title: Identification and validation of platelet low biological variation proteins, superior to GAPDH, actin and tubulin, as tools in clinical proteomics.
abstract: Accurate biomarker quantification requires carefully chosen normalisation procedures. When single proteins are used as loading controls (LCs), it is crucial that their expressional stability must be known. Platelets are an important biomarker source, especially for neurological diseases. We performed a systematical analysis of the platelet proteome to identify proteins suitable as LCs, using the 2-D DIGE system. We first screened a healthy population (n=137), aged between 18 and 104years, to find proteins with small coefficients of total variation (CVtot), herein termed low biological variation proteins (LBVP). Thereafter, expressional stability was verified in 101 patients suffering from Alzheimer's- (AD), Parkinson's- disease, vascular dementia or schizophrenia. Interestingly, traditional LCs such as tubulin beta-1 and GAPDH, were not found amongst LBVP. The least variable protein, calculated over all 238 individuals, was 14-3-3 gamma, with a CVtot of 9.3%, showing no gender, age or disease dependency. The normalisation capability of 14-3-3 gamma was superior to traditional LC in quantifying Western blot signals of the platelet AD-biomarker Monoamine Oxidase B of patient versus controls. Similar results were obtained with HepG2 cells, treated in vitro with DNA-methyltransferase inhibitor 5-aza-2'deoxicytidine. Finally, we provide a list of alternative normalisation candidates for accurate biomarker quantification.
  
  
  CONCLUSIONS
  This paper suggests a considerable list of platelet proteins with a lower biological variation than well known "housekeeping" proteins like GAPDH and tubulin. Spot abundances of found proteins are middle ranged and unaffected by gender, age and certain diseases. Hence, listed proteins might be valuable normalisation candidates used additionally or alternatively. Platelet's least variable protein 14-3-3 gamma is validated as normalisation protein in platelet biomarker quantification. Furthermore 14-3-3 gamma is demonstrated to be also stable expressed by in HepG2, cells others than platelets, when treated by DNA methylation inhibitor.
authors.affiliation: Institute of Physiology, Center of Physiology and Pharmacology, Medical University of Vienna, 1090 Vienna, Austria.
authors.forename: Roland
authors.initials: R
authors.lastname: Baumgartner
authors.forename: Ellen
authors.initials: E
authors.lastname: Umlauf
authors.forename: Michael
authors.initials: M
authors.lastname: Veitinger
authors.forename: Sheila
authors.initials: S
authors.lastname: Guterres
authors.forename: Eduard
authors.initials: E
authors.lastname: Rappold
authors.forename: Rita
authors.initials: R
authors.lastname: Babeluk
authors.forename: Goran
authors.initials: G
authors.lastname: Mitulović
authors.forename: Rudolf
authors.initials: R
authors.lastname: Oehler
authors.forename: Maria
authors.initials: M
authors.lastname: Zellner
mesh_terms: D048948:14-3-3 Proteins / Q000737:chemistry / Q000378:metabolism*; D000199:Actins / Q000737:chemistry / Q000378:metabolism; D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000544:Alzheimer Disease / Q000378:metabolism; D015415:Biomarkers / Q000737:chemistry / Q000378:metabolism; D001792:Blood Platelets / Q000737:chemistry / Q000378:metabolism*; D015153:Blotting, Western / Q000592:standards*; D015140:Dementia, Vascular / Q000378:metabolism; D005260:Female; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000737:chemistry / Q000378:metabolism; D056945:Hep G2 Cells; D006801:Humans; D008297:Male; D008875:Middle Aged; D008995:Monoamine Oxidase / Q000737:chemistry / Q000378:metabolism; D010300:Parkinson Disease / Q000378:metabolism; D040901:Proteomics / Q000592:standards*; D012559:Schizophrenia / Q000378:metabolism; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism
chemical_list: D048948:14-3-3 Proteins; D000199:Actins; D015415:Biomarkers; D014404:Tubulin; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); D008995:Monoamine Oxidase
keywords: 1-D WB; 14-3-3 gamma; 2-D DIGE; Alzheimer's disease; Biomarkers; DTT; GADPH; Glyceraldehyde 3-phosphate dehydrogenase; LBVP; LC; Loading controls; Mao-B; Platelets; RuBPS; SA; SD; TFA; dithiothreitol; loading control; low biological variation protein; monoamine oxidase B; one-dimensional Western blot; ruthenium(II) tris(bathophenthroline disulfonate); standard deviation; standardised abundance; trifluoroacetic acid; two-dimensional difference gel electrophoresis