Method Development And Application Of Mass Spectrometry For Metabolic Changes Analysis Of Model Mice

Mass spectrometry is a method to separate and detect charged ions according to the mass charge ratio under the action of electric field and magnetic field.It can accurately analyze compounds qualitatively and quantitatively,and has been widely used in the detection of biological samples.Tandem mass spectrometry and mass spectrometry imaging are currently the two most widely used mass spectrometry techniques.Tandem mass spectrometry is a combination of two or more mass analyzers connected in time or space to form a combined analysis method,which can be applied to therapeutic drug monitoring,toxicology,endocrinology,pediatric and other fields.Mass spectrometry imaging technology is an imaging method based on mass spectrometry technology,which can directly scan biological samples,and obtain a heat map containing the spatial distribution information of various molecules after special software processing,so as to realize the discovery of biomarkers and the monitoring of specific compounds.This method can achieve high throughput imaging and analysis of hundreds of different molecules on a single tissue slice or tissue chip.Mass spectrometry imaging technology can be applied to clinical biomarker capture,real-time surgery implementation,3D-MSI,single-cell imaging and other fields.It is currently an important analysis technology in life science research.Based on the powerful advantages of mass spectrometry technology,we first apply mass spectrometry imaging technology to explore the metabolic changes in the brain tissue of the circadian rhythm model mouse,and conduct an in-depth analysis of potential metabolism-related molecules and their effects.We successfully constructed circadian rhythm model mouse through 12 h/12 h lighting cycle,and then confirmed the imaging parameters of mouse brain tissue through instrument calibration,DESI signal optimization,and DESI-MSI condition screening.Finally,the brain tissue sections of circadian rhythm model mouse under 6 different illumination time points were scanned locally and whole-brain.Through manual screening of DESI-MSI data,we obtained two types of ions with characteristic distribution only in SCN nuclei and no signal only in SCN nuclei.Based on the vital function of SCN in the circadian rhythm system,we consider that they are closely related to the circadian rhythm.Then,through further quantitative analysis,we acquired 15 molecules with obvious diurnal oscillation and characteristic distribution only in SCN nuclei,including L-carnitine,adenosine,phospholipids,etc.Among these compounds,the intensity of L-camitine and acetyl-L-carnitine is consistent with the circadian oscillation phenomenon reported in the literature,and is consistent with the circadian oscillation law of circadian rhythm genes BMAl1 and Clock reported in the literature.Acetyl-L-carnitine is key intermediate for ?-oxidation of fatty acids in mitochondria.They participate in the oxidation of fatty acids by transporting fatty acids from the cytoplasm to the mitochondria,and their diurnal oscillations in the SCN nucleus again confirm their crucial role in metabolism.The diurnal oscillations of phospholipid signals indicate that these phospholipid molecules are closely related to the regulation and maintenance of the central circadian rhythm.In addition,adenosine is closely linked to circadian rhythms and is involved in several important signaling pathways.Thus,the diurnal oscillations of 5-methylcytidine and adenosine,which are specifically distributed in the SCN nucleus,show the relationship between them and the circadian rhythm-related signal pathways in the brain to a certain extent,and is also provides a basis for subsequent research on nucleosides and circadian rhythm.For tandem mass spectrometry,we used triple quadrupole linear ion trap mass spectrometer to quantitatively detect endogenous neuropeptides in model mice.Firstly,we constructed a quantitative detection method for specific neuropeptide using standard substances.Then,plasma samples were pretreated by the protein precipitation method to remove most of the impurities.After that,the endogenous neuropeptides in the plasma samples of model mice were quantitatively detected by the triple quadrupole linear ion trap mass spectrometer.Through these operation,we found that there was a lot of individual variation in the OT level of the mice plasma under the dark condition,and the exposure to light for a certain period of time would make the OT level of the mice plasma remain at a higher level as a whole,that is,to narrow the individual variation,and ultimately make the OT level of the mice plasma tend to a uniform and relatively high level.On the other hand,sodium,dehydration,and the drug IMP21 can regulate the level of AVP.High concentration of sodium and dehydration can increase the synthesis and secretion of AVP,while the drug IMP21 can compensate for the increase of AVP level caused by dehydration,and IMP21 can further reduce the increase of AVP level caused by dehydration when given with water after water exclusion treatment.This phenomenon is consistent with the current clinical application of IMP21.In addition to the development and application of mass spectrometry techniques,the role of mirror M2 proton channels in chiral drugs was explored using single-channel electrophysiological techniques.We first obtained L-M2 and D-M2 by chemical synthesis,and then restored the conformation of the chemically synthesized linear protein through the constructed phospholipid bilayer.Then we verified and compared the biological and pharmacological activities of L-M2 and D-M2 by single channel electrophysiology.The results showed that the biological activity of D-M2 protein synthesized by chemical synthesis was not significantly different from that of L-M2,and the non-chiral drug Amantadine could inhibit D-/L-M2 protein at the same time,but its effect on D-M2 was slightly slow,while Rimantadine with chiral center could inhibit L-M2 protein current,but its effect on D-M2 was very slow,and its inhibition effect was affected by voltage,so we speculated that there is a competitive relationship between pressures,which is incomplete inhibition.Compared with other chemical analysis methods,mass spectrometry technology has become an important means of life science research due to its unique characteristics of high sensitivity,high resolution,and high throughput.It will also further assist the detection and analysis of biological samples by continuously optimizing its own performance to provide more accurate and intuitive results for the analysis of peptides and biological macromolecules.