Preliminary Study Of The Purification Of Mitochondrial Subpopulations Via Recycling Free-flow Isoelectric Focusing

Mitochondria are one of the most important organelles in eukaryotic cells,which are involved in critical physiological activities such as energy supply,cell apoptosis and aging process.The dynamic and heterogeneous feature of mitochondria show close relationships with diseases,and the in-depth study concerning mitochondrial function,in this sense,has crucial value.With the rapid development of technology,it is no longer fresh to study organelles via imaging approaches.However,it poses difficulties in thorough analysis of total mitochondrial population.Since mitochondria are desired targets in proteomic research,the purity and quantity requirement are very important.But most current conventional methods are not fully qualified for this demand.Free-Flow Electrophoresis(FFE)combines the merits of preparation and analysis,which is based on none-stationary phase separation.The mild conditions make it quite suitable for mitochondria isolation.A plenty of researches have been reported on mitochondria separation in recent years.But most of them are carried out by Free-Flow Zonal Electrophoresis(FFZE),the simplest mode of FFE.Even though FFZE shows good performance in mitochondria isolation,it still encounters incomplete enrichment and a relatively low recovery.FFZE also imposes restriction on pI measurement of mitochondria.Since mitochondria are heterogeneous organelles with special features,we wonder whether a more efficient tool could provide more information on the subpopulations.In this work,we initially utilized traditional FFZE(mode 24)to isolate mitochondria from crude sample,and the optimized conditions were then performed on a novel Recycling Free-Flow Isoelectric Focusing(RFFIEF)apparatus.The RFFIEF device was independently developed and fabricated in our lab.Meanwhile,we for the first time improved the sample injection technique considering the fragility of mitochondria.The post-pH Gradient Sample Injection(post-PGSI)method was proposed to replace the common mixed sample injection,also known as pre-pH Gradient Sample Injection(pre-PGSI).Crude mitochondria of rabbit liver obtained from differential centrifugation were purified according to their pI values as six subpopulation fractions.Transmission electron microscope images revealed that intact mitochondria existed in two fractions of pH 6.24 and 6.61,degenerative mitochondria were in two fractions of pH 5.46 and 5.72,and inner membrane vesicles(IMVs)appeared in the fractions of pH 4.70 and 5.04.The membrane potential analysis confirmed that the mitochondria with higher pI values also have higher biological activity than the damaged ones in lower pI values.Particularly,proteomics analyses unveiled that 26% more proteins were detected in the intact fractions than those of IMVs,and 21% more proteins in intact mitochondria were identified than those of crude mitochondria.The significance of this work lies in:(i)the modified sample injection method greatly improved the performance of biological sample isolation;(ii)more intact mitochondria were obtained together with a high resolution between different subpopulations.Besides,high recovery was obtained in each independent trial(over 85%),which reflects the stability of our instrument;(iii)the predominance of pI measurement of mitochondria during the separation process might provide useful references for related study,and expands the application of RFFIEF.