应用定量蛋白质组学方法研究代谢综合征早期肾损害尿液蛋白标志物
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摘要
目的：探讨丙酮沉淀法提取尿蛋白的影响因素,优化丙酮沉淀法富集尿蛋白实验流程。
方法：(1)收集健康成年男性和女性各6例晨起第一次尿液50mL,将男性和女性的尿液标本分别混合,-80℃下保存备用。(2)实验分组：①根据是否加入防腐剂异噻唑啉酮分为两组；②根据尿液解冻方法不同分为两组；③根据尿液与丙酮的体积比不同分为两组,④根据尿液中加入丙酮后放置时间的不同分为五组。(3)主要步骤：解冻尿液标本,加入丙酮静置,4℃下离心,提取蛋白。Bradford法测量尿蛋白浓度,应用聚丙烯酰胺凝胶电泳(SDS-PAGE)较各组间蛋白有无差异。
结果：1、防腐剂对丙酮沉淀提取尿蛋白没有明显影响；2、冰冻尿液在37℃下解冻和在常温下解冻对丙酮沉淀提取的尿蛋白成分没有影响；3、尿液与丙酮体积比为1：3时提取的尿蛋白浓度高于1：1时提取的尿蛋白的浓度(P<0.05)；4、随着放置时间的延长,提取的尿蛋白浓度依次增加。
结论：我们优化了丙酮沉淀富集尿蛋白的实验流程,及尿液中是否加入防腐剂对丙酮沉淀没有显著影响,可在37℃下溶解尿液样品,按尿液和丙酮体积比1：3加入丙酮,4℃静置过夜。
目的：在尿液蛋白质组学研究中,建立一种应用硝酸纤维素(nitrocellulose, NC)膜快速富集和储存大量尿蛋白的样品处理方法。
方法：(1)收集健康男性和女性志愿者各2人晨起第一次尿液250mL,取等量尿液混合,平均分为20mL每管,—80℃冻存待用。(2)取20mL尿液,按尿液与丙酮体积比1：3在4℃下沉淀过夜富集尿蛋白,重复3次。(3)取20mL尿液,冰浴中将尿液按20mL/min循环通过NC膜1分钟(尿液一次通过NC膜的时间)、10分钟、30分钟、1小时、2小时和15小时,每个时间段重复3次。(4)取尿液20mL,按步骤(3)方法通过NC膜0分钟,将吸附有蛋白的NC膜置一80℃冻存6个月,重复3次。(5) Bradford法测量尿蛋白浓度,应用t检验的方法比较丙酮沉淀富集和NC膜各时间段吸附的尿蛋白含量。(6)应用SDS—PAGE初步比较丙酮沉淀富集和NC膜吸附的尿蛋白。(7)应用液相色谱分离串联质谱(LC-MS/MS)鉴定丙酮沉淀富集、NC膜吸附和冻存的尿蛋白。
结果：1、NC膜能够快速吸附尿蛋白,lmin-2h时蛋白量无显著差异(P>0.05),15小时时吸附的尿蛋白量最多(P<0.05)。丙酮沉淀法富集的蛋白量较NC膜吸附lmin-2h多(P<0.05),较NC膜吸附15小时无显著差异(P=0.953)。2、SDS-PAGE结果显示,与丙酮沉淀法相比,NC膜吸附法包含了丙酮沉淀的主要蛋白条带,但明显减少了白蛋白的富集。3、LC-MS/MS鉴定结果显示,丙酮沉淀法、NC膜吸附0分钟和NC膜吸附15小时三组分别鉴定到628、619和323个蛋白,三组质谱鉴定的重复率分别为82.7%、76.7%和60.5%。4、丙酮沉淀富集和NC膜吸附的尿蛋白分子量和蛋白等电点没有明显的差异。9、尿蛋白在NC膜上冰冻6个月后,共鉴定到蛋白656个,质谱鉴定的重复率为80.2%,与丙酮沉淀和NC膜吸附的新鲜尿蛋白数目相似。
结论：1、通过与丙酮沉淀法比较,我们建立了一种简便快捷的尿液蛋白吸附方法：NC膜吸附法；2、NC膜吸附法适用于处理大规模的临床尿液样品；3、NC膜吸附法可以用来储存大量尿液标本中的蛋白。
目的：随着蛋白质组学技术不断发展,我们可以通过尿液蛋白质组学研究来寻找疾病的尿液蛋白标志物。由于人类的尿液蛋白质组存在很大的动态变化,本研究通过分析正常人尿液蛋白质组来确定尿液蛋白质组学研究时所需最小样本量,这在临床蛋白质组学研究中具有重要意义。
方法：(1)样品收集：收集20名健康志愿者(10男,10女)过夜尿；(2)丙酮沉淀法富集尿液蛋白后,进行胰酶酶切；(3)应用1DL-MS/MS方法鉴定蛋白：为了全面鉴定尿液蛋白质组,对每个蛋白样品进行多次重复的1DL-MS/MS鉴定。(4)通过统计学分析,确定尿液蛋白质组学研究所需最小样本量。
结果：(1)在男性和女性组中,当样本量超过9时,每增加一个样品,新增蛋白或肽段的数目<5%。(2)在男性和女性样品混合组中,当样本量超过9时,每增加一个样品,新增蛋白或肽段的数目<5%。(3)统计学分析发现,男性和女性尿液蛋白质组表达谱有一定的差异。
结论：为了全面鉴定人类尿液蛋白质组表达谱,最小样本量可以为6例；为了达到更好的蛋白鉴定效果,样本量可以为10例；一组样本中,男性和女性样本量最好平衡。
目的：评估相对和绝对定量的等量异位标签技术(Isobaric Tags for Relative and Absolute Quantitation, iTRAQ)技术的定量效果；应用LC-MS/MS方法建立代谢综合征(metabolic syndrome, MetS)早期肾损害尿液蛋白质组表达谱；采用谱图数定量和iTRAQ标记定量两种方法鉴定MetS早期肾损害尿液差异蛋白；应用Western Blot方法验证候选蛋白标志物。
方法：(1)iTRAQ定量方法的评估：应用iTRAQ标记试剂114、115、116和117标记已知分子量的标准肽段混合物(tryptic peptide test mix4, TP4);由114-117标记后样品分别按1：1：1：1和1：2：4：6混合；应用基质辅助激光解析电离飞行时间质谱(MALDI-TOF/TOF)检测114、115、116和117之间的比例。(2)病例分组：将入选者分为健康对照(组Ⅰ：6例)、MetS合并正常白蛋白尿(组Ⅱ：6例)和MetS合并微量白蛋白尿(组Ⅲ：6例)三组。(3)尿液收集和处理：收集组Ⅰ、组Ⅱ和组Ⅲ患者的过夜尿；丙酮沉淀法富集尿液蛋白；将每组6例尿蛋白进行组内混合,后进行蛋白酶切及肽段除盐。(4)iTRAQ标记：取等量组Ⅰ、组Ⅱ和组Ⅲ酶切后肽段进行混合作为内标；iTRAQ标记试剂114、115、116和117分别标记内标、组Ⅰ组Ⅱ和组Ⅲ样品；标记后进行1：1：1：1混合。(5)质谱鉴定：应用LC-MS/MS分别鉴定组Ⅰ、组Ⅱ和组Ⅲ及iTRAQ标记后的混合蛋白样品。(6)蛋白相对定量及差异蛋白确定：采用谱图数定量法和iTRAQ标记定量法相对定量组Ⅰ、组Ⅱ和组Ⅲ中的蛋白；采用ANOVA检验比较三组间差异蛋白,应用Tukey检验进一步分析两两间差异蛋白。(7)蛋白功能分析：应用Panther软件分析两种定量方法共同鉴定到的差异蛋白的分子功能、参与的生物过程和分子通路。(8)蛋白验证：应用Western Blot方法在正常人、MetS合并正常白蛋白尿和MetS合并微量白蛋白尿患者的尿液标本中验证CP、APOA4和EGF蛋白的表达。
结果：(1)在MALDI-TOF/TOF质谱扫描中被检测到的标准多肽峰869、1262和1353均被iTRAQ试剂标记；标记试剂114-1]7标记的样品按1：1:1：1和1：2：4：6混合后,经MALDI-TOF/TOF扫描,114、115、116和117之间的比例基本符合上述比例。(2)非标记的组Ⅰ、组Ⅱ和组Ⅲ蛋白样品,分别鉴定到807、630和456个蛋白；iTRAQ标记后的混合蛋白样品共鉴定到566个蛋白。(3)通过谱图数定量,发现三组间差异蛋白共有156个,其中组Ⅰ与组Ⅱ间差异蛋白有88个,组Ⅱ和组Ⅲ间差异蛋白有64个：通过iTRAQ标记定量,发现三组间差异蛋白共有209个,其中组Ⅰ与组Ⅱ间差异蛋白有156个,组Ⅱ和组Ⅲ间差异蛋白有118个；两种定量方法,共同发现的三组间差异蛋白有73个,其中组Ⅰ与组Ⅱ间差异蛋白有37个,组Ⅱ与组Ⅲ间差异蛋白有21个。(4)蛋白功能分析结果显示,三组间差异蛋白的分子功能主要为结合功能、催化活性和酶调节活性；差异蛋白参与的生物过程主要为代谢过程、细胞过程和免疫过程；差异蛋白参与的分子通路最主要的为凝血通路。(5)Western Blot验证结果显示,CP和APOA4在组Ⅲ中的表达显著高于组Ⅰ和组Ⅱ；EGF在组Ⅱ和组Ⅲ中的表达显著低于组Ⅰ,与质谱鉴定结果相符。
结论：(1)我们应用LC-MS/MS鉴定,建立了正常人、MetS合并正常白蛋白尿和MetS合并微量白蛋白尿患者的尿液蛋白质组表达谱。(2)采用谱图数定量和iTRAQ标记两种定量两种方法鉴定到了三组之间的尿液差异蛋白。(3)通过验证,CP和APOA4可以作为MetS早期肾损害的候选蛋白标志物；EGF可作为MetS的候选蛋白标志物。
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.
