Development Of Methionine Methylation Profiling In Hunman Berast Cancer Cells Based On Metabolic Stable Isotope Laebling

One-carbon metabolism,involving the folate and methionine cycle,supports multiple physiological processes,such as the biosynthesis of lipids,nucleotides,and proteins;and the maintenance of redox,genetic,and epigenetic statuses.It is also associated with methylation reactions.Methylation of components involved in one-carbon metabolism is extremely important in investigations of various diseases and extremely important in cancer.This is especially true in the development of cancer,which can be promoted by various factors such as DNA methylation disorders and abnormal nucleotide synthesis.However,the majority of studies of methylation in cancer have focused on genomics and proteomics,but this phenomenon also has a very close relationship with metabolism.Comprehensive studies on methylation is essential and may provide us a better understanding of tumorigenesis,and lead to the discovery of potential biomarkers.The main content of this thesis includes the following three parts:1.Metabolic profiling analysis of methylated metabolitesFirst,we introduced 13CD3-Methionine as the only medium source into breast cancer growing cell cultures and extracted intracellular lipids and metabolites to improve the analytical coverage.Then a method for methylation metabolic profiling was constructed and optimized respectively.Relevant methylation labeling metabolites were found and identified through variation and comparison with their Full MS and MS/MS spectra.Pathway analysis was also applied to obtain a comprehensive view of the interconnection of methylated metabolites as well as potential metabolism disorder.Finally,20 metabolites consisting of amino acids,oligo-peptides,phosphatidylcholines,and some possible new small molecules were positively identified.The structures of some metabolites remained unknown and some metabolites have not been reported in the literature,such as S-adenosyl-(3-amino-4-oxopentyl)methyl sulfonium(SAAOMS).Based on pathway analysis,an integrative view of the potential active methyl unit metabolic pathway and relevant metabolic enzymes was established.Several methylation pathways were found possibly in disorder in breast cancer cells including histone methylation,biosynthesis of phosphatidylcholines,biosyntheisi of creatine,biosynthesis of 1-methyl nicotinamide,biosynthesis of spermidine and spermine and so on.2.Relative quantification analysis of labeled methylated metabolitesTo maintain the optimal state of labeling and cellular morphology.MDA-MB-231 cell extracts with 48 h of labeling via the same sample preparation were used as stable isotope internal standards for every corresponding CH3-metabolite and relative quantification can be rapidly performed using the differential mixing ratios of 13CD3-/CH3-cell samples based on isotope dilution assays.An optimized selected ion monitoring(SIM)assay consisting of one full MS experiment and three SIM experiments was established for the relative quantification of polar metabolite parts.Moreover,for the lipid parts,owing to their relatively high abundance,a relative quantification method was established using a full MS scan.Linearity was determined using a series of mixed 13CD3-/CH3-MDA-MB-231 cell samples at ratios ranging from 50:1 to 1:5.Accuracy was assessed using MDA-MB-231 cell samples mixed at three different ratios.The results showed that for over 80%of the metabolites,the average R-squared values were 0.99 and the average RSD values were below 20%,indicating good linearity achieved within the linear range.3.Methylation metabolic profiling analysis of human breast cancer cellsUsing the relative quantification methodology outlined above,we investigated the relative abundances of labeled methylated metabolites in five breast cancer cell lines,namely,MDA-MB-231,MCF-7,MDA-MB-468,SK-BR-3,and T47D,representing major molecular subtypes of breast cancer.We also analyzed a Homo sapiens mammary epithelial cell line(MEC),MCF-10A,to find methylation metabolites with significant differences or regular variations.The cell samples were mixed with an equal volume of,3CD3-labeled internal standard and this mixture was subjected to LC-MS for analysis.The findings showed that most methylated metabolites exhibited higher abundances in breast cancer cells than in MEC,which verified the increased methylation activities in cancer cells.The findings also showed the possession of different methylation characteristics in different cell lines,which may be due to their genetic and epigenetic changes.It is notable that 1-Methyl nicotinamide was found to exhibit significant change in this analysis,which were decreased to less than one-fifth of that in MEC.Further investigations and validation of the biological experiments are warranted to reveal the overall methylation metabolic network within breast cancer cells.