Study On Membrane Proteins And Proteomics Of The Rat Temporal Cortex And Hippocampus During Pregnancy And Lactation

The proteins on the biofilm are named membrane proteins.Membrane proteins of cells are the key components for the exchange of substances between cells and the surrounding environment and for the maintenance of physiological and important biological functions,such as signal identification and transduction,ion exchange,material transport,maintenance of cells and tissue structures,etc.Most membrane proteins are embedded within the lipid bilayer in different degrees,and the inner part of the membrane is composed of some non-polar amino acids,which is closely bound to the membrane.The outer membrane is composed of hydrophilic amino acids with high polarity.Although the traditional anionic surfactants SDS can denature and dissolve most of the membrane proteins,SDS is not easily removed during the subsequent sample processing,and the ion response strength is inhibited,which is not conducive to the detection of peptides.The critical micelle concentration?CMC?of the non-ionic surfactant Triton-X is relative lower,and the isolation of membrane protein is not very satisfied.The present study established a method based on TM-PEK kit extraction and nanoLC-MS/MS technology to analyze the rat membrane proteins from the rat temporal cortex.The extractive effects of two extraction buffers?extraction buffer 2A and extraction buffer 2B?in the TM-PEK kit on the cortex membrane proteins of neonatal and adult rats were compared.The health condition of women during pregnancy and lactation has been a subject of great concern.Women during pregnancy ang lactation have significant changes in behavior and physiology in comparion with the women in non-pregnancy.SD Rats grow fast and easy to feed.They are often used in the experiment of medical research,drug safety evaluation,life sciences and investigation involved in growth and development.In theory,the hippocampus and the temporal cortex of rats during the pregnancy and lactation should have great changes in body conditions in comparion with the rats in non-pregnancy.The present study also aimed to observe the expression of proteins in the hippocampus and the temporal cortex of rats during the pregnancy and lactation using tandem mass tags?TMT?.Part one The establishment of membrane proteomics method to analyzetemporal cortex in neonatal and adult ratsObjective:To establish a membrane proteomics method using Novagen Proteo Extract extraction and Nano LC-MS/MS technologies,and to analyze the difference of membrane proteins from temporal cortex tissue between neonatal and adult rats.Methods:The rat cerebral cortical membrane proteins were extracted by two extract buffers?extraction buffer 2A and extraction buffer 2B?in the Novagen Proteo Extract Transmembrane Protein Extraction Kit?TM-PEK?.After enzymatic hydrolysis by the filter aided proteome preparation?FASP?and membrane protein analysis by nano-liquid chromatography coupled with Orbitrap Fusion mass spectrometer,the proteins were identified by Proteome Discover software.The hydrophobicity,transmembrane status and bioinformatics of identified proteins were also analyzed.Results:1.The Nano LC-MS/MS identification results of the two membrane protein extractions are as follows.According to the Proteome Discover 2.1software search database,a total of 489 proteins and 1108 PMSs?Peptide matched spectra?were identified by the extraction buffer 2A,and 2590proteins and 8322 PMSs were identified by the extraction buffer 2B.2.The physical and chemical properties of the two extractions are as follows.Most of the MW of the identified proteins was in the range of 20-40kDa,and their pI distribution was between 5-7 and 8-9.In comparion with the extraction buffer 2A,the protein identified by the extraction buffer 2B was widely distributed in the range of MW and pI.The number of proteins identified as positive value of GRAVY by the extraction buffer of 2A and the extraction buffer 2B were 30?6%?and 272?11%?,respectively.In comparion with the extraction buffer 2A,extraction buffer 2B had higher identification efficiency for different hydrophobic proteins.There was a significant difference in TMDs distribution of the extracted proteins between the two extraction buffers,and the proteins extracted by extraction buffer 2B contained more transmembrane regions?proteins with TMDs more than 14existed only in the extract buffer 2B?.In comparion with the extraction buffer2A,the extraction buffer 2B was more suitable for enzymatic hydrolysis and identification of hydrophobic proteins with multiple transmembrane domains.3.The number of membrane proteins?1233?of adult SD rats extracted and identified by extraction buffer 2A was significantly greater than that of