方法：(1)收集健康成年男性和女性各6例晨起第一次尿液50mL,将男性和女性的尿液标本分别混合,-80℃下保存备用。(2)实验分组：①根据是否加入防腐剂异噻唑啉酮分为两组；②根据尿液解冻方法不同分为两组；③根据尿液与丙酮的体积比不同分为两组,④根据尿液中加入丙酮后放置时间的不同分为五组。(3)主要步骤：解冻尿液标本,加入丙酮静置,4℃下离心,提取蛋白。Bradford法测量尿蛋白浓度,应用聚丙烯酰胺凝胶电泳(SDS-PAGE)较各组间蛋白有无差异。
结果：1、防腐剂对丙酮沉淀提取尿蛋白没有明显影响；2、冰冻尿液在37℃下解冻和在常温下解冻对丙酮沉淀提取的尿蛋白成分没有影响；3、尿液与丙酮体积比为1：3时提取的尿蛋白浓度高于1：1时提取的尿蛋白的浓度(P<0.05)；4、随着放置时间的延长,提取的尿蛋白浓度依次增加。
结论：我们优化了丙酮沉淀富集尿蛋白的实验流程,及尿液中是否加入防腐剂对丙酮沉淀没有显著影响,可在37℃下溶解尿液样品,按尿液和丙酮体积比1：3加入丙酮,4℃静置过夜。
目的：在尿液蛋白质组学研究中,建立一种应用硝酸纤维素(nitrocellulose, NC)膜快速富集和储存大量尿蛋白的样品处理方法。
方法：(1)收集健康男性和女性志愿者各2人晨起第一次尿液250mL,取等量尿液混合,平均分为20mL每管,—80℃冻存待用。(2)取20mL尿液,按尿液与丙酮体积比1：3在4℃下沉淀过夜富集尿蛋白,重复3次。(3)取20mL尿液,冰浴中将尿液按20mL/min循环通过NC膜1分钟(尿液一次通过NC膜的时间)、10分钟、30分钟、1小时、2小时和15小时,每个时间段重复3次。(4)取尿液20mL,按步骤(3)方法通过NC膜0分钟,将吸附有蛋白的NC膜置一80℃冻存6个月,重复3次。(5) Bradford法测量尿蛋白浓度,应用t检验的方法比较丙酮沉淀富集和NC膜各时间段吸附的尿蛋白含量。(6)应用SDS—PAGE初步比较丙酮沉淀富集和NC膜吸附的尿蛋白。(7)应用液相色谱分离串联质谱(LC-MS/MS)鉴定丙酮沉淀富集、NC膜吸附和冻存的尿蛋白。
结果：1、NC膜能够快速吸附尿蛋白,lmin-2h时蛋白量无显著差异(P>0.05),15小时时吸附的尿蛋白量最多(P<0.05)。丙酮沉淀法富集的蛋白量较NC膜吸附lmin-2h多(P<0.05),较NC膜吸附15小时无显著差异(P=0.953)。2、SDS-PAGE结果显示,与丙酮沉淀法相比,NC膜吸附法包含了丙酮沉淀的主要蛋白条带,但明显减少了白蛋白的富集。3、LC-MS/MS鉴定结果显示,丙酮沉淀法、NC膜吸附0分钟和NC膜吸附15小时三组分别鉴定到628、619和323个蛋白,三组质谱鉴定的重复率分别为82.7%、76.7%和60.5%。4、丙酮沉淀富集和NC膜吸附的尿蛋白分子量和蛋白等电点没有明显的差异。9、尿蛋白在NC膜上冰冻6个月后,共鉴定到蛋白656个,质谱鉴定的重复率为80.2%,与丙酮沉淀和NC膜吸附的新鲜尿蛋白数目相似。
结论：1、通过与丙酮沉淀法比较,我们建立了一种简便快捷的尿液蛋白吸附方法：NC膜吸附法；2、NC膜吸附法适用于处理大规模的临床尿液样品；3、NC膜吸附法可以用来储存大量尿液标本中的蛋白。
目的：随着蛋白质组学技术不断发展,我们可以通过尿液蛋白质组学研究来寻找疾病的尿液蛋白标志物。由于人类的尿液蛋白质组存在很大的动态变化,本研究通过分析正常人尿液蛋白质组来确定尿液蛋白质组学研究时所需最小样本量,这在临床蛋白质组学研究中具有重要意义。
方法：(1)样品收集：收集20名健康志愿者(10男,10女)过夜尿；(2)丙酮沉淀法富集尿液蛋白后,进行胰酶酶切；(3)应用1DL-MS/MS方法鉴定蛋白：为了全面鉴定尿液蛋白质组,对每个蛋白样品进行多次重复的1DL-MS/MS鉴定。(4)通过统计学分析,确定尿液蛋白质组学研究所需最小样本量。
结果：(1)在男性和女性组中,当样本量超过9时,每增加一个样品,新增蛋白或肽段的数目<5%。(2)在男性和女性样品混合组中,当样本量超过9时,每增加一个样品,新增蛋白或肽段的数目<5%。(3)统计学分析发现,男性和女性尿液蛋白质组表达谱有一定的差异。
结论：为了全面鉴定人类尿液蛋白质组表达谱,最小样本量可以为6例；为了达到更好的蛋白鉴定效果,样本量可以为10例；一组样本中,男性和女性样本量最好平衡。
目的：评估相对和绝对定量的等量异位标签技术(Isobaric Tags for Relative and Absolute Quantitation, iTRAQ)技术的定量效果；应用LC-MS/MS方法建立代谢综合征(metabolic syndrome, MetS)早期肾损害尿液蛋白质组表达谱；采用谱图数定量和iTRAQ标记定量两种方法鉴定MetS早期肾损害尿液差异蛋白；应用Western Blot方法验证候选蛋白标志物。
方法：(1)iTRAQ定量方法的评估：应用iTRAQ标记试剂114、115、116和117标记已知分子量的标准肽段混合物(tryptic peptide test mix4, TP4);由114-117标记后样品分别按1：1：1：1和1：2：4：6混合；应用基质辅助激光解析电离飞行时间质谱(MALDI-TOF/TOF)检测114、115、116和117之间的比例。(2)病例分组：将入选者分为健康对照(组Ⅰ：6例)、MetS合并正常白蛋白尿(组Ⅱ：6例)和MetS合并微量白蛋白尿(组Ⅲ：6例)三组。(3)尿液收集和处理：收集组Ⅰ、组Ⅱ和组Ⅲ患者的过夜尿；丙酮沉淀法富集尿液蛋白；将每组6例尿蛋白进行组内混合,后进行蛋白酶切及肽段除盐。(4)iTRAQ标记：取等量组Ⅰ、组Ⅱ和组Ⅲ酶切后肽段进行混合作为内标；iTRAQ标记试剂114、115、116和117分别标记内标、组Ⅰ组Ⅱ和组Ⅲ样品；标记后进行1：1：1：1混合。(5)质谱鉴定：应用LC-MS/MS分别鉴定组Ⅰ、组Ⅱ和组Ⅲ及iTRAQ标记后的混合蛋白样品。(6)蛋白相对定量及差异蛋白确定：采用谱图数定量法和iTRAQ标记定量法相对定量组Ⅰ、组Ⅱ和组Ⅲ中的蛋白；采用ANOVA检验比较三组间差异蛋白,应用Tukey检验进一步分析两两间差异蛋白。(7)蛋白功能分析：应用Panther软件分析两种定量方法共同鉴定到的差异蛋白的分子功能、参与的生物过程和分子通路。(8)蛋白验证：应用Western Blot方法在正常人、MetS合并正常白蛋白尿和MetS合并微量白蛋白尿患者的尿液标本中验证CP、APOA4和EGF蛋白的表达。
结果：(1)在MALDI-TOF/TOF质谱扫描中被检测到的标准多肽峰869、1262和1353均被iTRAQ试剂标记；标记试剂114-1]7标记的样品按1：1:1：1和1：2：4：6混合后,经MALDI-TOF/TOF扫描,114、115、116和117之间的比例基本符合上述比例。(2)非标记的组Ⅰ、组Ⅱ和组Ⅲ蛋白样品,分别鉴定到807、630和456个蛋白；iTRAQ标记后的混合蛋白样品共鉴定到566个蛋白。(3)通过谱图数定量,发现三组间差异蛋白共有156个,其中组Ⅰ与组Ⅱ间差异蛋白有88个,组Ⅱ和组Ⅲ间差异蛋白有64个：通过iTRAQ标记定量,发现三组间差异蛋白共有209个,其中组Ⅰ与组Ⅱ间差异蛋白有156个,组Ⅱ和组Ⅲ间差异蛋白有118个；两种定量方法,共同发现的三组间差异蛋白有73个,其中组Ⅰ与组Ⅱ间差异蛋白有37个,组Ⅱ与组Ⅲ间差异蛋白有21个。(4)蛋白功能分析结果显示,三组间差异蛋白的分子功能主要为结合功能、催化活性和酶调节活性；差异蛋白参与的生物过程主要为代谢过程、细胞过程和免疫过程；差异蛋白参与的分子通路最主要的为凝血通路。(5)Western Blot验证结果显示,CP和APOA4在组Ⅲ中的表达显著高于组Ⅰ和组Ⅱ；EGF在组Ⅱ和组Ⅲ中的表达显著低于组Ⅰ,与质谱鉴定结果相符。
结论：(1)我们应用LC-MS/MS鉴定,建立了正常人、MetS合并正常白蛋白尿和MetS合并微量白蛋白尿患者的尿液蛋白质组表达谱。(2)采用谱图数定量和iTRAQ标记两种定量两种方法鉴定到了三组之间的尿液差异蛋白。(3)通过验证,CP和APOA4可以作为MetS早期肾损害的候选蛋白标志物；EGF可作为MetS的候选蛋白标志物。
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.
引文
[1]. Pisitkun T, Johnstone R, Knepper MA. Discovery of urinary biomarkers[J]. Mol Cell Proteomics. 2006;5:1760-71.
[2]. Barratt J, Topham P. Urine proteomics:the present and future of measuring urinary protein components in disease[J]. CMAJ.2007; 177:361-8.
[3]. Goligorsky MS, Addabbo F, O'Riordan E. Diagnostic potential of urine proteome:a broken mirror of renal diseases[J]. J Am Soc Nephrol.2007; 18:2233-9.
[4]. Lee RS, Monigatti F, Briscoe AC, Waldon Z, Freeman MR, Steen H. Optimizing sample handling for urinary proteomics[J]. J Proteome Res.2008;7:4022-30.
[5]. Thongboonkerd V, McLeish KR, Arthur JM, Klein JB. Proteomic analysis of normal human urinary proteins isolated by acetone precipitation or ultracentrifugation[J]. Kidney Int.2002;62:1461-9.
[6]. Thongboonkerd V, Chutipongtanate S, Kanlaya R. Systematic evaluation of sample preparation methods for gel-based human urinary proteomics:quantity, quality, and variability[J]. J Proteome Res. 2006;5:183-91.
[7]. Thongboonkerd V. Practical points in urinary proteomics[J]. J Proteome Res.2007;6:3881-90.
[8]. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Anal Biochem.1976;72:248-54.
[9]. Khan A, Packer NH. Simple urinary sample preparation for proteomic analysis[J]. J Proteome Res. 2006;5:2824-38.
[10]. Wei Sun YC, Fuxin Li, Ling Zhang, Ruifeng Yang, Zhi Zhang, Dexian Zheng, Youhe Gao. Dynamic urinary proteomic analysis reveals stable proteins to be potential biomarkers[J]. Proteomics Clin. 2009;3:370-82.
[11]. Nagaraj N, Mann M. Quantitative analysis of the intra-and inter-individual variability of the normal urinary proteome[J]. J Proteome Res.2011;10:637-45.
[12]. Selevsek N, Matondo M, Sanchez Carbayo M, Aebersold R, Domon B. Systematic quantification of peptides/proteins in urine using selected reaction monitoring[J]. Proteomics.2011;11:1135-47.
[13]. Pristoupil TI, Kramlova M, Sterbikova J. On the mechanism of adsorption of proteins to nitrocellulose in membrane chromatography[J]. J Chromatogr.1969;42:367-75.
[14]. Burnette WN. "Western blotting":electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A[J]. Anal Biochem.1981;112:195-203.
[15]. Wisniewski JR, Zougman A, Nagaraj N, Mann M. Universal sample preparation method for proteome analysis[J]. Nat Methods.2009;6:359-62.
[16]. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets:procedure and some applications[J]. Proc Natl Acad Sci USA.1979;76:4350-4.
[17]. Wang N, Xie C, Young JB, Li L. Off-line two-dimensional liquid chromatography with maximized sample loading to reversed-phase liquid chromatography-electrospray ionization tandem mass spectrometry for shotgun proteome analysis[J]. Anal Chem.2009;81:1049-60.
[18]. Pennica D, Kohr WJ, Kuang WJ, Glaister D, Aggarwal BB, Chen EY, et al. Identification of human uromodulin as the Tamm-Horsfall urinary glycoprotein[J]. Science.1987;236:83-8.
[1]. Spahr CS, Davis MT, McGinley MD, Robinson JH, Bures EJ, Beierle J, et al. Towards defining the urinary proteome using liquid chromatography-tandem mass spectrometry. I. Profiling an unfractionated tryptic digest[J]. Proteomics.2001; 1:93-107.
[2]. Adachi J, Kumar C, Zhang Y, Olsen JV, Mann M. The human urinary proteome contains more than 1500 proteins, including a large proportion of membrane proteins[J]. Genome Biol.2006;7:R80.
[3]. Gonzales PA, Pisitkun T, Hoffert JD, Tchapyjnikov D, Star RA, Kleta R, et al. Large-scale proteomics and phosphoproteomics of urinary exosomes[J]. J Am Soc Nephrol.2009;20:363-79.
[4]. Kentsis A, Monigatti F, Dorff K, Campagne F, Bachur R, Steen H. Urine proteomics for profiling of human disease using high accuracy mass spectrometry[J]. Proteomics Clin Appl.2009;3:1052-61.
[5]. O'Riordan E, Orlova TN, Podust VN, Chander PN, Yanagi S, Nakazato M, et al. Characterization of urinary peptide biomarkers of acute rejection in renal allografts[J]. Am J Transplant.2007;7:930-40.
[6]. Rossing K, Mischak H, Dakna M, Zurbig P, Novak J, Julian BA, et al. Urinary proteomics in diabetes and CKD[J].J Am Soc Nephrol.2008; 19:1283-90.
