title: Application of 2-D DIGE to formalin-fixed, paraffin-embedded tissues.
abstract: The ability to investigate the proteome of formalin-fixed, paraffin-embedded (FFPE) tissues can be considered a major recent achievement in the field of clinical proteomics. However, gel-based approaches to the investigation of FFPE tissue proteomes have lagged behind, mainly because of insufficient quality of full-length protein extracts. Here, the 2-D DIGE technology was investigated for applicability to FFPE proteins, for internal reproducibility among replicate FFPE extracts, and for comparability between FFPE and fresh-frozen tissue profiles. The 2-D DIGE patterns obtained upon labeling and electrophoresis of replicate FFPE tissue extracts were highly reproducible, with satisfactory resolution and complexity. Moreover, the implementation of DIGE enabled to highlight and characterize the consistent differences found in the FFPE profiles compared with fresh-frozen profiles, represented by an acidic shift, directly correlated to the protein pI value, and by a reduction in spot signal intensity, directly correlated to molecular weight and percentage of lysine residues. Being constantly and reproducibly present in all FFPE tissue extract replicates at similar extents, these modifications do not appear to hinder the comparative analysis of FFPE tissue extracts by 2-D DIGE, opening the way to its application for the differential proteomic investigation of archival tissue repositories.
authors.affiliation: Porto Conte Ricerche Srl, Alghero, Sassari, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Tanca
authors.forename: Daniela
authors.initials: D
authors.lastname: Pagnozzi
authors.forename: Giovanni
authors.initials: G
authors.lastname: Falchi
authors.forename: Roberto
authors.initials: R
authors.lastname: Tonelli
authors.forename: Stefano
authors.initials: S
authors.lastname: Rocca
authors.forename: Tonina
authors.initials: T
authors.lastname: Roggio
authors.forename: Sergio
authors.initials: S
authors.lastname: Uzzau
authors.forename: Maria Filippa
authors.initials: MF
authors.lastname: Addis
mesh_terms: D000818:Animals; D005404:Fixatives / Q000737:chemistry; D005557:Formaldehyde / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000737:chemistry; D008970:Molecular Weight; D016612:Paraffin Embedding / Q000706:statistics & numerical data; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D020543:Proteome / Q000032:analysis* / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D012756:Sheep; D014020:Tissue Extracts / Q000737:chemistry; D016707:Tissue Fixation / Q000706:statistics & numerical data; D057825:Two-Dimensional Difference Gel Electrophoresis / Q000706:statistics & numerical data*
chemical_list: D005404:Fixatives; D011506:Proteins; D020543:Proteome; D014020:Tissue Extracts; D005557:Formaldehyde; D008239:Lysine