The Research Of Urinary Protein Biomarkers Of Metabolic Syndrome With Early Renal Injury By Quantitative Proteomic Technique

Objective:To optimize acetone precipitation method by studing factors influencing acetone precipitation.Methods:(1) First morning urine samples were collected from healthy male (6people) and female (6people) donors. Urine samples were pooled together intra-gender.(2) Groups:①Samples were divided into two groups according to whether or not to add preservatives.②According to different thawing methods, samples were divided into two groups.④According the volume of acetone, samples were divided into two groups.④Samples were divided into five groups according to mixing time.(3) Major steps:thaw urine samples, add iced-acetone and mix, centrifuge at4℃, add1mL lysis buffer into precipitation, centrifuge for10minutes at4℃, and store the supernatant at-80℃. Bradford method was used to measure the concentration of urinary protein, and SDS-PAGE was used to evaluate the availability of inter-group differences in protein.Results:1. Comparing to1:1, when urine and acetone were at1:3volume ratio, the urinary protein concentration extracted was higher.2. Urinary protein concentration increase with the time.3. Whether thawed at37℃or at room temperature, urinary protein can not be affected.4. Preservatives did not significantly affect urinary protein extracted by acetone precipitation method.Conclution:We optimized acetone precipitation method:Adding preservatives had no significant effect on the acetone precipitation; Urine samples can be dissolved at37℃; The volume ratio of urine and acetone should be1:3; The mixture of urine and acetone should be placed at4℃overnight. Objective:To establish a rapid and simple urinary protein adsorption method for proteomic research:nitrocellulose membrane adsorption method, which can also store proteins of large amount of urine samples.Methods:(1) First morning urine samples were collected from healthy male (2people) and female (2people) donors. Urine samples were pooled together. The pooled urine was divided into the average of20mL per tube, and stored at-80℃.(2) Acetone precipitation method was used to precipitate proteins from20mL urine, and this was repeated three times.(3) In the ice bath, proteins were adsorbed by NC membrane for1min,10min,30min,1h,2h and15h from20mL urine each. For each time period, studies were repeated three times.(4) Proteins were adsorbed by NC membrane for lmin from20mL urine, and the NC membrane with urinary proteins was stored at-80℃for six months.(5) Bradford method was used to measure protein concentration, and the t-test was applied to compare the amount of proteins among acetone precipitation method and NC membrane adsorption method.(6) SDS-PAGE was used to preliminary compare of protein patterns among acetone precipitation method and NC membrane adsorption method.(7) LC-MS/MS method was applied to identify proteins enriched by acetone precipitation method and NC membrane adsorption method and that stored by NC membrane.Results:1. Urinary protein can be quickly absorbed by NC membrane. The amount of proteins absorbed by NC membrane for1min-2h was similar (P>0.05), and that absorbed by NC membrane for15h was most (P<0.05). Compared to that absorbed by NC membrane for lmin-2h, the amount of proteins enriched by acetone precipitation method was higher (P<0.05), and the the amount of proteins enriched by acetone precipitation method was similar with that absorbed by NC membrane for15h (P=0.953).2. Results of SDS-PAGE showed that, compared with acetone precipitation, the NC membrane adsorption method contains the major protein bands of acetone precipitation, but a significant reduction in albumin enrichment.3. By LC-MS/MS analysis, total of628,619and323proteins were identified in acetone precipitation, NC adsorption for lmin and NC adsorption for15h, and the reproducibilities of the three groups were82.7%,76.7%and60.5%.4. There is no obvious difference between acetone precipitation enrichment and the NC membrane adsorption method in protein molecular weight and isoelectric point.9. After stored in NC membrane for6months, a total of656urinary proteins were identified by LC-MS/MS, and the reproducibility was80.2%, which was similar to resules of fresh urinary proteins.Conclution:1. We established a simple and rapid urinary protein adsorption method:NC membrane adsorption method.2. This method is suitable for handling large-scale clinical urine samples.3. This method can be used to store a large number of urinary protein samples. Objective:With the development of proteomic techniques, many clinical diseases were found to induce changes in the urinary proteome. Due to the great variations in the urinary proteome, it is especially important to define the minimal sample size for clinical research.Methods:In this study1DL-MS/MS was used to analyze overnight urinary samples from20healthy volunteers (10males and10females). To obtain a comprehensive analysis of the urinary proteome, replicate1DLCMS/MS runs were performed