[7]. Bellei E, Rossi E, Lucchi L, Uggeri S, Albertazzi A, Tomasi A, et al. Proteomic analysis of early urinary biomarkers of renal changes in type 2 diabetic patients[J]. Proteomics Clin Appl.2008;2:478-91.
[8]. Decramer S, Wittke S, Mischak H, Zurbig P, Walden M, Bouissou F, et al. Predicting the clinical outcome of congenital unilateral ureteropelvic junction obstruction in newborn by urinary proteome analysis[J]. Nat Med.2006; 12:398-400.
[9]. Irmak S, Tilki D, Heukeshoven J, Oliveira-Ferrer L, Friedrich M, Huland H, et al. Stage-dependent increase of orosomucoid and zinc-alpha2-glycoprotein in urinary bladder cancer[J]. Proteomics. 2005;5:4296-304.
[10]. Theodorescu D, Schiffer E, Bauer HW, Douwes F, Eichhorn F, Polley R, et al. Discovery and validation of urinary biomarkers for prostate cancer[J]. Proteomics Clin Appl.2008;2:556-70.
[11]. Kaiser T, Kamal H, Rank A, Kolb HJ, Holler E, Ganser A, et al. Proteomics applied to the clinical follow-up of patients after allogeneic hematopoietic stem cell transplantation [J]. Blood.2004; 104:340-9.
[12]. Zimmerli LU, Schiffer E, Zurbig P, Good DM, Kellmann M, Mouls L, et al. Urinary proteomic biomarkers in coronary artery disease[J]. Mol Cell Proteomics.2008;7:290-8.
[13]. Thongboonkerd V, Chutipongtanate S, Kanlaya R. Systematic evaluation of sample preparation methods for gel-based human urinary proteomics:quantity, quality, and variability[J]. J Proteome Res. 2006;5:183-91.
[14]. Oh J, Pyo JH, Jo EH, Hwang SI, Kang SC, Jung JH, et al. Establishment of a near-standard two-dimensional human urine proteomic map[J]. Proteomics.2004;4:3485-97.
[15]. Khan A, Packer NH. Simple urinary sample preparation for proteomic analysis[J]. J Proteome Res. 2006;5:2824-38.
[16].Nagaraj N, Mann M. Quantitative analysis of the intra-and inter-individual variability of the normal urinary proteome[J]. J Proteome Res.2011;10:637-45.
[17]. Wei Sun YC, Fuxin Li, Ling Zhang, Ruifeng Yang, Zhi Zhang, Dexian Zheng, Youhe Gao. Dynamic urinary proteomic analysis reveals stable proteins to be potential biomarkers[J]. Proteomics Clin. 2009;3:370-82.
[18]. Zhang X, Guo Y, Song Y, Sun W, Yu C, Zhao X, et al. Proteomic analysis of individual variation in normal livers of human beings using difference gel electrophoresis[J]. Proteomics.2006;6:5260-8.
[19]. Kersey PJ, Duarte J, Williams A, Karavidopoulou Y, Birney E, Apweiler R. The International Protein Index:an integrated database for proteomics experiments[J]. Proteomics.2004;4:1985-8.
[20]. Nesvizhskii AI, Aebersold R. Interpretation of shotgun proteomic data:the protein inference problem[J]. Mol Cell Proteomics.2005;4:1419-40.
[21]. Sun W, Wu S, Wang X, Zheng D, Gao Y. An analysis of protein abundance suppression in data dependent liquid chromatography and tandem mass spectrometry with tryptic peptide mixtures of five known proteins[J]. Eur J Mass Spectrom (Chichester, Eng).2005;11:575-80.
[22]. Elias JE, Gibbons FD, King OD, Roth FP, Gygi SP. Intensity-based protein identification by machine learning from a library of tandem mass spectra[J]. Nat Biotechnol.2004;22:214-9.
[23]. Sun W, Li F, Wu S, Wang X, Zheng D, Wang J, et al. Human urine proteome analysis by three separation approaches[J]. Proteomics.2005;5:4994-5001.
[1], Wu WW, Wang G, Baek SJ, Shen RF. Comparative study of three proteomic quantitative methods, DIGE, cICAT, and iTRAQ, using 2D gel-or LC-MALDI TOF/TOF[J]. J Proteome Res.2006;5:651-8.
[2]. Chong PK, Gan CS, Pham TK, Wright PC. Isobaric tags for relative and absolute quantitation (iTRAQ) reproducibility:Implication of multiple injections[J]. J Proteome Res.2006;5:1232-40.
[3]. Ong SE, Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, et al. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics[J]. Mol Cell Proteomics.2002; 1:376-86.
[4]. Gygi SP, Rist B, Gerber SA, Turecek F, Gelb MH, Aebersold R. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags[J]. Nat Biotechnol.1999; 17:994-9.
[5]. Li J, Steen H, Gygi SP. Protein profiling with cleavable isotope-coded affinity tag (cICAT) reagents: the yeast salinity stress response[J]. Mol Cell Proteomics.2003;2:1198-204.
[6]. Ross PL, Huang YN, Marchese JN, Williamson B, Parker K, Hattan S, et al. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents[J]. Mol Cell Proteomics.2004;3:1154-69.
[7]. Choe L, D'Ascenzo M, Relkin NR, Pappin D, Ross P, Williamson B, et al.8-plex quantitation of changes in cerebrospinal fluid protein expression in subjects undergoing intravenous immunoglobulin treatment for Alzheimer's disease[J]. Proteomics.2007;7:3651-60.
[8]. WHO. Definition, diagnosis and classification of diabetes mellitus and its complication[J]. WHOYNCD/NCS.1999:31-2.
[9]. Kahn R. Metabolic syndrome:is it a syndrome? Does it matter? [J] Circulation.2007; 115:1806-10; discussion 11.
[10]. Ford ES, Giles WH, Mokdad AH. Increasing prevalence of the metabolic syndrome among u.s. Adults[J]. Diabetes Care.2004;27:2444-9.
[11].陶世冰,任艳,田浩明,等.2007年成都地区成人代谢综合征患病率的流行病学调查[J].四川大学学报.2007；40：1062-5.
[12].樊晓红,蔡建芳,李学旺等.中国汉族人以尿白蛋白肌酐比值诊断微量白蛋白尿的界值研究[J].中华肾脏病杂志.2010；26：807-11
[13]. Laaksonen DE, Lakka HM, Niskanen LK, Kaplan GA, Salonen JT, Lakka TA. Metabolic syndrome and development of diabetes mellitus:application and validation of recently suggested definitions of the metabolic syndrome in a prospective cohort study [J]. Am J Epidemiol.2002; 156:1070-7.
[14]. Lakka HM, Laaksonen DE, Lakka TA, Niskanen LK, Kumpusalo E, Tuomilehto J, et al. The metabolic syndrome and total and cardiovascular disease mortality in middle-aged men [J]. JAMA. 2002;288:2709-16.
[15]. Panagiotis K MEaHJM. Multifactorial intervention in metabolic syndrome targeting at prevention of chronic kidney disease-ready for prime time [J]? Nephrol Dial Transplant.2007;22:2768-74.
[16]. Chen J, Muntner P, Hamm LL, Jones DW, Batuman V, Fonseca V, et al. The metabolic syndrome and chronic kidney disease in U.S. adults [J]. Ann Intern Med.2004; 140:167-74.
[17]. Tanaka H, Shiohira Y, Uezu Y, Higa A, Iseki K. Metabolic syndrome and chronic kidney disease in Okinawa, Japan [J]. Kidney Int.2006;69:369-74.
[18]. Agrawal V, Shah A, Rice C, Franklin BA, McCullough PA. Impact of treating the metabolic syndrome on chronic kidney disease [J].Nat Rev Nephrol.2009;5:520-8.
[19]. Ryu S, Chang Y, Woo HY, Lee KB, Kim SG, Kim DI, et al. Time-dependent association between metabolic syndrome and risk of CKD in Korean men without hypertension or diabetes [J]. Am J Kidney Dis.2009;53:59-69.
[20]. Chen J, Muntner P, Hamm LL, Jones DW, Batuman V, Fonseca V, et al. The metabolic syndrome and chronic kidney disease in U.S. adults [J]. Ann Intern Med.2004;140:167-74.
[21]. Koroshi A. Microalbuminuria, is it so important [J]? Hippokratia.2007;11:105-7.
[22]. Goligorsky MS, Addabbo F, O'Riordan E. Diagnostic potential of urine proteome:a broken mirror of renal diseases [J]. J Am Soc Nephrol.2007;18:2233-9.
[23]. Alberti KG, Zimmet P, Shaw J. The metabolic syndrome--a new worldwide definition [J]. Lancet. 2005;366:1059-62.
[24]. Thongboonkerd V. Practical points in urinary proteomics [J]. J Proteome Res.2007;6:3881-90.
[25].Nagaraj N, Mann M. Quantitative analysis of the intra- and inter-individual variability of the normal urinary proteome [J]. J Proteome Res.2011;10:637-45.
[26]. Wei Sun YC, Fuxin Li, Ling Zhang, Ruifeng Yang, Zhi Zhang, Dexian Zheng, Youhe Gao. Dynamic urinary proteomic analysis reveals stable proteins to be potential biomarkers [J]. Proteomics Clin. 2009;3:370-82.
[27]. Patel VJ, Thalassinos K, Slade SE, Connolly JB, Crombie A, Murrell JC, et al. A comparison of labeling and label-free mass spectrometry-based proteomics approaches [J]. J Proteome Res. 2009;8:3752-9.
[28]. Choi H, Fermin D, Nesvizhskii Al. Significance analysis of spectral count data in label-free shotgun proteomics [J]. Mol Cell Proteomics.2008;7:2373-85.
[29]. Usaite R, Wohlschlegel J, Venable JD, Park SK, Nielsen J, Olsson L, et al. Characterization of global yeast quantitative proteome data generated from the wild-type and glucose repression saccharomyces cerevisiae strains:the comparison of two quantitative methods [J]. J Proteome Res.2008;7:266-75.
[30].Neilson KA, Mariani M, Haynes PA. Quantitative proteomic analysis of cold-responsive proteins in rice [J]. Proteomics.2011;11:1696-706.
[31]. Old WM, Meyer-Arendt K, Aveline-Wolf L, Pierce KG, Mendoza A, Sevinsky JR, et al. Comparison of label-free methods for quantifying human proteins by shotgun proteomics [J]. Mol Cell Proteomics. 2005;4:1487-502.
[32]. Zybailov B, Mosley AL, Sardiu ME, Coleman MK, Florens L, Washburn MP. Statistical analysis of membrane proteome expression changes in Saccharomyces cerevisiae [J]. J Proteome Res.2006;5:2339-47.
[33]. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets:procedure and some applications [J]. Proc Natl Acad Sci U S A.1979;76:4350-4.
[34]. Hellman NE, Gitlin JD. Ceruloplasmin metabolism and function [J]. Annu Rev Nutr.2002;22:439-58.
[35]. Kosman DJ. FET3P, ceruloplasmin, and the role of copper in iron metabolism [J]. Adv Protein Chem. 2002;60:221-69.
[36]. Yamazaki M, Ito S, Usami A, Tani N, Hanyu O, Nakagawa O, et al. Urinary excretion rate of ceruloplasmin in non-insulin-dependent diabetic patients with different stages of nephropathy [J]. Eur J Endocrinol.1995;132:681-7.
[37].Narita T, Sasaki H, Hosoba M, Miura T, Yoshioka N, Morii T, et al. Parallel increase in urinary excretion rates of immunoglobulin G, ceruloplasmin, transferrin, and orosomucoid in normoalbuminuric type 2 diabetic patients [J]. Diabetes Care.2004;27:1176-81.
[38].Narita T, Fujita H, Koshimura J, Meguro H, Kitazato H, Shimotomai T, et al. Glycemic control reverses increases in urinary excretions of immunoglobulin G and ceruloplasmin in type 2 diabetic patients with normoalbuminuria [J]. Horm Metab Res.2001;33:370-8.
[39]. Ece A, Kelekci S, Hekimoglu A, Kocamaz H, Balik H, Yolbas I, et al. Neutrophil activation, protein oxidation and ceruloplasmin levels in children with Henoch-Schonlein purpura [J]. Pediatr Nephrol. 2007;22:1151-7.
[40]. Rao PV, Lu X, Standley M, Pattee P, Neelima G, Girisesh G, et al. Proteomic identification of urinary biomarkers of diabetic nephropathy [J]. Diabetes Care.2007;30:629-37.
