title: Differential denaturation of serum proteome reveals a significant amount of hidden information in complex mixtures of proteins.
abstract: Recently developed proteomic technologies allow to profile thousands of proteins within a high-throughput approach towards biomarker discovery, although results are not as satisfactory as expected. In the present study we demonstrate that serum proteome denaturation is a key underestimated feature; in fact, a new differential denaturation protocol better discriminates serum proteins according to their electrophoretic mobility as compared to single-denaturation protocols. Sixty nine different denaturation treatments were tested and the 3 most discriminating ones were selected (TRIDENT analysis) and applied to human sera, showing a significant improvement of serum protein discrimination as confirmed by MALDI-TOF/MS and LC-MS/MS identification, depending on the type of denaturation applied. Thereafter sera from mice and patients carrying cutaneous melanoma were analyzed through TRIDENT. Nine and 8 protein bands were found differentially expressed in mice and human melanoma sera, compared to healthy controls (p<0.05); three of them were found, for the first time, significantly modulated: α2macroglobulin (down-regulated in melanoma, p<0.001), Apolipoprotein-E and Apolipoprotein-A1 (both up-regulated in melanoma, p<0.04), both in mice and humans. The modulation was confirmed by immunological methods. Other less abundant proteins (e.g. gelsolin) were found significantly modulated (p<0.05).
  
  
  CONCLUSIONS
  i) serum proteome contains a large amount of information, still neglected, related to proteins folding; ii) a careful serum denaturation may significantly improve analytical procedures involving complex protein mixtures; iii) serum differential denaturation protocol highlights interesting proteomic differences between cancer and healthy sera.
authors.affiliation: Dip. Ematologia, Oncologia e Medicina Molecolare, Istituto Superiore di Sanità, Roma, Italy.
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Verdoliva
authors.forename: Cinzia
authors.initials: C
authors.lastname: Senatore
authors.forename: Maria Letizia
authors.initials: ML
authors.lastname: Polci
authors.forename: Stefania
authors.initials: S
authors.lastname: Rossi
authors.forename: Martina
authors.initials: M
authors.lastname: Cordella
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Carlucci
authors.forename: Paolo
authors.initials: P
authors.lastname: Marchetti
authors.forename: Giancarlo
authors.initials: G
authors.lastname: Antonini-Cappellini
authors.forename: Antonio
authors.initials: A
authors.lastname: Facchiano
authors.forename: Daniela
authors.initials: D
authors.lastname: D'Arcangelo
authors.forename: Francesco
authors.initials: F
authors.lastname: Facchiano
mesh_terms: D000818:Animals; D001798:Blood Proteins / Q000737:chemistry* / Q000378:metabolism*; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D019295:Computational Biology; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008297:Male; D013058:Mass Spectrometry; D051379:Mice; D008810:Mice, Inbred C57BL; D017510:Protein Folding; D040901:Proteomics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D001798:Blood Proteins