for each sample. The minimal sample size for a group was estimated by statistical analysis.Results:When the sample size for a male/female group exceeded9, less than5%new proteins/peptides were identified by adding a new sample to the existing sample pool. When the group sample size reached10, the newly identified proteins/peptides percentage was less than5%upon adding a new sample, and statistical analysis indicated that urinary proteomes from males and females showed different patterns.Conclusion:To obtain a comprehensive analysis of the human urinary proteome, the recommended minimal sample size is10, and the number of male and female samples had better be balanced. Objective:To assess the iTRAQ technology, to generate urine proteome profiling of MetS with early renal injury based on LC-MS/MS method, to find potential urine biomarkers by spectral counting and iTRAQ quantitative proteomic approaches, and to explore the pathogenesis of MetS and its early renal damage by analyzing functions of differential proteins.Methods:1. The establishment and assessment of iTRAQ method:The reagents of114,115,116and117were used to lable the mixture of four known molecular weight fpeptides; Labeled samples were mixed at the ratio of1:1:1:1and1:2:4:6; MALDI-TOF/TOF method was applicated to test the proportion between114-117.2. Patient selection:Urine samples were collected from epidemiologic study of MS and renal involvement in Pinggu district, Beijing during the period of2008and2009. MS was diagnosed by IDF criteria, while MetS patients with early renal injury were defined as efined as20μg/min≤UAE<200μg/min and eGFR≥60mL/min.1.73m2. Participants were divided into three group:group I (healthy subjects), group Ⅱ (MetS patients with normoalbuminuria) and group III (MS patients with microalbuminuria).3. Urine collection and preparation:Overnight urine were collected from three groups. Acetone precipitation was used to precipitate urinary proteins. Equal amount of6protein samples intra-group were mixed together. After mixture, proteins were digested by trypsin and the salt were removed from peptides.4. iTRAQ labelling:Equal amount of peptides in three groups were mixted together to be intral contral. Intral contral, group Ⅰ, group Ⅱ and group Ⅲ were labeled by iTRAQ reagent labels114,115,116and117, and were mixed together at the ratio of1:1:1:1.5. Protein identification:Proteins of three groups and proteins in mixed samples labeled by iTRAQ were identified by LC-MS/MS.6. Relative quantification of proteins:Spectral counting approach was used to quantify free labeled proteins; Relative intensity of different isotope-labeled proteins were compared in iTRAQ method.7. Definition of differencial proteins:ANOVA was used to compare differentially expressed proteins among three groups in spectral counting and iTRAQ labeling approaches. Differentially expressed proteins identified in both methods were considered to be candidate protein biomarkers of the MetS early kidney damage.8. Analysis of protein function:Panther software was applied to analyze protein functions.9. Protein validation:Western Blot method was applied to validate differentially expressed proteins CP, APOA4and EGF; Image J software was used to measure protein expression intensity in images.Results:1. By MALDI-TOF/TOF scan, the ratio between iTRAQ report groups114,115,116and117were similar with1:1:1:1and1:2:4:6.2. In label-free method, total of807,630,456proteins were identified respectively in group Ⅰ, group Ⅱ and group Ⅲ, and a total of566proteins were identified in mixed iTRAQ labeled sample.3. In spectral counting method, there are89differentially expressed proteins among group I and group Ⅱ and106among group Ⅱ and group Ⅲ; In iTRAQ labeling method,165proteins were identified differentially expressed among group Ⅰ and group Ⅱ and173among group Ⅱ and group Ⅲ; In both methods,47protiens were differentially expressed among group Ⅰ and35protiens among group Ⅱ and group Ⅲ.4. Western Blot and Image J image analysis showed that the expression of APOA4in group III was significantly higher than that in group Ⅰ and group Ⅱ, and the expression of EGF in Group Ⅱ and Group Ⅲ was significantly lower than that in group Ⅰ. These results were consistent with LC-MS/MS results.5. Protein function analysis revealed that the molecular function of differentially expressed proteins among three groups mainly included binding, catalytic activity and enzyme regulator activity, they were mainly involved in metabolic processes, cellular processes, transport and immune processes, and the pathways that they participated in were blood coagulation, Wnt signaling pathway and Cadherin signaling pathway.Conclusion:1. We have established urinary proteome profiling of MetS with early renal injury identified by LC-MS/MS.2. Differencial proteins of MetS with early renal injury were identified by LC-MS/MS method in spectral counting and iTRAQ approachs.3. CP and APOA4were candidate protein biomarkers of MetS with early renal damage; EGF was a candidate protein biomarkers of MetS.