[41]. Moon PG, Lee JE, You S, Kim TK, Cho JH, Kim IS, et al. Proteomic analysis of urinary exosomes from patients of early IgA nephropathy and thin basement membrane nephropathy [J]. Proteomics. 2011;11:2459-75.
[42]. Duverger N, Ghalim N, Ailhaud G, Steinmetz A, Fruchart JC, Castro G. Characterization of apoA-IV-containing lipoprotein particles isolated from human plasma and interstitial fluid [J]. Arterioscler Thromb.1993; 13:126-32.
[43]. von Eckardstein A, Huang Y, Wu S, Sarmadi AS, Schwarz S, Steinmetz A, et al. Lipoproteins containing apolipoprotein A-IV but not apolipoprotein A-I take up and esterify cell-derived cholesterol in plasma [J]. Arterioscler Thromb Vase Biol.1995;15:1755-63.
[44]. Duverger N, Tremp G, Caillaud JM, Emmanuel F, Castro G, Fruchart JC, et al. Protection against atherogenesis in mice mediated by human apolipoprotein A-IV [J]. Science.1996;273:966-8.
[45]. Cohen RD, Castellani LW, Qiao JH, Van Lenten BJ, Lusis AJ, Reue K. Reduced aortic lesions and elevated high density lipoprotein levels in transgenic mice overexpressing mouse apolipoprotein A-IV [J]. J Clin Invest.1997;99:1906-16.
[46]. Kronenberg F, Kuen E, Ritz E, Konig P, Kraatz G, Lhotta K, et al. Apolipoprotein A-IV serum concentrations are elevated in patients with mild and moderate renal failure [J]. J Am Soc Nephrol. 2002;13:461-9.
[47]. Lingenhel A, Lhotta K, Neyer U, Heid IM, Rantner B, Kronenberg MF, et al. Role of the kidney in the metabolism of apolipoprotein A-Ⅳ:influence of the type of proteinuria [J]. J Lipid Res.2006;47:2071-9.
[48]. Fisher DA, Salido EC, Barajas L. Epidermal growth factor and the kidney [J]. Annu Rev Physiol. 1989;51:67-80.
[49]. Gilbert RE, Cox A, McNally PG, Wu LL, Dziadek M, Cooper ME, et al. Increased epidermal growth factor in experimental diabetes related kidney growth in rats [J]. Diabetologia.1997;40:778-85.
[50]. Mathiesen ER, Nexo E, Hommel E, Parving HH. Reduced urinary excretion of epidermal growth factor in incipient and overt diabetic nephropathy [J]. Diabet Med.1989;6:121-6.
[51]. Stangou M, Alexopoulos E, Papagianni A, Pantzaki A, Bantis C, Dovas S, et al. Urinary levels of epidermal growth factor, interleukin-6 and monocyte chemoattractant protein-1 may act as predictor markers of renal function outcome in immunoglobulin A nephropathy [J]. Nephrology (Carlton). 2009; 14:613-20.
[52]. Cutillas PR, Chalkley RJ, Hansen KC, Cramer R, Norden AG, Waterfield MD, et al. The urinary proteome in Fanconi syndrome implies specificity in the reabsorption of proteins by renal proximal tubule cells [J]. Am J Physiol Renal Physiol.2004;287:F353-64.
[53]. Yang L, Li X, Wang H. Possible mechanisms explaining the tendency towards interstitial fibrosis in aristolochic acid-induced acute tubular necrosis [J]. Nephrol Dial Transplant.2007;22:445-56.
[54].Baer PC, Geiger H. Different effects of growth factors on human renal early distal tubular cells in vitro [J]. Kidney Blood Press Res.2006;29:225-30.
[55]. Haussler U, von Wichert G, Schmid RM, Keller F, Schneider G. Epidermal growth factor activates nuclear factor-kappaB in human proximal tubule cells [J]. Am J Physiol Renal Physiol.2005;289:F808-15.
[1]. WHO. Definition, diagnosis and classification of diabetes mellitus and its complication [J]. WHO/NCD/NCS.1999:31-2.
[2]. Kahn R. Metabolic syndrome:is it a syndrome? Does it matter [J]? Circulation.2007;115:1806-10; discussion 11.
[3]. Ford ES, Giles WH, Mokdad AH. Increasing prevalence of the metabolic syndrome among u.s. Adults [J]. Diabetes Care.2004;27:2444-9.
[4].陶世冰,任艳,田浩明,等.2007年成都地区成人代谢综合征患病率的流行病学调查[J].四川大学学报.2007;40：1062-5.
[5].樊晓红,蔡建芳,李学旺等.中国汉族人以尿白蛋白肌酐比值诊断微量白蛋白尿的界值研究[J].中华肾脏病杂志.2010;26：807-11
[6]. Laaksonen DE, Lakka HM, Niskanen LK, Kaplan GA, Salonen JT, Lakka TA. Metabolic syndrome and development of diabetes mellitus:application and validation of recently suggested definitions of the metabolic syndrome in a prospective cohort study [J]. Am J Epidemiol.2002; 156:1070-7.
[7]. Lakka HM, Laaksonen DE, Lakka TA, Niskanen LK, Kumpusalo E, Tuomilehto J, et al. The metabolic syndrome and total and cardiovascular disease mortality in middle-aged men [J]. JAMA. 2002;288:2709-16.
[8]. Panagiotis K MEaHJM. Multifactorial intervention in metabolic syndrome targeting at prevention of chronic kidney disease-ready for prime time [J]? Nephrol Dial Transplant.2007;22:2768-74.
[9]. Chen J, Muntner P, Hamm LL, Jones DW, Batuman V, Fonseca V, et al. The metabolic syndrome and chronic kidney disease in U.S. adults [J]. Ann Intern Med.2004; 140:167-74.
[10]. Tanaka H, Shiohira Y, Uezu Y, Higa A, Iseki K. Metabolic syndrome and chronic kidney disease in Okinawa, Japan [J]. Kidney Int.2006;69:369-74.
[11]. Koroshi A. Microalbuminuria, is it so important [J]? Hippokratia.2007; 11:105-7.
[12]. Ruan X, Guan Y. Metabolic syndrome and chronic kidney disease [J]. J Diabetes.2009;1:236-45.
[13]. Cornier MA, Dabelea D, Hernandez TL, Lindstrom RC, Steig AJ, Stob NR, et al. The metabolic syndrome [J]. Endocr Rev.2008;29:777-822.
[14]. Bravata DM, Wells CK, Concato J, Kernan WN, Brass LM, Gulanski BI. Two measures of insulin sensitivity provided similar information in a U.S. population [J]. J Clin Epidemiol.2004;57:1214-7.
[15]. St-Onge MP, Janssen I, Heymsfield SB. Metabolic syndrome in normal-weight Americans:new definition of the metabolically obese, normal-weight individual [J]. Diabetes Care.2004;27:2222-8.
[16]. Mayer-Davis EJ, D'Agostino R, Jr., Karter AJ, Haffner SM, Rewers MJ, Saad M, et al. Intensity and amount of physical activity in relation to insulin sensitivity:the Insulin Resistance Atherosclerosis Study [J]. JAMA.1998;279:669-74.
[17]. Eckel RH, Grundy SM, Zimmet PZ. The metabolic syndrome [J]. Lancet.2005;365:1415-28.
[18]. Tooke JE, Hannemann MM. Adverse endothelial function and the insulin resistance syndrome [J]. J Intern Med.2000;247:425-31.
[19]. Ferrannini E, Buzzigoli G, Bonadonna R, Giorico MA, Oleggini M, Graziadei L, et al. Insulin resistance in essential hypertension [J]. N Engl J Med.1987;317:350-7.
[20]. Bonora E, Capaldo B, Perin PC, Del Prato S, De Mattia G, Frittitta L, et al. Hyperinsulinemia and insulin resistance are independently associated with plasma lipids, uric acid and blood pressure in non-diabetic subjects [J]. The GISIR database. Nutr Metab Cardiovasc Dis.2008;18:624-31.
[21]. Anderson EA, Hoffman RP, Balon TW, Sinkey CA, Mark AL. Hyperinsulinemia produces both sympathetic neural activation and vasodilation in normal humans [J]. J Clin Invest.1991;87:2246-52.
[22]. Hall JE, Henegar JR, Dwyer TM, Liu J, Da Silva AA, Kuo JJ, et al. Is obesity a major cause of chronic kidney disease [J]? Adv Ren Replace Ther.2004; 11:41-54.
[23]. Roden M, Krssak M, Stingl H, Gruber S, Hofer A, Furnsinn C, et al. Rapid impairment of skeletal muscle glucose transport/phosphorylation by free fatty acids in humans [J]. Diabetes.1999;48:358-64.
[24].Norata GD, Ongari M, Garlaschelli K, Raselli S, Grigore L, Catapano AL. Plasma resistin levels correlate with determinants of the metabolic syndrome [J]. Eur J Endocrinol.2007;156:279-84.
[25]. Bahia L, Aguiar LG, Villela N, Bottino D, Godoy-Matos AF, Geloneze B, et al. Relationship between adipokines, inflammation, and vascular reactivity in lean controls and obese subjects with metabolic syndrome [J]. Clinics (Sao Paulo).2006;61:433-40.
[26].Xydakis AM, Case CC, Jones PH, Hoogeveen RC, Liu MY, Smith EO, et al. Adiponectin, inflammation, and the expression of the metabolic syndrome in obese individuals:the impact of rapid weight loss through caloric restriction [J]. J Clin Endocrinol Metab.2004;89:2697-703.
[27]. Chagnac A, Weinstein T, Korzets A, Ramadan E, Hirsch J, Gafter U. Glomerular hemodynamics in severe obesity [J]. Am J Physiol Renal Physiol.2000;278:F817-22.
[28]. Wolf G, Chen S, Han DC, Ziyadeh FN. Leptin and renal disease [J]. Am J Kidney Dis.2002;39:1-11.
[29]. Zhang R, Liao J, Morse S, Donelon S, Reisin E. Kidney disease and the metabolic syndrome [J]. Am J Med Sci.2005;330:319-25.
[30]. Kamijo A, Kimura K, Sugaya T, Yamanouchi M, Hase H, Kaneko T, et al. Urinary free fatty acids bound to albumin aggravate tubulointerstitial damage [J]. Kidney Int.2002;62:1628-37.
[31]. Cusumano AM, Bodkin NL, Hansen BC, lotti R, Owens J, Klotman PE, et al. Glomerular hypertrophy is associated with hyperinsulinemia and precedes overt diabetes in aging rhesus monkeys [J]. Am J Kidney Dis.2002;40:1075-85.
[32]. Abrass CK, Spicer D, Raugi GJ. Insulin induces a change in extracellular matrix glycoproteins synthesized by rat mesangial cells in culture [J]. Kidney Int.1994;46:613-20.
[33]. Palmer BF. Impaired renal autoregulation:implications for the genesis of hypertension and hypertension-induced renal injury [J]. Am J Med Sci.2001;321:388-400.
[34]. Sowers JR. Insulin resistance and hypertension [J]. Am J Physiol Heart Circ Physiol. 2004;286:H1597-602.
[35]. Bonventre JV. The kidney proteome:a hint of things to come [J]. Kidney Int.2002;62:1470-1.
[36]. O'Farrell PH. High resolution two-dimensional electrophoresis of proteins [J]. J Biol Chem. 1975;250:4007-21.
[37]. Timms JF, Cramer R. Difference gel electrophoresis [J]. Proteomics.2008;8:4886-97.
[38]. Unlu M, Morgan ME, Minden JS. Difference gel electrophoresis:a single gel method for detecting changes in protein extracts [J]. Electrophoresis.1997;18:2071-7.
[39]. Domon B, Aebersold R. Options and considerations when selecting a quantitative proteomics strategy [J]. Nat Biotechnol.2010;28:710-21.
[40]. Choi H, Fermin D, Nesvizhskii Al. Significance analysis of spectral count data in label-free shotgun proteomics [J]. Mol Cell Proteomics.2008;7:2373-85.
[41]. Usaite R, Wohlschlegel J, Venable JD, Park SK, Nielsen J, Olsson L, et al. Characterization of global yeast quantitative proteome data generated from the wild-type and glucose repression saccharomyces cerevisiae strains:the comparison of two quantitative methods [J]. J Proteome Res.2008;7:266-75.
