The Applications Of Fe₃O₄/TiO₂ Composites-Electrospray Ionization Mass Spectrometry In The Rapid Analysis Of Phospholipids In Single Cell

Cell is the basic structural and functional unit of living biological system.Because of the heterogeneity of cell,the changes and differences of metabolites among different cells and even different subcellular compartments may lead to the variability and differential susceptibility of biological organisms.Therefore,there has been a growing focus of modern scientific research in the qualitative and quantitative analysis of components and metabolites from single cells.Phospholipids?PLs?,as important component of the basic skeleton of cell membrane,play an important role in many important life activities such as signal transduction,energy fueling,cell proliferation and apoptosis.The deregulations of phospholipids metabolisms may cause a variety of pathophysiological processes?diabetes,Alzheimer's disease and cancer,etc.?.Therefore,it is necessary to analyze and quantify the phospholipids metabolites at the single cell level.In recent years,various analytical methods including electrophoresis,liquid chromatography-mass spectrometry and mass spectrometry have been known for the analysis of phospholipids in complex biological samples.However,these analytical methods are difficult to meet the needs of fast,high-throughput,high specificity and high sensitivity analysis of phospholipids in micro-volume biological samples due to the disadvantages of complex sample pretreatments and large sample consumption.With the development of metal oxide affinity materials?MOA?,it is possible to rapidly and specifically enrich phospholipids in micro-volume samples.In this paper,a novel method was developed for the rapid and sensitivity qualitative and quantitative determination of phospholipids in single cells based on the characteristics of easy separation and selective enrichment of phospholipids by the Fe₃O₄/TiO₂ composite material combined with electrospray ionization mass spectrometry?Fe₃O₄/TiO₂-ESI-MS?.The main contents of this paper are as follows:1.A brief introduction to the structure,category and main functions of phospholipids.The existing analysis methods of phospholipids and their deficiencies in complex biological samples are summarized.2.The principle characteristics,device structure and analysis performance of Fe₃O₄/TiO₂-ESI-MS technology were studied.3.Fe₃O₄/TiO₂-ESI-MS was applied to the study of phospholipids metabolism at single cell levels.In this study,we have achieved the differentiation of 6 types of cancer single cells by analyzing single cell-specific metabolites?phospholipids?using Fe₃O₄/TiO₂-electrospray ionization mass spectrometry?Fe₃O₄/TiO₂-ESI-MS?combined with partial least squares discriminant analysis?PLS-DA?.In this experiment,the parameters such as the ratio of eluent,amounts of Fe₃O₄/TiO₂composites,adsorption time and elution time were optimized.Under the optimal experimental conditions,eighteen phospholipids in single cell samples were detected and identified by MS/MS experiments.PLS-DA score chart revealed that the six types of single cells were clearly differentiated.The proposed method without complicated pretreatment of single-cell samples,has the advantages of high analysis speed,high selectivity and minimal sample consumption,which provides a new analysis method for the study of phosphorylated substances in single-cell samples.4.The Fe₃O₄/TiO₂-ESI-MS was applied to the quantitative analysis of phospholipids in single cells.In this study,phospholipids in single cells were selectively enriched and easily separated using the magnetic Fe₃O₄/TiO₂ composites,and then rapidly quantitative determined coupled with electrospray ionization mass spectrometry?ESI-MS?.The limits of detections?LODs?were 0.012?g/L for PC?34:1?and 0.014?g/L for PC?36:2?in PBS matrix,with the linear range of 0.05-50?g/L?R²?0.999?.This proposed method also provides good recoveries?PC?34:1?:95.90-104.00%,PC?36:2?:94.90-103.00%?with the relative standard deviations?RSDs?less than 6.92%.In addition,quantitative determination of PCs in real unicellular samples was also achieved,with the concentration of 1.82-2.11?g/L for PC?34:1?and 1.25-1.65?g/L for PC?36:2?in six types of single cell.The experimental results demonstrated that Fe₃O₄/TiO₂-ESI-MS contains the advantages of high selectivity,high sensitivity and low limit of detection,which provides an effective method for the quantitative determination of phospholipids in micro-volume biological samples.