Study Of Novel Mass Spectrometry Methods For Analysis Of Cellular Metabolites

Cells are the basic units of the morphological structure and life activities of the organism.Different cells have cell-to-cell heterogeneity.Single-cell analysis can detect the information carried by individual cells,which can be covered by a means result.Single-cell chemical profiling could further the current understanding of biological metabolic and life process.In recent years,mass spectrometry(MS)-based cell analysis has received extensive attention due to its high sensitivity,high throughput and chemical specificity.However,there is a big issue when using MS to analyze intracellular metabolites,especially lipids.Rich structural information of lipids in complex biological samples can now be routinely obtained,including the classes of the lipids,fatty acyl/alkyl composition,and even the sn positions of the fatty acyl/alkyl chains.The locations of the carbon-carbon double bonds(C=C)in the lipids,however,have rarely been identified using commercial MS systems,due to the complexity of the lipid structure.Furthermore,as for sampling methods of MS-based single cell analysis,solid probe sampling could be free from clogging problem,but it is difficult to further increase the sensitivity of MS.To solve the above problems,here,we designed two mass spectrometry cell analysis methods for detection of cellular metabolites.Details are as follows:1.A method of identification of C=C location and relative quantitation of unsaturated phosphatidylcholine(PC)isomers in breast cells by online photochemical reaction-pulsed directed current electrospray-tandem mass spectrometry(PB-pulsed-dc-ESI-MS/MS)was established,using benzophenone(BP)as a photochemical reactant.The C=C in the unsaturated PC and the carbonyl in BP was specifically cycled under the irradiation of 254 nm ultraviolet light(Paterno-Buchi,PB reaction).The PB products are ionized and mass-isolated for low-energy collision dissociation through the non-contact pulsed-dc-ESI ionization method.The double bond position and the relative content of the location isomers can be obtained from the resulting ions in the MS/MS.Due to the use of the new photochemical reactant BP,the PB product can be separated from the original reactant phospholipids on the mass axis in the mass spectra.In addition,the difference of the detected ions is between 150 Da,which is large and can avoid the overlap problem.The pulsed-dc-ESI ionization method is a non-contact one,avoiding the electrical contact of electrode with sample solution.Its flow rate is low and it can boost sample economy.But at the same time,it will not reduce the sensitivity.The C=C location of 8 kinds of unsaturated PCs in MCF-7 and MCF-10A was detected,and the relative contents of 4 kinds of C=C location isoforms were analyzed.The establishment of such a rapid and easy mass spectrometry method can analyze the C=C location and the relative content of location isomers,and it is expected to be a powerful tool to identify different cell states and different disease states.2.A method for detection of metabolites in single onion epidemic cell by coupling Solid-Phase Microextraction with polarization induced electrospray ionization(PIESI)was established,using a polypyrrole(PPY)coated stainless steel as sampling probe.The electrochemical corrosion method was used to obtain the metal needle with smaller tip diameter,and the PPY layer was electrodeposited at the tip of the needle by cyclic voltammetry.The microstructure was used as a small SPME device.The probe was controlled by a three-dimensional manipulator to precisely insert into target cell for the enrichment of metabolites.Afterwards,the enrichment metabolites were desorbed for a certain period of time in the glass electrode pre-incubated with the desorption solvent.The metal probe was removed and the glass electrode was placed at a distance of 10 mm from the MS inlet.The metabolites were ionized by PIESI.The detection of metabolites in single onion epidemic cell had been successfully achieved and significant differences in internal and external epidermal cells were observed.Compared with the traditional probe sampling technology,due to the modification of polypyrrole adsorbent,there is a better enrichment sampling effect.The method provides a more sensitive way of sampling single cell with a solid probe and analyzing the enrichment metabolites with MS.