[42]. Anderson L, Hunter CL. Quantitative mass spectrometric multiple reaction monitoring assays for major plasma proteins [J]. Mol Cell Proteomics.2006;5:573-88.
[43]. Gao J, Opiteck GJ, Friedrichs MS, Dongre AR, Hefta SA. Changes in the protein expression of yeast as a function of carbon source [J]. J Proteome Res.2003;2:643-9.
[44]. Ono M, Shitashige M, Honda K, Isobe T, Kuwabara H, Matsuzuki H, et al. Label-free quantitative proteomics using large peptide data sets generated by nanoflow liquid chromatography and mass spectrometry [J]. Mol Cell Proteomics.2006;5:1338-47.
[45].Neilson KA, Mariani M, Haynes PA. Quantitative proteomic analysis of cold-responsive proteins in rice [J]. Proteomics.2011;11:1696-706.
[46]. Gygi SP, Rist B, Gerber SA, Turecek F, Gelb MH, Aebersold R. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags [J]. Nat Biotechnol.1999;17:994-9.
[47]. Li J, Steen H, Gygi SP. Protein profiling with cleavable isotope-coded affinity tag (cICAT) reagents: the yeast salinity stress response [J]. Mol Cell Proteomics.2003;2:1198-204.
[48]. Ong SE, Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, et al. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics [J]. Mol Cell Proteomics.2002; 1:376-86.
[49]. Goligorsky MS, Addabbo F, O'Riordan E. Diagnostic potential of urine proteome:a broken mirror of renal diseases [J]. J Am Soc Nephrol.2007; 18:2233-9.
[50]. Mischak H, Apweiler R, Banks RE, Conaway M, Coon J, Dominiczak A, et al. Clinical proteomics:A need to define the field and to begin to set adequate standards [J]. Proteomics Clin Appl.2007; 1:148-56.
[51]. Thongboonkerd V. Current status of renal and urinary proteomics:ready for routine clinical application? [J]. Nephrol Dial Transplant.2009;25:11-6.
[52]. Pejcic M, Stojnev S, Stefanovic V. Urinary proteomics-a tool for biomarker discovery [J]. Ren Fail. 2010;32:259-68.
[53]. Smith MP, Banks RE, Wood SL, Lewington AJ, Selby PJ. Application of proteomic analysis to the study of renal diseases [J].Nat Rev Nephrol.2009;5:701-12.
[54]. Goligorsky MS, Addabbo F, O'Riordan E. Diagnostic potential of urine proteome:a broken mirror of renal diseases [J]. J Am Soc Nephrol.2007; 18:2233-9.
[55]. Woroniecki RP, Orlova TN, Mendelev N, Shatat IF, Hailpern SM, Kaskel FJ, et al. Urinary proteome of steroid-sensitive and steroid-resistant idiopathic nephrotic syndrome of childhood [J]. Am J Nephrol. 2006;26:258-67.
[56]. Merchant ML, Perkins BA, Boratyn GM, Ficociello LH, Wilkey DW, Barati MT, et al. Urinary peptidome may predict renal function decline in type 1 diabetes and microalbuminuria [J]. J Am Soc Nephrol.2009;20:2065-74.
[57]. Ho J, Lucy M, Krokhin O, Hayglass K, Pascoe E, Darroch G, et al. Mass spectrometry-based proteomic analysis of urine in acute kidney injury following cardiopulmonary bypass:a nested case-control study [J]. Am J Kidney Dis.2009;53:584-95.
[58]. Clarke W, Silverman BC, Zhang Z, Chan DW, Klein AS, Molmenti EP. Characterization of renal allograft rejection by urinary proteomic analysis [J]. Ann Surg.2003;237:660-4; discussion 4-5.
[59]. Mischak H, Kaiser T, Walden M, Hillmann M, Wittke S, Herrmann A, et al. Proteomic analysis for the assessment of diabetic renal damage in humans [J]. Clin Sci (Lond).2004; 107:485-95.
[60]. Thongboonkerd V, Klein JB, Jevans AW, McLeish KR. Urinary proteomics and biomarker discovery for glomerular diseases [J]. Contrib Nephrol.2004;141:292-307.
[61]. Haider H, Jiang SJ, Ye L, Aziz S, Law PK, Sim EK. Effectiveness of transient immunosuppression using cyclosporine for xenomyoblast transplantation for cardiac repair [J]. Transplant Proc. 2004;36:232-5.
[62]. Rao PV, Lu X, Standley M, Pattee P, Neelima G, Girisesh G, et al. Proteomic identification of urinary biomarkers of diabetic nephropathy [J]. Diabetes Care.2007;30:629-37.
[63]. Dihazi H, Asif AR, Agarwal NK, Doncheva Y, Muller GA. Proteomic analysis of cellular response to osmotic stress in thick ascending limb of Henle's loop (TALH) cells [J]. Mol Cell Proteomics. 2005;4:1445-58.
[64]. Rossing K, Mischak H, Dakna M, Zurbig P, Novak J, Julian BA, et al. Urinary proteomics in diabetes and CKD [J]. J Am Soc Nep hrol.2008;19:1283-90.
[65]. Chutipongtanate S, Nakagawa Y, Sritippayawan S, Pittayamateekul J, Parichatikanond P, Westley BR, et al. Identification of human urinary trefoil factor 1 as a novel calcium oxalate crystal growth inhibitor [J]. J Clin Invest.2005; 115:3613-22.
[66]. Beck LH, Jr., Bonegio RG, Lambeau G, Beck DM, Powell DW, Cummins TD, et al. M-type phospholipase A2 receptor as target antigen in idiopathic membranous nephropathy [J]. N Engl J Med. 2009;361:11-21.
[67]. Wasinger VC, Cordwell SJ, Cerpa-Poljak A, Yan JX, Gooley AA, Wilkins MR, et al. Progress with gene-product mapping of the Mollicutes:Mycoplasma genitalium [J]. Electrophoresis.1995; 16:1090-4.
[68]. Wilkins MR, Sanchez JC, Gooley AA, Appel RD, Humphery-Smith I, Hochstrasser DF, et al. Progress with proteome projects:why all proteins expressed by a genome should be identified and how to do it [J]. Biotechnol Genet Eng Rev.1996; 13:19-50.
[69]. Wilkins MR, Pasquali C, Appel RD, Ou K, Golaz O, Sanchez JC, et al. From proteins to proteomes: Large scale protein identification by two-dimensional electrophoresis and amino acid analysis [J]. Bio-Technol.1996; 14:61-5.
[70]. Ross PL, Huang YN, Marchese JN, Williamson B, Parker K, Hattan S, et al. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents [J]. Mol Cell Proteomics.2004;3:1154-69.
[71]. Dayon L, Hainard A, Licker V, Turck N, Kuhn K, Hochstrasser DF, et al. Relative quantification of proteins in human cerebrospinal fluids by MS/MS using 6-plex isobaric tags [J]. Anal Chem. 2008;80:2921-31.
[72]. Unwin RD, Evans CA, Whetton AD. Relative quantification in proteomics:new approaches for biochemistry [J]. Trends Biochem Sci.2006;31:473-84.
[73]. Wu WW, Wang G, Baek SJ, Shen RF. Comparative study of three proteomic quantitative methods, DIGE, cICAT, and iTRAQ, using 2D gel-or LC-MALDI TOF/TOF [J]. J Proteome Res.2006;5:651-8.
[74]. Chong PK, Gan CS, Pham TK, Wright PC. Isobaric tags for relative and absolute quantitation (iTRAQ) reproducibility:Implication of multiple injections [J]. J Proteome Res.2006;5:1232-40.
[75]. Brewis IA, Brennan P. Proteomics technologies for the global identification and quantification of proteins [J]. Adv Protein Chem Struct Biol.2010;80:l-44.
[76]. Pieper R, Gatlin CL, McGrath AM, Makusky AJ, Mondal M, Seonarain M, et al. Characterization of the human urinary proteome:a method for high-resolution display of urinary proteins on two-dimensional electrophoresis gels with a yield of nearly 1400 distinct protein spots [J]. Proteomics.2004;4:1159-74.
[77]. Afkarian M, Bhasin M, Dillon ST, Guerrero MC, Nelson RG, Knowler WC, et al. Optimizing a proteomics platform for urine biomarker discovery [J]. Mol Cell Proteomics.2010;9:2195-204.
[78]. Akkina SK, Zhang Y, Nelsestuen GL, Oetting WS, Ibrahim HN. Temporal stability of the urinary proteome after kidney transplant:more sensitive than protein composition [J]? J Proteome Res. 2009;8:94-103.
[79]. Wenderfer SE, Dubinsky WP, Hernandez-Sanabria M, Braun MC. Urine proteome analysis in murine nephrotoxic serum nephritis [J]. Am J Nephrol.2009;30:450-8.
[80]. Chen YT, Chen CL, Chen HW, Chung T, Wu CC, Chen CD, et al. Discovery of novel bladder cancer biomarkers by comparative urine proteomics using iTRAQ technology [J]. J Proteome Res. 2010;9:5803-15.
[81]. Chen G, Zhang Y, Jin X, Zhang L, Zhou Y, Niu J, et al. Urinary proteomics analysis for renal injury in hypertensive disorders of pregnancy with iTRAQ labeling and LC-MS/MS [J]. Proteomics Clin Appl. 2011;5:300-10.
[82]. Kaspar H, Dettmer K, Chan Q, Daniels S, Nimkar S, Daviglus ML, et al. Urinary amino acid analysis: a comparison of iTRAQ-LC-MS/MS, GC-MS, and amino acid analyzer [J]. J Chromatogr B Analyt Technol Biomed Life Sci.2009;877:1838-46.
[83]. DeSouza LV, Taylor AM, Li W, Minkoff MS, Romaschin AD, Colgan TJ, et al. Multiple reaction monitoring of mTRAQ-Iabeled peptides enables absolute quantification of endogenous levels of a potential cancer marker in cancerous and normal endometrial tissues [J]. J Proteome Res.2008;7:3525-34.
[2]. Barratt J, Topham P. Urine proteomics:the present and future of measuring urinary protein components in disease[J]. CMAJ.2007; 177:361-8.
[3]. Goligorsky MS, Addabbo F, O'Riordan E. Diagnostic potential of urine proteome:a broken mirror of renal diseases[J]. J Am Soc Nephrol.2007; 18:2233-9.
[4]. Lee RS, Monigatti F, Briscoe AC, Waldon Z, Freeman MR, Steen H. Optimizing sample handling for urinary proteomics[J]. J Proteome Res.2008;7:4022-30.
[5]. Thongboonkerd V, McLeish KR, Arthur JM, Klein JB. Proteomic analysis of normal human urinary proteins isolated by acetone precipitation or ultracentrifugation[J]. Kidney Int.2002;62:1461-9.
[6]. Thongboonkerd V, Chutipongtanate S, Kanlaya R. Systematic evaluation of sample preparation methods for gel-based human urinary proteomics:quantity, quality, and variability[J]. J Proteome Res. 2006;5:183-91.
[7]. Thongboonkerd V. Practical points in urinary proteomics[J]. J Proteome Res.2007;6:3881-90.
[8]. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Anal Biochem.1976;72:248-54.
[9]. Khan A, Packer NH. Simple urinary sample preparation for proteomic analysis[J]. J Proteome Res. 2006;5:2824-38.
[10]. Wei Sun YC, Fuxin Li, Ling Zhang, Ruifeng Yang, Zhi Zhang, Dexian Zheng, Youhe Gao. Dynamic urinary proteomic analysis reveals stable proteins to be potential biomarkers[J]. Proteomics Clin. 2009;3:370-82.
[11]. Nagaraj N, Mann M. Quantitative analysis of the intra-and inter-individual variability of the normal urinary proteome[J]. J Proteome Res.2011;10:637-45.
[12]. Selevsek N, Matondo M, Sanchez Carbayo M, Aebersold R, Domon B. Systematic quantification of peptides/proteins in urine using selected reaction monitoring[J]. Proteomics.2011;11:1135-47.
[13]. Pristoupil TI, Kramlova M, Sterbikova J. On the mechanism of adsorption of proteins to nitrocellulose in membrane chromatography[J]. J Chromatogr.1969;42:367-75.
[14]. Burnette WN. "Western blotting":electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A[J]. Anal Biochem.1981;112:195-203.
