Differential Proteomic Analysis Based On 1DE,Whole-gel Slicing And Quantitative LC-MS/MS:Methodological Establishment And Applications

Proteomics is large-scale study of proteomes.With quantitative and qualitative analysis of all the proteins in a given organism(or an organ,tissue or cell),proteomics ultimately aims to gain comprehensive understanding of the composition,functions,processes and the biochemical mechanisms of the system.Differential proteomics explores physiological and pathological mechanisms by comparing the differences of a distinct proteome in different states and has been widely used for understanding disease development,therapeutic method development and clinical biomarker discovery.However,the present differential proteomic works mainly focus on detecting the differences of protein quantities,though it has been widely agreed that the functions of all the proteins and the biological processes they participitate in could be directly affected by changes of structures and protein-protein interactions.Currently,the mainstream differential proteomic analysis mostly uses the shotgun strategy,the core of which is to enzymatically resolve complex protein mixtures into peptide mixtures and perform all the analysis at peptide level,including separation,detection and differential analysis by high performance liquid chromatography-tandem mass spectrometry(LC-MS/MS).Shotgun strategy has shown very high performance in both identification and quantitation for global and in-depth proteomic analysis,but its principle would inevitably cause significant loss of information on protein structures and interactions.In this dissertation,we report on the development of a new differential proteomic strategy,1DE-LC-MS,by combining onedimensional gel electrophoresis(1DE),whole-gel slicing and LC-MS/MS analysis.1DE is the most widely used technique in protein science,providing high-resolution and high-efficiency separation of complex protein samples.As the separation is by molecular mass,a property directly associated with structure,it is possible to retrieve the structure or even interaction information(e.g.using nondenaturing separation)with appropriate subsequent analysis.Whole-gel slicing followed with quantitative LC-MS/MS guarantees total and in-depth analysis of the gel-separated proteins and reconstruction of the quantity distribution on the gel for each protein.Briefly,the strategy consists of five main steps,namely,1)separating the proteomic samples by 1DE,employing nondenaturing or denaturing conditions according to the aim of the research and the nature of samples;2)slicing the gel lanes into squares of the same size;3)treating all the gel squares with standardized procedures of in-gel digestion and quantitative LC-MS/MS analysis;4)analyzing the data for identity,quantity and differential information;5)for each detected protein,reconstructing the quantity distribution along the gel lane(named here as "native 1DE-MS blots" when nondenaturing 1DE is used,and "SDS-PAGE-MS blots " when SDS-PAGE is used),and further mining the hundreds or thousands of blottings for structure and interaction associated information.Nondenaturing 1DE-LC-MS and SDS-PAGE-LCMS methods were applied to study of human plasma/serum and focal cerebral ischemia-reperfusion(I/R)injury in rat,respectively.The major contents are as follows:1.The main procedures of the strategy were optimized to meet the requirement of differential proteomic analysis,e.g.parallel gel making,parallel electrophoresis,and simultaneous gel slicing using multi-blade cutters.The necessity of gel staining was especially examined since in this strategy all the gel lanes are equally sliced without referring to the staining pattern,and a considerable amount of labor and time could be saved if the step could be skipped.Unexpectedly,the results showed that staining with CBB was beneficial: more proteins were detected and for most proteins their quantity distribution were better reconstructed.The underlying mechanism was discussed and the staing step was kept in the subsequent experiments.Then,“native 1DE-MS blots” or “SDS-PAGE-MS blots”were plotted for proein structure-related analysis.2.Establishment of nondenatuirng 1DE-based 1DE-LC-MS method.In nondenaturing 1DE,proteins are separated with their structures and interactions retained in the gel and we expected these information would be retrieved and reflected in the final differential analysis results.We applied the method to proteomic analysis of human plasma and serum.The results showed satisfactory performance in both proteomic coverage and differential analysis.A total of 315 proteins were identified without depletion of abundant proteins.Thrity-three proteins showed significant differences between their native 1DE-MS-blots of plasma and serum.Many of the differences matched with accumulated knowledge on protein interactions and proteolysis involved in blood coagulation,complement and wound healing processes,proving the method could be used to detect structure-and interaction-associated differences for mechanism study.3.Establishment of SDS-PAGE-based 1DE-LC-MS method.SDS-PAGE is the most commonly used protein separation method in biochemical analysis and especially suitable for samples containing water-insoluble proteins.Although the interactions between proteins or polypeptide chains are disrupted with the denaturation treatment,the distribution of proteins in the gel lanes may still contain abundant structural information,e.g.presence of isoforms,function-related enzymatic cleavage and degradation.For analysis of whole tissue or cell lysates that contain many insoluble proteins,we established a differential proteomic method based on SDS-PAGE-LC-MS systematic analysis and applied it to the proteomic analysis of rat cerebral cortex in the subacute to long-term phases of focal cerebral ischemia-reperfusion(I/R)injury.In this study,totally six groups,i.e.three I/R groups(ischemia 2 hours + reperfusion 1 day,7 days,and 14 days)and three sham-operated groups were examined.The proteins of cerebral cortex were analyzed by SDS-PAGE,whole-gel slicing,and quantitative LC-MS/MS.Totally,5621 proteins were identified,among which 568,755,and 492 proteins were detected to have significant dys-regulation in the model groups with 1,7,and 14 days of reperfusion,respectively,when compared with the corresponding sham groups(n = 4,fold change ? 1.5 or ? 0.67 and p ? 0.05).Bioinformatic analysis on the functions and reperfusion time-dependent dys-regulation profiles of the proteins exhibited changes of structures and biological processes in cytoskeleton,synaptic plasticity,energy metabolism,inflammation,and lysosome.In addition,the quantity distributions on the gels were reconstructed for all the proteins and the obtained SDS-PAGE-MS blots showed many proteins had more than one form on the gel and many of the forms are related to in vivo biological processes(e.g.protein maturation,enzymatic cleavage and degradation).The results fully demonstrated the performance and unique advantages of this strategy in large-scale differential proteomic analysis.In summary,we established a differential proteomic strategy based on 1DE-LC-MS systematic analysis,and applied it to practical cases.Compared with the shotgun strategy that only focused on protein quantification,the new method we established can obtain not only the protein quantification but also protein structure and interaction associated differential information according 1DE-MS blots.We believe this work would provide new approaches to obtain comprehensive understanding of complex protein systems.