Preliminary Study Of Comparative Proteomics Between Sensory And Motor Neurons

ObjectiveUsing the isobaric tags for relative and absolute quantitation(iTRAQ)coupled with online two-dimensional liquid chromatography tandem mass spectrometry(2D LC-MS/MS)protemics technique to compare the protein profile of sensory and motor neurons in the rat.The analysis of functional classification and signal pathway of differentially expressed proteins by bioinformatics analysis provide theoretical and experimental basis for the search for studying mechanism of neural chemotaxis growth.Methods1.The dorsal root ganglions and spinal cord of the same fetus in a 13.5d embryo were taken to obtain sensory and motor neurons.Immunopanning and gradient centrifugation are used to obtain purified sensory and motor neurons respectively.The culture protocols of sensory and motor neuron are established,and the purity of neurons was identified by flow cytometry with related specific antibody labels and ICC.2.The total proteins of sensory and motor neuron total protein were extracted,and the BCA method was used to detect the protein concentration.Silver staining was performed after SDS-PAGE electrophoresis to detect protein quality.3.After precipitation by acetone?enzymatic hydrolysis and iTRAQ labeling,the protein samples were separated by HPLC(UltiMate 3000-HPLC)and detected by mass spectrometry,the data was collected.4.Differentially expressed proteins were obtained by quantitative analysis of proteins obtained by mass spectrometry detection,and differentially expressed proteins were subjected to functional classification and subcellular localization using bioinformatics methods.5.The expression of some different proteins was verified by Western blot,IHC and ICC.6.By means of bioinformatics analysis of signal transduction pathways for differentially expressed proteins,a significant signaling pathway that is differentially expressed is screened out.7.By adding Sema3 A recombinant protein in sensory and motor neurons to detect the changes of neurite length and number and study different effects of Sema3 A on sensory and motor neurons.Results1.Purified sensory and motor neurons were obtained.The expression of NF in sensory neurons and ChAT expression in motor neurons were detected by flow cytometry.The results showed that the purity of sensory and motor neuron were both above 90%,and immunocytochemistry was used to identify purity.The results showed that sensory and motor neurons were of good purity2.After SDS-PAGE electrophoresis and silver staining,protein bands are clear.The result shows that extracted protein of sensory and motor neuron samples were of good quality.3.A total of 3005 proteins were identified(Unused ? 1.3(Confidence ? 95%),FDR <1%),of which 2986 proteins have quantitative information.A total of 227 differentially expressed proteins were analyzed.The sensory/motor ratio was 2 or more times,that is,there were 122 high-expressing proteins in sensory neurons,0.5,that is,high-expressed proteins in motor neurons,and about 7% of differentially expressed proteins,suggesting that there is a big difference between the two kinds of neurons.4.Biological function classification of sensory and motor neuron differential protein shows that highly expressed sensory neurons involved in mitochondrial energy,lipid biosynthesis,protein synthesis,cytoskeleton,receptors and signals,cell cycle,cytoplasmic vesicles and so on;motor neurons are involved in metabolism,RNA processing,chromatin modification,vesicles,axons and cytoskeleton,receptors and signals,Cell cycle,protein folding and modification,material transport and so on.By the subcellular localization of highly expressed proteins in sensory and motor neurons,61% proteins in highly expressed sensory neurons are located in the cytoplasm,17% in the plasma membrane and 15% in the nucleus;and 50% of the highly expressed proteins in motor neurons are localized in the cytoplasm,32% in the nucleus,14% in the plasma membrane.5.Western blot analysis of some of the differentially expressed proteins showed that Neuropillin1(Nrp1),Advillin,Annexin V,Neurofilament Medium(NF M),Neurofilament Light(NF L),Peripherin are higly expressed in sensory neurons;Glia Maturation Factor Beta(GMFB)and Neural Cell Adhesion Factor(NCAM)are highly expressed in motor neurons,results were consistent with mass spectrometry test results.6.ICC shows that NF M,Advillin and PlexinA1 were higher expressed in sensory neurons than that of motor.7.IHC shows that NF M was higher expressed in DRG than that of anterior horn of spinal cord.8.The signal transduction pathways of sensory and motor neuron differential proteins have shown that there are more interactions and roles between Sema3 A and its receptors in differentially expressed pathways related to axon guidance.8.Sema3 A recombinant protein acts on sensory and motor neuron respectively,the numer of average neuritis and the reduced length are higher in sensory neurons than that of motor neurons.Conclusion1.Based on the establishment of sensory and motor neuron purification methods,iTRAQ conjugated 2D LC-MS/MS quantitative proteomics technology was used to compare the protein expression profiles of sensory and motor neurons.A total of 227 differentially expressed proteins were found through bioinformatics analysis.Among them,there were 122 highly expressed proteins in sensory neurons and 105 highly expressed proteins in motor neurons.Signaling pathway analysis of differentially expressed proteins revealed that they interact with SemaA and its receptors in axon guidance-related signaling pathways.2.Sema3 A may have selective differences in sensory and motor neuron and have a stronger effect on sensory neurons.