neonatal rats?277?.The KEGG signal transduction pathway analysis showed that the differentially expressed proteins in adult rats were significantly enriched in the proteasome(P=1.2E^-21),synaptic vesicle cycle(P=4.0E^-13),carbon metabolism(P=1.6E^-13)pathway and pyruvate metabolism(P=7.0E^-8)pathway.The differentially expressed proteins in the neonatal rats were significantly enriched in the spliceosome(P=3.4E^-11)and ribosome(P=1.7E^-6)pathway.4.The membrane proteins of adult rats extracted and identified by extraction buffer 2B were 1679,and the membrane proteins in neonatal rats were 1708.The KEGG signal transduction pathway analysis showed that the differentially expressed proteins in adult rats were significantly enriched in the GABAergic synapse(P=2.6E^-7),glutamatergic synapse(P=8.0E^-7),and dopaminergic synapse(P=1.8E^-5)pathway.The the differentially expressed proteins in neonatal rats were significantly enriched in the RNA transport(P=4.4E^-14),protein processing in endoplasmic reticulum(P=7.0E^-6)and ribosome biogenesis in eukaryotes(P=6.0E^-4)pathway,which were associated with protein synthesis.Conclusion:The study established a membrane proteomics method using Novagen ProteoExtract extraction and Nano LC-MS/MS technologies to analyze the difference in membrane proteins of temporal cortex tissue between neonatal and adult rats.There were much more proteins related to the synapses of the brain neurons in adult rats in comparion with the neonatal ones,whereas the protein synthesis was more active in the neonatal rats.Part two Analysis of membrane proteomics of rat temporal cortex andhippocampus during late pregnancy and at the end of lactation.Objectives:To establish a membrane proteomics methodusing Percoll density gradient centrifugation and Nano LC-MS/MS to analyze the difference of membrane proteins from the rat temporal cortex and hippocampus during the late pregnancy and at the end of lactation.Methods:The rat brain membrane proteins were extracted by extraction buffer 2B from the rat temporal cortex and hippocampus,and TMT?Tandem Mass Tags?was used to quantitatively mark the proteins.After membrane protein analysis by nano-liquid chromatography coupled with Orbitrap Fusion mass spectrometer,the proteins were identified by Proteome Discover software.The bioinformatics of identified proteins were also analyzed.Results:1.The differentially expressed proteins obtained from the hippocampus between the rats during the late pregnancy and the rats at the end of lactation are as follows.There were 236 proteins were identified in the two kind of rats,439 proteins were identified only in the rats during the late pregnancy,and1007 proteins were identified only in the rats at the end of lactation.The differentially expressed proteins obtained from the temporal cortex between the rats during the late pregnancy and the rats at the end of lactation are as follows.There were 318 proteins were identified in the two kind of rats,930proteins were identified only in the rats during the late pregnancy,and 558proteins were identified only in the rats at the end of lactation.2.The KEGG signal transduction pathway analysis showed that the differentially expressed proteins obtained from the hippocampus in the rats during the late pregnancy were significantly enriched in the oxidative phosphorylation pathway(P=1.03E^-4).The differentially expressed proteins obtained from the hippocampus in the rats at the end of lactation were significantly enriched in the oxytocin signaling pathway(P=6.78E^-4),the oocyte meiosis?P=0.003?and the Wnt signaling pathway?P=0.006?.3.The KEGG signal transduction pathway analysis showed that the differentially expressed proteins obtained from the temporal cortex in the rats during the late pregnancy were significantly enriched in the estrogen signaling pathway(P=1.03E^-4)and oxytocin signaling pathway?P=0.005?.The differentially expressed proteins obtained from the temporal cortex in the rats at the end of lactation were significantly enriched in the progesterone-mediated oocyte maturation?P=0.005?and prolactin signaling pathway(P=1.43E^-4).Conclusion:There were significant difference in the expressed proteins obtained from the temporal cortex and hippocampus between the rats during the late pregnancy and the rats at the end of lactation.The differentially expressed proteins obtained from the temporal cortex in the rats during the late pregnancy were significantly enriched in the estrogen signaling pathway.The differentially expressed proteins obtained from the temporal cortex in the rats at the end of lactation were significantly enriched in prolactin signaling pathway,and that from the hippocampus in the same rats were significantly enriched in the oxytocin signaling pathway and the Wnt signaling pathway.