古菌脂质的前处理方法比较及超高效液相色谱-高分辨质谱分析

Comparison of pretreatment methods in lipid analysis and ultra-performance liquid chromatography-mass spectrometry analysis of archaea

古菌是一类兼具真菌及细菌细胞特征、大多生活在极端环境下的单细胞微生物。由于膜结构及代谢通路的特殊性,研究古菌脂质对深入探究极端环境下生命的正常活动具有重要意义。前处理方法在组学分析中会极大影响最终结果的准确度及可靠性。该文基于超高效液相色谱-高分辨质谱联用(ultra-performance liquid chromatography-high resolution mass spectrometry,UPLC-HRMS)技术,以嗜热嗜压古菌Pyrococcus yayanosii为模式生物,采用Bligh-Dyer加酸法、Folch法、甲基叔丁基醚(methyl tert-butyl ether,MTBE)法及固相萃取(SPE)法提取脂质成分,从提取效率、重复性、歧视性等角度进行方法评价,并最终确定SPE法和MTBE法的提取重复性和提取效率最好,适合高通量古菌全脂质提取。利用SPE提取、高分辨质谱分析对P.yayanosii的常规脂质进行了全面分析,共鉴定到了1402种脂质。本研究旨在为古菌和其他极端微生物的非靶向脂质组学分析和脂质代谢研究提供方法参考。

Archaea are single-cell microorganisms,structurally and biochemically similar to bacteria and fungi.Most of them live in extreme environments,such as high salt,extremely acidic,extremely hot,and anaerobicenvironments.The membrane structure and related metabolic pathways of archaea are different from those of other microorganisms.Therefore,studying the lipid metabolism of archaea is of great significance for exploring the life activities in extreme environments.As the first step in lipidomic analysis,lipid extraction and pretreatment methods play an important role,as they influence the accuracy and reliability of the final results.We harnessed ultra-performance liquid chromatography coupled with high-resolution mass spectrometry(UPLC-HRMS)to detect the total normal lipids.The hyperthermophilic archaeon Pyrococcus yayanosii was selected as the model.The Bligh-Dyer acidic method,Folch method,methyl tert-butyl ether(MTBE)method,and solid-phase extraction(SPE)method were compared by multi-component analysis in terms of extraction efficiency,reproducibility,and extraction discrimination.Comprehensive analysis revealed that the SPE and MTBE methods showed the best extraction repeatability and extraction efficiency,and were suitable for high-throughput microbial lipid extraction.Finally,normal lipid components of P.yayanosii were comprehensively analyzed by SPE coupled with UPLC-HRMS.A total of 1402 lipid components were identified.This article aims to provide a reference for non-targeted lipidomic analysis of archaea and other microorganisms towards understanding their lipid metabolism.