Uncovering suitable reference proteins for expression studies in human adipose tissue with relevance to obesity.
Uncovering suitable reference proteins for expression studies in human adipose tissue with relevance to obesity.
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DOI：
10.1371/journal.pone.0030326
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Peral B
中科院分区：
文献类型：
--
作者：
Pérez-Pérez R;López JA;García-Santos E;Camafeita E;Gómez-Serrano M;Ortega-Delgado FJ;Ricart W;Fernández-Real JM;Peral B
蛋白质表达研究的基础上的两个主要的腹内人体脂肪库，皮下和网膜脂肪，可以揭示肥胖及其并发症的机制。在这里，我们解决，第一次，参考蛋白的数据标准化，这是必不可少的准确比较蛋白质水平的表达研究的基础上，从肥胖和非肥胖个体的脂肪的识别和验证。为了揭示在网膜和皮下脂肪库（研究1）或非肥胖和肥胖个体（研究2）的网膜脂肪中同等表达的脂肪组织蛋白，我们重新分析了我们以前发表的基于二维荧光差异凝胶电泳的数据。选择在所有测试条件下具有相似表达水平的24种蛋白质（研究1中的12种和研究2中的12种），并通过质谱法鉴定。免疫印迹分析用于确认脂肪组织中潜在参考蛋白的表达模式，并验证了三种蛋白：PARK 7、ENOA和FAA。还使用蛋白质印迹分析来测试常规上样对照蛋白。ENOA、PARK 7和常规上样对照蛋白β-肌动蛋白在来自非肥胖和肥胖个体的脂肪中显示稳定的表达谱，而FAA在配对的网膜和皮下脂肪样品中保持稳定的表达水平。ENOA、PARK 7和β-肌动蛋白是基于网膜脂肪的肥胖研究中的适当参考标准，而FAA是肥胖和非肥胖受试者中网膜和皮下脂肪组织比较分析的最佳负荷对照。在脂肪组织的差异表达研究中，常规上样对照蛋白GAPDH和TBB 5以及CALX都不是适当的标准。使用拟议的参考蛋白将有助于充分分析蛋白质差异表达的背景下，肥胖，一个目标，难以实现本研究之前。
Protein expression studies based on the two major intra-abdominal human fat depots, the subcutaneous and the omental fat, can shed light into the mechanisms involved in obesity and its co-morbidities. Here we address, for the first time, the identification and validation of reference proteins for data standardization, which are essential for accurate comparison of protein levels in expression studies based on fat from obese and non-obese individuals. To uncover adipose tissue proteins equally expressed either in omental and subcutaneous fat depots (study 1) or in omental fat from non-obese and obese individuals (study 2), we have reanalyzed our previously published data based on two-dimensional fluorescence difference gel electrophoresis. Twenty-four proteins (12 in study 1 and 12 in study 2) with similar expression levels in all conditions tested were selected and identified by mass spectrometry. Immunoblotting analysis was used to confirm in adipose tissue the expression pattern of the potential reference proteins and three proteins were validated: PARK7, ENOA and FAA. Western Blot analysis was also used to test customary loading control proteins. ENOA, PARK7 and the customary loading control protein Beta-actin showed steady expression profiles in fat from non-obese and obese individuals, whilst FAA maintained steady expression levels across paired omental and subcutaneous fat samples. ENOA, PARK7 and Beta-actin are proper reference standards in obesity studies based on omental fat, whilst FAA is the best loading control for the comparative analysis of omental and subcutaneous adipose tissues either in obese and non-obese subjects. Neither customary loading control proteins GAPDH and TBB5 nor CALX are adequate standards in differential expression studies on adipose tissue. The use of the proposed reference proteins will facilitate the adequate analysis of proteins differentially expressed in the context of obesity, an aim difficult to achieve before this study.
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影响因子：
7.7
作者：
Mantovani G;Bondioni S;Alberti L;Gilardini L;Invitti C;Corbetta S;Zappa MA;Ferrero S;Lania AG;Bosari S;Beck-Peccoz P;Spada A
通讯作者：
Spada A
影响因子：
4.4
作者：
Rasche A;Al-Hasani H;Herwig R
通讯作者：
Herwig R
DOI：
10.1073/pnas.0601752103
发表时间：
2006-04-25
影响因子：
11.1
作者：
Gesta, S;Blüher, M;Kahn, CR
通讯作者：
Kahn, CR
影响因子：
4.4
作者：
Perez-Perez, Rafael;Ortega-Delgado, Francisco J.;Peral, Belen
通讯作者：
Peral, Belen
影响因子：
50.3
作者：
Kim, RH;Peters, M;Mak, TW
通讯作者：
Mak, TW