title: Uncovering suitable reference proteins for expression studies in human adipose tissue with relevance to obesity.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Instituto de Investigaciones Biomédicas, Alberto Sols, Consejo Superior de Investigaciones Científicas & Universidad Autónoma de Madrid, Madrid, Spain.
authors.forename: Rafael
authors.initials: R
authors.lastname: Pérez-Pérez
authors.forename: Juan A
authors.initials: JA
authors.lastname: López
authors.forename: Eva
authors.initials: E
authors.lastname: García-Santos
authors.forename: Emilio
authors.initials: E
authors.lastname: Camafeita
authors.forename: María
authors.initials: M
authors.lastname: Gómez-Serrano
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Ortega-Delgado
authors.forename: Wifredo
authors.initials: W
authors.lastname: Ricart
authors.forename: José M
authors.initials: JM
authors.lastname: Fernández-Real
authors.forename: Belén
authors.initials: B
authors.lastname: Peral
mesh_terms: D000199:Actins / Q000378:metabolism; D000328:Adult; D015415:Biomarkers / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000378:metabolism; D015153:Blotting, Western; D004268:DNA-Binding Proteins / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008875:Middle Aged; D009765:Obesity / Q000378:metabolism*; D009852:Omentum / Q000378:metabolism*; D015513:Oncogene Proteins / Q000378:metabolism; D010751:Phosphopyruvate Hydratase / Q000378:metabolism; D000071617:Protein Deglycase DJ-1; D011506:Proteins / Q000378:metabolism*; D020543:Proteome / Q000378:metabolism; D040901:Proteomics / Q000379:methods / Q000592:standards; D012015:Reference Standards; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D050151:Subcutaneous Fat / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000378:metabolism
chemical_list: D000199:Actins; D015415:Biomarkers; D014408:Biomarkers, Tumor; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D015513:Oncogene Proteins; D011506:Proteins; D020543:Proteome; D025521:Tumor Suppressor Proteins; C105131:PARK7 protein, human; D000071617:Protein Deglycase DJ-1; C095157:ENO1 protein, human; D010751:Phosphopyruvate Hydratase