[15]. Wisniewski JR, Zougman A, Nagaraj N, Mann M. Universal sample preparation method for proteome analysis[J]. Nat Methods.2009;6:359-62.
[16]. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets:procedure and some applications[J]. Proc Natl Acad Sci USA.1979;76:4350-4.
[17]. Wang N, Xie C, Young JB, Li L. Off-line two-dimensional liquid chromatography with maximized sample loading to reversed-phase liquid chromatography-electrospray ionization tandem mass spectrometry for shotgun proteome analysis[J]. Anal Chem.2009;81:1049-60.
[18]. Pennica D, Kohr WJ, Kuang WJ, Glaister D, Aggarwal BB, Chen EY, et al. Identification of human uromodulin as the Tamm-Horsfall urinary glycoprotein[J]. Science.1987;236:83-8.
[1]. Spahr CS, Davis MT, McGinley MD, Robinson JH, Bures EJ, Beierle J, et al. Towards defining the urinary proteome using liquid chromatography-tandem mass spectrometry. I. Profiling an unfractionated tryptic digest[J]. Proteomics.2001; 1:93-107.
[2]. Adachi J, Kumar C, Zhang Y, Olsen JV, Mann M. The human urinary proteome contains more than 1500 proteins, including a large proportion of membrane proteins[J]. Genome Biol.2006;7:R80.
[3]. Gonzales PA, Pisitkun T, Hoffert JD, Tchapyjnikov D, Star RA, Kleta R, et al. Large-scale proteomics and phosphoproteomics of urinary exosomes[J]. J Am Soc Nephrol.2009;20:363-79.
[4]. Kentsis A, Monigatti F, Dorff K, Campagne F, Bachur R, Steen H. Urine proteomics for profiling of human disease using high accuracy mass spectrometry[J]. Proteomics Clin Appl.2009;3:1052-61.
[5]. O'Riordan E, Orlova TN, Podust VN, Chander PN, Yanagi S, Nakazato M, et al. Characterization of urinary peptide biomarkers of acute rejection in renal allografts[J]. Am J Transplant.2007;7:930-40.
[6]. Rossing K, Mischak H, Dakna M, Zurbig P, Novak J, Julian BA, et al. Urinary proteomics in diabetes and CKD[J].J Am Soc Nephrol.2008; 19:1283-90.
[7]. Bellei E, Rossi E, Lucchi L, Uggeri S, Albertazzi A, Tomasi A, et al. Proteomic analysis of early urinary biomarkers of renal changes in type 2 diabetic patients[J]. Proteomics Clin Appl.2008;2:478-91.
[8]. Decramer S, Wittke S, Mischak H, Zurbig P, Walden M, Bouissou F, et al. Predicting the clinical outcome of congenital unilateral ureteropelvic junction obstruction in newborn by urinary proteome analysis[J]. Nat Med.2006; 12:398-400.
[9]. Irmak S, Tilki D, Heukeshoven J, Oliveira-Ferrer L, Friedrich M, Huland H, et al. Stage-dependent increase of orosomucoid and zinc-alpha2-glycoprotein in urinary bladder cancer[J]. Proteomics. 2005;5:4296-304.
[10]. Theodorescu D, Schiffer E, Bauer HW, Douwes F, Eichhorn F, Polley R, et al. Discovery and validation of urinary biomarkers for prostate cancer[J]. Proteomics Clin Appl.2008;2:556-70.
[11]. Kaiser T, Kamal H, Rank A, Kolb HJ, Holler E, Ganser A, et al. Proteomics applied to the clinical follow-up of patients after allogeneic hematopoietic stem cell transplantation [J]. Blood.2004; 104:340-9.
[12]. Zimmerli LU, Schiffer E, Zurbig P, Good DM, Kellmann M, Mouls L, et al. Urinary proteomic biomarkers in coronary artery disease[J]. Mol Cell Proteomics.2008;7:290-8.
[13]. Thongboonkerd V, Chutipongtanate S, Kanlaya R. Systematic evaluation of sample preparation methods for gel-based human urinary proteomics:quantity, quality, and variability[J]. J Proteome Res. 2006;5:183-91.
[14]. Oh J, Pyo JH, Jo EH, Hwang SI, Kang SC, Jung JH, et al. Establishment of a near-standard two-dimensional human urine proteomic map[J]. Proteomics.2004;4:3485-97.
[15]. Khan A, Packer NH. Simple urinary sample preparation for proteomic analysis[J]. J Proteome Res. 2006;5:2824-38.
[16].Nagaraj N, Mann M. Quantitative analysis of the intra-and inter-individual variability of the normal urinary proteome[J]. J Proteome Res.2011;10:637-45.
[17]. Wei Sun YC, Fuxin Li, Ling Zhang, Ruifeng Yang, Zhi Zhang, Dexian Zheng, Youhe Gao. Dynamic urinary proteomic analysis reveals stable proteins to be potential biomarkers[J]. Proteomics Clin. 2009;3:370-82.
[18]. Zhang X, Guo Y, Song Y, Sun W, Yu C, Zhao X, et al. Proteomic analysis of individual variation in normal livers of human beings using difference gel electrophoresis[J]. Proteomics.2006;6:5260-8.
[19]. Kersey PJ, Duarte J, Williams A, Karavidopoulou Y, Birney E, Apweiler R. The International Protein Index:an integrated database for proteomics experiments[J]. Proteomics.2004;4:1985-8.
[20]. Nesvizhskii AI, Aebersold R. Interpretation of shotgun proteomic data:the protein inference problem[J]. Mol Cell Proteomics.2005;4:1419-40.
[21]. Sun W, Wu S, Wang X, Zheng D, Gao Y. An analysis of protein abundance suppression in data dependent liquid chromatography and tandem mass spectrometry with tryptic peptide mixtures of five known proteins[J]. Eur J Mass Spectrom (Chichester, Eng).2005;11:575-80.
[22]. Elias JE, Gibbons FD, King OD, Roth FP, Gygi SP. Intensity-based protein identification by machine learning from a library of tandem mass spectra[J]. Nat Biotechnol.2004;22:214-9.
[23]. Sun W, Li F, Wu S, Wang X, Zheng D, Wang J, et al. Human urine proteome analysis by three separation approaches[J]. Proteomics.2005;5:4994-5001.
[1], Wu WW, Wang G, Baek SJ, Shen RF. Comparative study of three proteomic quantitative methods, DIGE, cICAT, and iTRAQ, using 2D gel-or LC-MALDI TOF/TOF[J]. J Proteome Res.2006;5:651-8.
[2]. Chong PK, Gan CS, Pham TK, Wright PC. Isobaric tags for relative and absolute quantitation (iTRAQ) reproducibility:Implication of multiple injections[J]. J Proteome Res.2006;5:1232-40.
[3]. Ong SE, Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, et al. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics[J]. Mol Cell Proteomics.2002; 1:376-86.
[4]. Gygi SP, Rist B, Gerber SA, Turecek F, Gelb MH, Aebersold R. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags[J]. Nat Biotechnol.1999; 17:994-9.
[5]. Li J, Steen H, Gygi SP. Protein profiling with cleavable isotope-coded affinity tag (cICAT) reagents: the yeast salinity stress response[J]. Mol Cell Proteomics.2003;2:1198-204.
[6]. Ross PL, Huang YN, Marchese JN, Williamson B, Parker K, Hattan S, et al. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents[J]. Mol Cell Proteomics.2004;3:1154-69.
[7]. Choe L, D'Ascenzo M, Relkin NR, Pappin D, Ross P, Williamson B, et al.8-plex quantitation of changes in cerebrospinal fluid protein expression in subjects undergoing intravenous immunoglobulin treatment for Alzheimer's disease[J]. Proteomics.2007;7:3651-60.
[8]. WHO. Definition, diagnosis and classification of diabetes mellitus and its complication[J]. WHOYNCD/NCS.1999:31-2.
[9]. Kahn R. Metabolic syndrome:is it a syndrome? Does it matter? [J] Circulation.2007; 115:1806-10; discussion 11.
[10]. Ford ES, Giles WH, Mokdad AH. Increasing prevalence of the metabolic syndrome among u.s. Adults[J]. Diabetes Care.2004;27:2444-9.
[11].陶世冰,任艳,田浩明,等.2007年成都地区成人代谢综合征患病率的流行病学调查[J].四川大学学报.2007；40：1062-5.
[12].樊晓红,蔡建芳,李学旺等.中国汉族人以尿白蛋白肌酐比值诊断微量白蛋白尿的界值研究[J].中华肾脏病杂志.2010；26：807-11
[13]. Laaksonen DE, Lakka HM, Niskanen LK, Kaplan GA, Salonen JT, Lakka TA. Metabolic syndrome and development of diabetes mellitus:application and validation of recently suggested definitions of the metabolic syndrome in a prospective cohort study [J]. Am J Epidemiol.2002; 156:1070-7.
[14]. Lakka HM, Laaksonen DE, Lakka TA, Niskanen LK, Kumpusalo E, Tuomilehto J, et al. The metabolic syndrome and total and cardiovascular disease mortality in middle-aged men [J]. JAMA. 2002;288:2709-16.
[15]. Panagiotis K MEaHJM. Multifactorial intervention in metabolic syndrome targeting at prevention of chronic kidney disease-ready for prime time [J]? Nephrol Dial Transplant.2007;22:2768-74.
[16]. Chen J, Muntner P, Hamm LL, Jones DW, Batuman V, Fonseca V, et al. The metabolic syndrome and chronic kidney disease in U.S. adults [J]. Ann Intern Med.2004; 140:167-74.
[17]. Tanaka H, Shiohira Y, Uezu Y, Higa A, Iseki K. Metabolic syndrome and chronic kidney disease in Okinawa, Japan [J]. Kidney Int.2006;69:369-74.
[18]. Agrawal V, Shah A, Rice C, Franklin BA, McCullough PA. Impact of treating the metabolic syndrome on chronic kidney disease [J].Nat Rev Nephrol.2009;5:520-8.
[19]. Ryu S, Chang Y, Woo HY, Lee KB, Kim SG, Kim DI, et al. Time-dependent association between metabolic syndrome and risk of CKD in Korean men without hypertension or diabetes [J]. Am J Kidney Dis.2009;53:59-69.
[20]. Chen J, Muntner P, Hamm LL, Jones DW, Batuman V, Fonseca V, et al. The metabolic syndrome and chronic kidney disease in U.S. adults [J]. Ann Intern Med.2004;140:167-74.
[21]. Koroshi A. Microalbuminuria, is it so important [J]? Hippokratia.2007;11:105-7.
[22]. Goligorsky MS, Addabbo F, O'Riordan E. Diagnostic potential of urine proteome:a broken mirror of renal diseases [J]. J Am Soc Nephrol.2007;18:2233-9.
[23]. Alberti KG, Zimmet P, Shaw J. The metabolic syndrome--a new worldwide definition [J]. Lancet. 2005;366:1059-62.
[24]. Thongboonkerd V. Practical points in urinary proteomics [J]. J Proteome Res.2007;6:3881-90.
[25].Nagaraj N, Mann M. Quantitative analysis of the intra- and inter-individual variability of the normal urinary proteome [J]. J Proteome Res.2011;10:637-45.
[26]. Wei Sun YC, Fuxin Li, Ling Zhang, Ruifeng Yang, Zhi Zhang, Dexian Zheng, Youhe Gao. Dynamic urinary proteomic analysis reveals stable proteins to be potential biomarkers [J]. Proteomics Clin. 2009;3:370-82.
[27]. Patel VJ, Thalassinos K, Slade SE, Connolly JB, Crombie A, Murrell JC, et al. A comparison of labeling and label-free mass spectrometry-based proteomics approaches [J]. J Proteome Res. 2009;8:3752-9.
[28]. Choi H, Fermin D, Nesvizhskii Al. Significance analysis of spectral count data in label-free shotgun proteomics [J]. Mol Cell Proteomics.2008;7:2373-85.
[29]. Usaite R, Wohlschlegel J, Venable JD, Park SK, Nielsen J, Olsson L, et al. Characterization of global yeast quantitative proteome data generated from the wild-type and glucose repression saccharomyces cerevisiae strains:the comparison of two quantitative methods [J]. J Proteome Res.2008;7:266-75.
[30].Neilson KA, Mariani M, Haynes PA. Quantitative proteomic analysis of cold-responsive proteins in rice [J]. Proteomics.2011;11:1696-706.
