title: Critical comparison of sample preparation strategies for shotgun proteomic analysis of formalin-fixed, paraffin-embedded samples: insights from liver tissue.
abstract: BACKGROUND
  The growing field of formalin-fixed paraffin-embedded (FFPE) tissue proteomics holds promise for improving translational research. Direct tissue trypsinization (DT) and protein extraction followed by in solution digestion (ISD) or filter-aided sample preparation (FASP) are the most common workflows for shotgun analysis of FFPE samples, but a critical comparison of the different methods is currently lacking.
  
  
  METHODS
  DT, FASP and ISD workflows were compared by subjecting to the same label-free quantitative approach three independent technical replicates of each method applied to FFPE liver tissue. Data were evaluated in terms of method reproducibility and protein/peptide distribution according to localization, MW, pI and hydrophobicity.
  
  
  RESULTS
  DT showed lower reproducibility, good preservation of high-MW proteins, a general bias towards hydrophilic and acidic proteins, much lower keratin contamination, as well as higher abundance of non-tryptic peptides. Conversely, FASP and ISD proteomes were depleted in high-MW proteins and enriched in hydrophobic and membrane proteins; FASP provided higher identification yields, while ISD exhibited higher reproducibility.
  
  
  CONCLUSIONS
  These results highlight that diverse sample preparation strategies provide significantly different proteomic information, and present typical biases that should be taken into account when dealing with FFPE samples. When a sufficient amount of tissue is available, the complementary use of different methods is suggested to increase proteome coverage and depth.
authors.affiliation: Porto Conte Ricerche, S.P. 55 Porto Conte/Capo Caccia Km 8.400, Tramariglio, 07041 Alghero, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Tanca
authors.affiliation: Porto Conte Ricerche, S.P. 55 Porto Conte/Capo Caccia Km 8.400, Tramariglio, 07041 Alghero, Italy.
authors.forename: Marcello
authors.initials: M
authors.lastname: Abbondio
authors.affiliation: Porto Conte Ricerche, S.P. 55 Porto Conte/Capo Caccia Km 8.400, Tramariglio, 07041 Alghero, Italy.
authors.forename: Salvatore
authors.initials: S
authors.lastname: Pisanu
authors.affiliation: Porto Conte Ricerche, S.P. 55 Porto Conte/Capo Caccia Km 8.400, Tramariglio, 07041 Alghero, Italy.
authors.forename: Daniela
authors.initials: D
authors.lastname: Pagnozzi
authors.affiliation: Porto Conte Ricerche, S.P. 55 Porto Conte/Capo Caccia Km 8.400, Tramariglio, 07041 Alghero, Italy ; Dipartimento di Scienze Biomediche, Università di Sassari, Viale San Pietro 43/B, 07100, Sassari, Italy.
authors.forename: Sergio
authors.initials: S
authors.lastname: Uzzau
authors.affiliation: Porto Conte Ricerche, S.P. 55 Porto Conte/Capo Caccia Km 8.400, Tramariglio, 07041 Alghero, Italy.
authors.forename: Maria Filippa
authors.initials: MF
authors.lastname: Addis
keywords: Archival tissues; FASP; FFPE; LC-MS/MS; Protein extraction
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