通过引入空间手臂分子,设计合成了多肽亲和配基。以表面富含醛基的聚合物基质微球(matrix beads,MB)为亲和基质,建立了基于胺甲基化反应的多肽功能化亲和微球制备方法,该方法具有修饰反应条件温和、配基修饰效率高的特点。将所构建的亲和微球MB@MPP用于细胞破碎液中线粒体的直接分离,实现了完整线粒体的快速、高选择性和无损捕获。与商品化分离试剂盒相比,MB@MPP捕获的线粒体中标志蛋白富集效率更高,纯度更好。基于多肽功能化微球对线粒体高选择性分离能力,以线粒体代谢所需关键辅酶的前体小分子色氨酸和核黄素为目标,进一步建立了基于液相色谱-串联质谱的线粒体中活性小分子的分析检测方法,实现了激酶激动剂刺激前后线粒体中活性小分子含量及其动态变化分析,为线粒体功能解析和相关疾病分子机制研究提供了方法基础。Mitochondria perform various metabolic processes that significantly affect cell differentiation,proliferation,signal transduction,and programmed cell death.The disruption of mitochondrial bioenergetic and metabolic functions is closely related to many disorders.The specific isolation and purification of intact,high-purity,and functional mitochondria are central to the understanding of their mechanism of action but remain challenging tasks.In this study,a mitochondrial penetrating peptide(MPP)with the sequence FrFKFrFK(Ac)was used as a mitochondrial recognition motif to construct a peptide-guided affinity separation material.The multiple aromatic phenylalanine(F)residues in this amphiphilic peptide can confer lipophilicity to the mitochondrial membrane,whereas the basic residues(D-arginine and lysine)render the MPP surface positively charged,thereby promoting the binding of negatively charged mitochondria.After the derivatization of the N terminal of MPP with an oligoglycine spacer,the peptide ligands were conjugated to matrix beads(MB)with surface aldehyde functional groups.Peptide functionalization was performed via a condensation reaction between the amino group in the peptide ligand and the aldehyde group on the beads.The generated Schiff bases were reduced,affording stable covalent bonds.The dense and stable functionalization of the beads with the mitochondria-targeting peptides was demonstrated using high performance liquid chromatography(HPLC),zeta potential assay,and scanning electron microscopy(SEM).The immobilization efficiency of the peptide ligands was 1.47μmol/g,and the surface potential of MB@MPP was 11 mV.MB@MPP was used for the direct isolation of mitochondria after cell homogenization.As observed by SEM,mitochondria with a cross-sectional diameter of 500 nm were efficiently captured on the MB@MPP surface.Because the mitochondrial membrane potential is an important marker of mitochondrial function and the driving force behind the staining of mitochondria with Mito Tracker dyes,the specific binding

关 键 词：多肽 亲和识别 分离材料 线粒体 复杂体系 液相色谱-串联质谱 

分 类 号：O658[理学—分析化学]