多肽功能化亲和微球的制备与线粒体高选择性分离分析OA北大核心CSTPCDMEDLINE

Mitochondria perform various metabolic processes that significantly affect cell differ-entiation,proliferation,signal transduction,and programmed cell death.The disruption of mi-tochondrial bioenergetic and metabolic functions is closely related to many disorders.The spe-cific 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 con-fer lipophilicity to the mitochondrial membrane,whereas the basic residues(D-arginine and ly-sine)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 functionaliza-tion of the beads with the mitochondria-targeting peptides was demonstrated using high per-formance liquid chromatography(HPLC),zeta potential assay,and scanning electron micros-copy(SEM).The immobilization efficiency of the peptide ligands was 1.47 μmol/g,and the surface potential of MB@MPP was 11 mV.MB@MPP was used for the direct isolation of mito-chondria 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 mitochon-drial 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 and separation of fluorescent mitochondria from the cell samples revealed that the proposed MB@MPP-based isolation approach can keep mitochondria intact and retain their functions.Western blot assays were employed to characterize the protein markers of the mitochondria(citrate synthase(CS)and voltage-dependent anion channel protein(VDAC))and cytoplasmic protein(vinculin),and examine the integrity and purity of the captured mitochondria.The results showed that the lysates released from MB@MPP had high CS and VDAC contents.By contrast,vinculin,which is highly abundant in whole-cell lysates,was barely detected in the lysates from MB@MPP.These results suggest that MB@MPP isolates mitochondria with high affinity,specificity,and antifouling ability by using the targeting peptide as the capture handle.A comparison with a commercial mitochondrial isolation kit demonstrated that MB@MPP can separate mitochondria with higher CS and VDAC abundance and purity.Given the superior separation performance of MB@MPP,the molecular profiles of the isolated mitochondria under stress were subjected to further analysis of their molecular profiles under stress. A liquid chromatography-tandem mass spectrometry(LC-MS/MS)method was established to detect tryptophan(Trp)and riboflavin in the mitochondria.Quantification was performed in multiple-reaction monitoring(MRM)mode.Owing to the high purity of the mitochondria,the Trp and riboflavin contents were determined to be 265 and 0.67 nmol/mg,respectively.The metabolic response of mitochondria to external stimuli was further examined using acadesine,an adenosine 5'-monophosphate(AMP)-activated protein kinase activator with a wide range of metabolic effects,to treat cells.After cell homogenization,MB@MPP was used to separate the mitochondria from the cell samples with and without acadesine treatment,followed by LC-MS/MS analysis.The quantification results demonstrated that acadesine induced a 14％upregulation of Trp content in the mitochondria.By contrast,the riboflavin content decreased to 0.48 nmol/mg,which is 72％of that in untreated mitochondria.The changes in Trp and riboflavin contents could influence their metabolic pathways and,thus,the levels of their metabolites,such as nicotinamide adenine dinucleotide,flavin mononucleotide,and flavin adenine dinucle-otide,which are essential coenzymes in mitochondria.Peptide-functionalized affinity mi-crobeads with high affinity and specificity for mitochondria are promising for the efficient isolation of high-quality mitochondria,and offer a useful tool for understanding the complicated functions and dynamics of this unique organelle.