The Nondenaturing Proteomic Analysis Of Rat Plasma: A Global Identification And The Establishment Of Visible Nondenaturing Protein Dataset

Proteins are physiological function performers and direct reflectors of biological phenomena,which determine the important role of studies on protein structures,functions and protein-protein interaction in demonstrating changing mechanisms when living entities in the state of physiology or pathology.Most animal disease models of relevant studies on preclinical pharmacology,pharmacological effect and drug toxicity in the procedure of research and development of drugs rely on rats.In addition,change of component species and contents of plasma are major biomarkers in clinical test benefit from availability and stability of plasma,which make studies on rat plasma proteomics significant.This paper was first globally identified plasma proteins of rats separated by twodimensional non-denaturing gel electrophoresis.Using the analytic method of quantitative LC/LC-MS,we obtained the species and amount of proteins on which gel spots were stained with CBB.In 136 gel spots,199 non-redundant proteins were detected,and almost every spot contained multiple proteins,nearly 40 percent proteins were detected in several spots,it was demonstrated that double complexity of overlapping location and polymorphic distribution of proteins.Thus,we reconstructed the distribution of every protein in 136 gel spots using qualitative and quantitative information from analysis of MS/MS,and we obtained 199 nondenaturing protein maps.Comparing maps showed that every protein had unique distribution on gel spots,and one protein could be separated from others through location of gel spots(isoelectric points and molecular weight)and different amount of proteins,and these protein distributions should be directly related to the structure and interactions of proteins under physiological conditions.Not only the global analysis of rat plasma protein non-denaturing two-dimensional gel electrophoresis have been done the first time,and 199 non-denatured protein spectra consist the data set of rat plasma protein,which makes distribution of protein content visualized.All of this will be valuable for the studies which use rats as animal model.In addition,the data of nondenaturing spectrum also contains a lot of information about protein-protein interaction,bioinformatics and statistical analysis work should be integrated in data mining process,some work of this part also have been done in this paper.In details,global identification and nondenaturing protein maps drawing of rat plasma proteins comprised of three parts that were performed sequentially,namely,1)preparation of rat plasma sample followed with protein separation by nondenaturing two-dimensional electrophoresis;2)number,calculate apparent molecular weight and isoelectric spots of the 136 gel spots on the nondenaturing micro two-dimensional electrophoretic gel,follow with gelcutting,destaining,reduction,alkylation and in-gel trypsin digestion.Acquired Peptides were analyzed by nano ultra-performance liquid chromatography-tandem mass spectrometry(nanoUPLC-MS/MS)and obtain label free quantitativeanalysis results after processing of proteomic software.The results showed that 199 species of proteins were detected in all 136 gel spots.There was one to tween five kinds of proteins(average about eight kinds)on single protein spots.Quantity information of each protein distributed from one to ninety four gel spots;3)program Excel Visual Basic(VB)macros to draw the distribution of each protein in the grid cutting area which were reconstructed as a color density pattern.In total 199 native protein maps were obtained.Every protein spectrtum showed specific protein location and quantity distribution in the non-denaturing gel.Visualization and bioinformatic analysis of these information are valuable to deeply dig protein-protein interaction and function analysis.Based on above work,we had a primary try to apply the data sets of rat plasma:(1)analysis of rat plasma proteins in models of C-reactive protein knock-out and induced left ventricular hypertrophy.Benefiting from the existence of rat plasma datasets,we realize fastersearching of targeted protein spots or differential protein spots.(2)Building up research approaches to carry out relevant analysis between nondenaturing protein spectrums and prediction methods to screen protein-protein interaction pairs.(3)Through primary comparison with the results of human plasma,finding out several different protein distribution spots between above two species,which may provide extra thought and reference datasets to study on relevant human diseases based on animal model of rats.