The Proteomic Research On The Peripheral Blood And The Amygdala Of The CNS

Part One The Proteomic Research on Human Plasma by Integrating Polyethylene Glycol Fractionation and Immunoaffinity DepletionBackground The enormous depth complexity of the human plasma proteome poses a significant challenge for current mass spectrometry-based proteomic technologies in terms of detecting low-level proteins in plasma,which is essential for successful biomarker discovery efforts.Typically,a single-step analytical approach cannot reduce this intrinsic complexity.Current simplex immunodepletion techniques offer limited capacity for detecting low-abundance proteins,and integrated strategies are thus desirable.In this respect,we developed an improved strategy for analyzing the human plasma proteome by integrating polyethylene glycol(PEG)fractionation with immunoaffinity depletion.Methods 1.The whole-plasma proteins were precipitated with using PEG at various concentrations(4,8,12,16,20,24,and 30%).2.After precipitation,the protein samples were immunodepleted of seven plasma HAPs.The flow-through fractions were collected and applied to a liquid chromatography(LC)-MS/MS-based plasma proteome.3.Data analysis,and assess the efficiency of the integrate strategy.Results 1.Human plasma proteins were effectively fractionated by PEG.Fibrinogen group were fractionated by 4% PEG,Ig G group group were fractionated by 12% PEG,albumin group were fractionated by 30% PEG.2.PEG fractionation effect the distribution of the immunodepleted plasma proteins.In the BF image,most protein spots were spread over a relatively high MW range(>45.0 k Da)and a p I range of 5–8,while the protein spots in the CF image tended to reside within a p I range of 4–7.3.A side-by-side comparison indicated that our integrated strategy was increased by average 43.0% in the identification rate of low-abundance proteins,relying on an average 65.8% increase of the corresponding unique peptides.Conclusion The newly developed strategy of incorporating PEG fractionation to immunodepletion methods could effectively alleviate the signal suppressive effects of the major high-abundance proteins,thereby greatly enhancing the detection of low-abundance proteins.Part Two Proteomic Analysis of Human Serum Albuminome by Integrated Use of Quick Crosslinking and Two-Step PrecipitationBackground Depletion of high-abundant proteins such as human serum albumin(HSA)is a routine first step in blood-based proteomics.However,HSA-binding peptides and proteins,collectively called the albuminome and including potential biomarkers,can be lost during the depletion steps.Affinity-and chemical-based methods are usually employed to extract HSA-enriched fractions;however,these methods remain technically challenging.Herein,we report the development of a two-step precipitation(TSP)method by combined use of the readily available inexpensive reagents polyethylene glycol(PEG)and ethanol.Methods 1.Quick fixation of human serum samples was initially performed by formaldehyde of different concentrations.2.HSA-enriched fractions were collected by ethanol of of different concentrations,and the bested concentration of ethanol was assessed by immunoblotting.3.Bioinformatics analysis and network-based analysis of human serum albuminome.Results 1.The best crosslink condition is a 5-s crosslinking period in 10% formaldehyde.2.The Ig G was depleted when the concentration of PEG is 12%,and the serum albuminome can be produced by simply using ethanol concentrations of 57% and 60% after precipitation with 12% PEG4000 and PEG6000 respectively.3.A total of 171 proteins excluding HSA were identified from the fraction obtained with FC-TSP.Further interaction network analyses revealed 125 HSA-interacting proteins,including 21 direct and 104 indirect binders.The number of low-abundance proteins was 28,in which 12 were identified only by our strategy.Conclusion The new strategy has the potential to effectively survey the human albuminome,especially low-abundance proteins of clinical interest.Part Three Proteomic Analysis in the Amygdala of Rats Susceptible to Chronic Mild StressBackground The enormous depth complexity of the human plasma proteome poses a significant challenge for current mass spectrometry-based proteomic technologies in terms of detecting low-level proteins in plasma,which is essential for successful biomarker discovery efforts.Typically,a single-step analytical approach cannot reduce this intrinsic complexity.Current simplex immunodepletion techniques offer limited capacity for detecting low-abundance proteins,and integrated strategies are thus desirable.In this respect,we developed an improved strategy for analyzing the human plasma proteome by integrating polyethylene glycol(PEG)fractionation with immunoaffinity depletion.As a part of the limbic system,amygdala is involved in emotional responses,cue-associated memory and memory reconsolidation processes.Currently,many clinical and preclinical studies of depression have demonstrated multiple changes in critical brain regions including the hippocampus and prefrontal cortex,but the researches about amygdala are relatively few.Accumulating evidence shows that stress-induced structural and functional impairments in the hippocampus and prefrontal cortex are very different from the amygdala.The molecular mechanisms underlying amygdalar hyperactivity in depression remain unclear.Methods 1.Establish animal models by an 8-week CMS procedure,and identified susceptible and insusceptible subpopulations by the SPT and FST assessments.2.Differential proteomes in the amygdala were analyzed based on isobaric tags for relative and absolute quantitation(i TRAQ)labeling combined with mass spectrometry.3.Immunoblots were performed about the interesting proteins,and cell models were constructed to explore the potential molecular mechanism.Results 1.In the SPT assessment,8-week exposure to CMS resulted in significantly decreased sucrose preference of the susceptible group when compared to the control and insusceptible groups.In the FST assessment,compared with the control and insusceptible groups,the susceptible group had a greater immobility time.2.By the quantitative proteomic screening,a total of 2562 distinct proteins were identified,102 were differentially expressed,of which 25 synapse-associated proteins participated in biological processes underlying synaptic plasticity.3.Immunoblot analysis identified changes in NMDA and AMPA receptors,suggesting that CMS perturbs glutamatergic transmission in the amygdala.The expression levels of PSD-95 and Ca MKIIβ,which involved in the regulation of glutamatergic signaling,were significantly increased in the susceptible group.Additionally,an increase in VGlu T1,which is involved in glutamate uptake,was observed in both stressed groups.Conclusion The CMS susceptibility-related factors(including Glu N2A/B,PSD-95 and Ca MKIIβ)are potential antidepressant targets.Our findings should contribute to a better understanding of amygdalar synaptic plasticity in depression.