[31]. Old WM, Meyer-Arendt K, Aveline-Wolf L, Pierce KG, Mendoza A, Sevinsky JR, et al. Comparison of label-free methods for quantifying human proteins by shotgun proteomics [J]. Mol Cell Proteomics. 2005;4:1487-502.
[32]. Zybailov B, Mosley AL, Sardiu ME, Coleman MK, Florens L, Washburn MP. Statistical analysis of membrane proteome expression changes in Saccharomyces cerevisiae [J]. J Proteome Res.2006;5:2339-47.
[33]. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets:procedure and some applications [J]. Proc Natl Acad Sci U S A.1979;76:4350-4.
[34]. Hellman NE, Gitlin JD. Ceruloplasmin metabolism and function [J]. Annu Rev Nutr.2002;22:439-58.
[35]. Kosman DJ. FET3P, ceruloplasmin, and the role of copper in iron metabolism [J]. Adv Protein Chem. 2002;60:221-69.
[36]. Yamazaki M, Ito S, Usami A, Tani N, Hanyu O, Nakagawa O, et al. Urinary excretion rate of ceruloplasmin in non-insulin-dependent diabetic patients with different stages of nephropathy [J]. Eur J Endocrinol.1995;132:681-7.
[37].Narita T, Sasaki H, Hosoba M, Miura T, Yoshioka N, Morii T, et al. Parallel increase in urinary excretion rates of immunoglobulin G, ceruloplasmin, transferrin, and orosomucoid in normoalbuminuric type 2 diabetic patients [J]. Diabetes Care.2004;27:1176-81.
[38].Narita T, Fujita H, Koshimura J, Meguro H, Kitazato H, Shimotomai T, et al. Glycemic control reverses increases in urinary excretions of immunoglobulin G and ceruloplasmin in type 2 diabetic patients with normoalbuminuria [J]. Horm Metab Res.2001;33:370-8.
[39]. Ece A, Kelekci S, Hekimoglu A, Kocamaz H, Balik H, Yolbas I, et al. Neutrophil activation, protein oxidation and ceruloplasmin levels in children with Henoch-Schonlein purpura [J]. Pediatr Nephrol. 2007;22:1151-7.
[40]. Rao PV, Lu X, Standley M, Pattee P, Neelima G, Girisesh G, et al. Proteomic identification of urinary biomarkers of diabetic nephropathy [J]. Diabetes Care.2007;30:629-37.
[41]. Moon PG, Lee JE, You S, Kim TK, Cho JH, Kim IS, et al. Proteomic analysis of urinary exosomes from patients of early IgA nephropathy and thin basement membrane nephropathy [J]. Proteomics. 2011;11:2459-75.
[42]. Duverger N, Ghalim N, Ailhaud G, Steinmetz A, Fruchart JC, Castro G. Characterization of apoA-IV-containing lipoprotein particles isolated from human plasma and interstitial fluid [J]. Arterioscler Thromb.1993; 13:126-32.
[43]. von Eckardstein A, Huang Y, Wu S, Sarmadi AS, Schwarz S, Steinmetz A, et al. Lipoproteins containing apolipoprotein A-IV but not apolipoprotein A-I take up and esterify cell-derived cholesterol in plasma [J]. Arterioscler Thromb Vase Biol.1995;15:1755-63.
[44]. Duverger N, Tremp G, Caillaud JM, Emmanuel F, Castro G, Fruchart JC, et al. Protection against atherogenesis in mice mediated by human apolipoprotein A-IV [J]. Science.1996;273:966-8.
[45]. Cohen RD, Castellani LW, Qiao JH, Van Lenten BJ, Lusis AJ, Reue K. Reduced aortic lesions and elevated high density lipoprotein levels in transgenic mice overexpressing mouse apolipoprotein A-IV [J]. J Clin Invest.1997;99:1906-16.
[46]. Kronenberg F, Kuen E, Ritz E, Konig P, Kraatz G, Lhotta K, et al. Apolipoprotein A-IV serum concentrations are elevated in patients with mild and moderate renal failure [J]. J Am Soc Nephrol. 2002;13:461-9.
[47]. Lingenhel A, Lhotta K, Neyer U, Heid IM, Rantner B, Kronenberg MF, et al. Role of the kidney in the metabolism of apolipoprotein A-Ⅳ:influence of the type of proteinuria [J]. J Lipid Res.2006;47:2071-9.
[48]. Fisher DA, Salido EC, Barajas L. Epidermal growth factor and the kidney [J]. Annu Rev Physiol. 1989;51:67-80.
[49]. Gilbert RE, Cox A, McNally PG, Wu LL, Dziadek M, Cooper ME, et al. Increased epidermal growth factor in experimental diabetes related kidney growth in rats [J]. Diabetologia.1997;40:778-85.
[50]. Mathiesen ER, Nexo E, Hommel E, Parving HH. Reduced urinary excretion of epidermal growth factor in incipient and overt diabetic nephropathy [J]. Diabet Med.1989;6:121-6.
[51]. Stangou M, Alexopoulos E, Papagianni A, Pantzaki A, Bantis C, Dovas S, et al. Urinary levels of epidermal growth factor, interleukin-6 and monocyte chemoattractant protein-1 may act as predictor markers of renal function outcome in immunoglobulin A nephropathy [J]. Nephrology (Carlton). 2009; 14:613-20.
[52]. Cutillas PR, Chalkley RJ, Hansen KC, Cramer R, Norden AG, Waterfield MD, et al. The urinary proteome in Fanconi syndrome implies specificity in the reabsorption of proteins by renal proximal tubule cells [J]. Am J Physiol Renal Physiol.2004;287:F353-64.
[53]. Yang L, Li X, Wang H. Possible mechanisms explaining the tendency towards interstitial fibrosis in aristolochic acid-induced acute tubular necrosis [J]. Nephrol Dial Transplant.2007;22:445-56.
[54].Baer PC, Geiger H. Different effects of growth factors on human renal early distal tubular cells in vitro [J]. Kidney Blood Press Res.2006;29:225-30.
[55]. Haussler U, von Wichert G, Schmid RM, Keller F, Schneider G. Epidermal growth factor activates nuclear factor-kappaB in human proximal tubule cells [J]. Am J Physiol Renal Physiol.2005;289:F808-15.
[1]. WHO. Definition, diagnosis and classification of diabetes mellitus and its complication [J]. WHO/NCD/NCS.1999:31-2.
[2]. Kahn R. Metabolic syndrome:is it a syndrome? Does it matter [J]? Circulation.2007;115:1806-10; discussion 11.
[3]. Ford ES, Giles WH, Mokdad AH. Increasing prevalence of the metabolic syndrome among u.s. Adults [J]. Diabetes Care.2004;27:2444-9.
[4].陶世冰,任艳,田浩明,等.2007年成都地区成人代谢综合征患病率的流行病学调查[J].四川大学学报.2007;40：1062-5.
[5].樊晓红,蔡建芳,李学旺等.中国汉族人以尿白蛋白肌酐比值诊断微量白蛋白尿的界值研究[J].中华肾脏病杂志.2010;26：807-11
[6]. Laaksonen DE, Lakka HM, Niskanen LK, Kaplan GA, Salonen JT, Lakka TA. Metabolic syndrome and development of diabetes mellitus:application and validation of recently suggested definitions of the metabolic syndrome in a prospective cohort study [J]. Am J Epidemiol.2002; 156:1070-7.
[7]. Lakka HM, Laaksonen DE, Lakka TA, Niskanen LK, Kumpusalo E, Tuomilehto J, et al. The metabolic syndrome and total and cardiovascular disease mortality in middle-aged men [J]. JAMA. 2002;288:2709-16.
[8]. Panagiotis K MEaHJM. Multifactorial intervention in metabolic syndrome targeting at prevention of chronic kidney disease-ready for prime time [J]? Nephrol Dial Transplant.2007;22:2768-74.
[9]. Chen J, Muntner P, Hamm LL, Jones DW, Batuman V, Fonseca V, et al. The metabolic syndrome and chronic kidney disease in U.S. adults [J]. Ann Intern Med.2004; 140:167-74.
[10]. Tanaka H, Shiohira Y, Uezu Y, Higa A, Iseki K. Metabolic syndrome and chronic kidney disease in Okinawa, Japan [J]. Kidney Int.2006;69:369-74.
[11]. Koroshi A. Microalbuminuria, is it so important [J]? Hippokratia.2007; 11:105-7.
[12]. Ruan X, Guan Y. Metabolic syndrome and chronic kidney disease [J]. J Diabetes.2009;1:236-45.
[13]. Cornier MA, Dabelea D, Hernandez TL, Lindstrom RC, Steig AJ, Stob NR, et al. The metabolic syndrome [J]. Endocr Rev.2008;29:777-822.
[14]. Bravata DM, Wells CK, Concato J, Kernan WN, Brass LM, Gulanski BI. Two measures of insulin sensitivity provided similar information in a U.S. population [J]. J Clin Epidemiol.2004;57:1214-7.
[15]. St-Onge MP, Janssen I, Heymsfield SB. Metabolic syndrome in normal-weight Americans:new definition of the metabolically obese, normal-weight individual [J]. Diabetes Care.2004;27:2222-8.
[16]. Mayer-Davis EJ, D'Agostino R, Jr., Karter AJ, Haffner SM, Rewers MJ, Saad M, et al. Intensity and amount of physical activity in relation to insulin sensitivity:the Insulin Resistance Atherosclerosis Study [J]. JAMA.1998;279:669-74.
[17]. Eckel RH, Grundy SM, Zimmet PZ. The metabolic syndrome [J]. Lancet.2005;365:1415-28.
[18]. Tooke JE, Hannemann MM. Adverse endothelial function and the insulin resistance syndrome [J]. J Intern Med.2000;247:425-31.
[19]. Ferrannini E, Buzzigoli G, Bonadonna R, Giorico MA, Oleggini M, Graziadei L, et al. Insulin resistance in essential hypertension [J]. N Engl J Med.1987;317:350-7.
[20]. Bonora E, Capaldo B, Perin PC, Del Prato S, De Mattia G, Frittitta L, et al. Hyperinsulinemia and insulin resistance are independently associated with plasma lipids, uric acid and blood pressure in non-diabetic subjects [J]. The GISIR database. Nutr Metab Cardiovasc Dis.2008;18:624-31.
[21]. Anderson EA, Hoffman RP, Balon TW, Sinkey CA, Mark AL. Hyperinsulinemia produces both sympathetic neural activation and vasodilation in normal humans [J]. J Clin Invest.1991;87:2246-52.
[22]. Hall JE, Henegar JR, Dwyer TM, Liu J, Da Silva AA, Kuo JJ, et al. Is obesity a major cause of chronic kidney disease [J]? Adv Ren Replace Ther.2004; 11:41-54.
[23]. Roden M, Krssak M, Stingl H, Gruber S, Hofer A, Furnsinn C, et al. Rapid impairment of skeletal muscle glucose transport/phosphorylation by free fatty acids in humans [J]. Diabetes.1999;48:358-64.
[24].Norata GD, Ongari M, Garlaschelli K, Raselli S, Grigore L, Catapano AL. Plasma resistin levels correlate with determinants of the metabolic syndrome [J]. Eur J Endocrinol.2007;156:279-84.
[25]. Bahia L, Aguiar LG, Villela N, Bottino D, Godoy-Matos AF, Geloneze B, et al. Relationship between adipokines, inflammation, and vascular reactivity in lean controls and obese subjects with metabolic syndrome [J]. Clinics (Sao Paulo).2006;61:433-40.
[26].Xydakis AM, Case CC, Jones PH, Hoogeveen RC, Liu MY, Smith EO, et al. Adiponectin, inflammation, and the expression of the metabolic syndrome in obese individuals:the impact of rapid weight loss through caloric restriction [J]. J Clin Endocrinol Metab.2004;89:2697-703.
[27]. Chagnac A, Weinstein T, Korzets A, Ramadan E, Hirsch J, Gafter U. Glomerular hemodynamics in severe obesity [J]. Am J Physiol Renal Physiol.2000;278:F817-22.
[28]. Wolf G, Chen S, Han DC, Ziyadeh FN. Leptin and renal disease [J]. Am J Kidney Dis.2002;39:1-11.
