Instituto de Investigaciones Biomédicas, Alberto Sols, Consejo Superior de Investigaciones Científicas & Universidad Autónoma de Madrid, Madrid, Spain.
PLoS One. 2012;7(1):e30326. doi: 10.1371/journal.pone.0030326. Epub 2012 Jan 17.
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.
基于人体两个主要的腹部脂肪储存部位，即皮下脂肪和网膜脂肪，进行蛋白质表达研究可以揭示肥胖及其合并症相关的机制。在这里，我们首次确定和验证了用于数据标准化的参考蛋白，这对于准确比较肥胖和非肥胖个体的脂肪表达研究中的蛋白质水平至关重要。
为了发现网膜和皮下脂肪组织中表达水平相同的脂肪组织蛋白（研究 1）或非肥胖和肥胖个体的网膜脂肪中表达水平相同的蛋白（研究 2），我们重新分析了以前基于二维荧光差异凝胶电泳发表的数据。选择并通过质谱鉴定了在所有测试条件下表达水平相似的 24 种蛋白质（研究 1 中 12 种，研究 2 中 12 种）。免疫印迹分析用于确认潜在参考蛋白在脂肪组织中的表达模式，其中 3 种蛋白得到验证：PARK7、ENOA 和 FAA。Western Blot 分析还用于测试常规的加载对照蛋白。ENOA、PARK7 和常规的加载对照蛋白 Beta-actin 在非肥胖和肥胖个体的脂肪中表现出稳定的表达谱，而 FAA 在配对的网膜和皮下脂肪样本中保持稳定的表达水平。
ENOA、PARK7 和 Beta-actin 是基于网膜脂肪的肥胖研究中的合适参考标准，而 FAA 是肥胖和非肥胖受试者网膜和皮下脂肪组织比较分析的最佳加载对照。在脂肪组织差异表达研究中，常规的加载对照蛋白 GAPDH 和 TBB5 以及 CALX 都不是合适的标准。使用建议的参考蛋白将有助于在肥胖背景下对差异表达的蛋白质进行适当分析，这是在本研究之前难以实现的目标。