[29]. Zhang R, Liao J, Morse S, Donelon S, Reisin E. Kidney disease and the metabolic syndrome [J]. Am J Med Sci.2005;330:319-25.
[30]. Kamijo A, Kimura K, Sugaya T, Yamanouchi M, Hase H, Kaneko T, et al. Urinary free fatty acids bound to albumin aggravate tubulointerstitial damage [J]. Kidney Int.2002;62:1628-37.
[31]. Cusumano AM, Bodkin NL, Hansen BC, lotti R, Owens J, Klotman PE, et al. Glomerular hypertrophy is associated with hyperinsulinemia and precedes overt diabetes in aging rhesus monkeys [J]. Am J Kidney Dis.2002;40:1075-85.
[32]. Abrass CK, Spicer D, Raugi GJ. Insulin induces a change in extracellular matrix glycoproteins synthesized by rat mesangial cells in culture [J]. Kidney Int.1994;46:613-20.
[33]. Palmer BF. Impaired renal autoregulation:implications for the genesis of hypertension and hypertension-induced renal injury [J]. Am J Med Sci.2001;321:388-400.
[34]. Sowers JR. Insulin resistance and hypertension [J]. Am J Physiol Heart Circ Physiol. 2004;286:H1597-602.
[35]. Bonventre JV. The kidney proteome:a hint of things to come [J]. Kidney Int.2002;62:1470-1.
[36]. O'Farrell PH. High resolution two-dimensional electrophoresis of proteins [J]. J Biol Chem. 1975;250:4007-21.
[37]. Timms JF, Cramer R. Difference gel electrophoresis [J]. Proteomics.2008;8:4886-97.
[38]. Unlu M, Morgan ME, Minden JS. Difference gel electrophoresis:a single gel method for detecting changes in protein extracts [J]. Electrophoresis.1997;18:2071-7.
[39]. Domon B, Aebersold R. Options and considerations when selecting a quantitative proteomics strategy [J]. Nat Biotechnol.2010;28:710-21.
[40]. Choi H, Fermin D, Nesvizhskii Al. Significance analysis of spectral count data in label-free shotgun proteomics [J]. Mol Cell Proteomics.2008;7:2373-85.
[41]. Usaite R, Wohlschlegel J, Venable JD, Park SK, Nielsen J, Olsson L, et al. Characterization of global yeast quantitative proteome data generated from the wild-type and glucose repression saccharomyces cerevisiae strains:the comparison of two quantitative methods [J]. J Proteome Res.2008;7:266-75.
[42]. Anderson L, Hunter CL. Quantitative mass spectrometric multiple reaction monitoring assays for major plasma proteins [J]. Mol Cell Proteomics.2006;5:573-88.
[43]. Gao J, Opiteck GJ, Friedrichs MS, Dongre AR, Hefta SA. Changes in the protein expression of yeast as a function of carbon source [J]. J Proteome Res.2003;2:643-9.
[44]. Ono M, Shitashige M, Honda K, Isobe T, Kuwabara H, Matsuzuki H, et al. Label-free quantitative proteomics using large peptide data sets generated by nanoflow liquid chromatography and mass spectrometry [J]. Mol Cell Proteomics.2006;5:1338-47.
[45].Neilson KA, Mariani M, Haynes PA. Quantitative proteomic analysis of cold-responsive proteins in rice [J]. Proteomics.2011;11:1696-706.
[46]. Gygi SP, Rist B, Gerber SA, Turecek F, Gelb MH, Aebersold R. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags [J]. Nat Biotechnol.1999;17:994-9.
[47]. Li J, Steen H, Gygi SP. Protein profiling with cleavable isotope-coded affinity tag (cICAT) reagents: the yeast salinity stress response [J]. Mol Cell Proteomics.2003;2:1198-204.
[48]. Ong SE, Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, et al. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics [J]. Mol Cell Proteomics.2002; 1:376-86.
[49]. Goligorsky MS, Addabbo F, O'Riordan E. Diagnostic potential of urine proteome:a broken mirror of renal diseases [J]. J Am Soc Nephrol.2007; 18:2233-9.
[50]. Mischak H, Apweiler R, Banks RE, Conaway M, Coon J, Dominiczak A, et al. Clinical proteomics:A need to define the field and to begin to set adequate standards [J]. Proteomics Clin Appl.2007; 1:148-56.
[51]. Thongboonkerd V. Current status of renal and urinary proteomics:ready for routine clinical application? [J]. Nephrol Dial Transplant.2009;25:11-6.
[52]. Pejcic M, Stojnev S, Stefanovic V. Urinary proteomics-a tool for biomarker discovery [J]. Ren Fail. 2010;32:259-68.
[53]. Smith MP, Banks RE, Wood SL, Lewington AJ, Selby PJ. Application of proteomic analysis to the study of renal diseases [J].Nat Rev Nephrol.2009;5:701-12.
[54]. Goligorsky MS, Addabbo F, O'Riordan E. Diagnostic potential of urine proteome:a broken mirror of renal diseases [J]. J Am Soc Nephrol.2007; 18:2233-9.
[55]. Woroniecki RP, Orlova TN, Mendelev N, Shatat IF, Hailpern SM, Kaskel FJ, et al. Urinary proteome of steroid-sensitive and steroid-resistant idiopathic nephrotic syndrome of childhood [J]. Am J Nephrol. 2006;26:258-67.
[56]. Merchant ML, Perkins BA, Boratyn GM, Ficociello LH, Wilkey DW, Barati MT, et al. Urinary peptidome may predict renal function decline in type 1 diabetes and microalbuminuria [J]. J Am Soc Nephrol.2009;20:2065-74.
[57]. Ho J, Lucy M, Krokhin O, Hayglass K, Pascoe E, Darroch G, et al. Mass spectrometry-based proteomic analysis of urine in acute kidney injury following cardiopulmonary bypass:a nested case-control study [J]. Am J Kidney Dis.2009;53:584-95.
[58]. Clarke W, Silverman BC, Zhang Z, Chan DW, Klein AS, Molmenti EP. Characterization of renal allograft rejection by urinary proteomic analysis [J]. Ann Surg.2003;237:660-4; discussion 4-5.
[59]. Mischak H, Kaiser T, Walden M, Hillmann M, Wittke S, Herrmann A, et al. Proteomic analysis for the assessment of diabetic renal damage in humans [J]. Clin Sci (Lond).2004; 107:485-95.
[60]. Thongboonkerd V, Klein JB, Jevans AW, McLeish KR. Urinary proteomics and biomarker discovery for glomerular diseases [J]. Contrib Nephrol.2004;141:292-307.
[61]. Haider H, Jiang SJ, Ye L, Aziz S, Law PK, Sim EK. Effectiveness of transient immunosuppression using cyclosporine for xenomyoblast transplantation for cardiac repair [J]. Transplant Proc. 2004;36:232-5.
[62]. Rao PV, Lu X, Standley M, Pattee P, Neelima G, Girisesh G, et al. Proteomic identification of urinary biomarkers of diabetic nephropathy [J]. Diabetes Care.2007;30:629-37.
[63]. Dihazi H, Asif AR, Agarwal NK, Doncheva Y, Muller GA. Proteomic analysis of cellular response to osmotic stress in thick ascending limb of Henle's loop (TALH) cells [J]. Mol Cell Proteomics. 2005;4:1445-58.
[64]. Rossing K, Mischak H, Dakna M, Zurbig P, Novak J, Julian BA, et al. Urinary proteomics in diabetes and CKD [J]. J Am Soc Nep hrol.2008;19:1283-90.
[65]. Chutipongtanate S, Nakagawa Y, Sritippayawan S, Pittayamateekul J, Parichatikanond P, Westley BR, et al. Identification of human urinary trefoil factor 1 as a novel calcium oxalate crystal growth inhibitor [J]. J Clin Invest.2005; 115:3613-22.
[66]. Beck LH, Jr., Bonegio RG, Lambeau G, Beck DM, Powell DW, Cummins TD, et al. M-type phospholipase A2 receptor as target antigen in idiopathic membranous nephropathy [J]. N Engl J Med. 2009;361:11-21.
[67]. Wasinger VC, Cordwell SJ, Cerpa-Poljak A, Yan JX, Gooley AA, Wilkins MR, et al. Progress with gene-product mapping of the Mollicutes:Mycoplasma genitalium [J]. Electrophoresis.1995; 16:1090-4.
[68]. Wilkins MR, Sanchez JC, Gooley AA, Appel RD, Humphery-Smith I, Hochstrasser DF, et al. Progress with proteome projects:why all proteins expressed by a genome should be identified and how to do it [J]. Biotechnol Genet Eng Rev.1996; 13:19-50.
[69]. Wilkins MR, Pasquali C, Appel RD, Ou K, Golaz O, Sanchez JC, et al. From proteins to proteomes: Large scale protein identification by two-dimensional electrophoresis and amino acid analysis [J]. Bio-Technol.1996; 14:61-5.
[70]. Ross PL, Huang YN, Marchese JN, Williamson B, Parker K, Hattan S, et al. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents [J]. Mol Cell Proteomics.2004;3:1154-69.
[71]. Dayon L, Hainard A, Licker V, Turck N, Kuhn K, Hochstrasser DF, et al. Relative quantification of proteins in human cerebrospinal fluids by MS/MS using 6-plex isobaric tags [J]. Anal Chem. 2008;80:2921-31.
[72]. Unwin RD, Evans CA, Whetton AD. Relative quantification in proteomics:new approaches for biochemistry [J]. Trends Biochem Sci.2006;31:473-84.
[73]. Wu WW, Wang G, Baek SJ, Shen RF. Comparative study of three proteomic quantitative methods, DIGE, cICAT, and iTRAQ, using 2D gel-or LC-MALDI TOF/TOF [J]. J Proteome Res.2006;5:651-8.
[74]. Chong PK, Gan CS, Pham TK, Wright PC. Isobaric tags for relative and absolute quantitation (iTRAQ) reproducibility:Implication of multiple injections [J]. J Proteome Res.2006;5:1232-40.
[75]. Brewis IA, Brennan P. Proteomics technologies for the global identification and quantification of proteins [J]. Adv Protein Chem Struct Biol.2010;80:l-44.
[76]. Pieper R, Gatlin CL, McGrath AM, Makusky AJ, Mondal M, Seonarain M, et al. Characterization of the human urinary proteome:a method for high-resolution display of urinary proteins on two-dimensional electrophoresis gels with a yield of nearly 1400 distinct protein spots [J]. Proteomics.2004;4:1159-74.
[77]. Afkarian M, Bhasin M, Dillon ST, Guerrero MC, Nelson RG, Knowler WC, et al. Optimizing a proteomics platform for urine biomarker discovery [J]. Mol Cell Proteomics.2010;9:2195-204.
[78]. Akkina SK, Zhang Y, Nelsestuen GL, Oetting WS, Ibrahim HN. Temporal stability of the urinary proteome after kidney transplant:more sensitive than protein composition [J]? J Proteome Res. 2009;8:94-103.
[79]. Wenderfer SE, Dubinsky WP, Hernandez-Sanabria M, Braun MC. Urine proteome analysis in murine nephrotoxic serum nephritis [J]. Am J Nephrol.2009;30:450-8.
[80]. Chen YT, Chen CL, Chen HW, Chung T, Wu CC, Chen CD, et al. Discovery of novel bladder cancer biomarkers by comparative urine proteomics using iTRAQ technology [J]. J Proteome Res. 2010;9:5803-15.
[81]. Chen G, Zhang Y, Jin X, Zhang L, Zhou Y, Niu J, et al. Urinary proteomics analysis for renal injury in hypertensive disorders of pregnancy with iTRAQ labeling and LC-MS/MS [J]. Proteomics Clin Appl. 2011;5:300-10.
[82]. Kaspar H, Dettmer K, Chan Q, Daniels S, Nimkar S, Daviglus ML, et al. Urinary amino acid analysis: a comparison of iTRAQ-LC-MS/MS, GC-MS, and amino acid analyzer [J]. J Chromatogr B Analyt Technol Biomed Life Sci.2009;877:1838-46.
[83]. DeSouza LV, Taylor AM, Li W, Minkoff MS, Romaschin AD, Colgan TJ, et al. Multiple reaction monitoring of mTRAQ-Iabeled peptides enables absolute quantification of endogenous levels of a potential cancer marker in cancerous and normal endometrial tissues [J]. J Proteome Res.2008;7:3525-34.
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