Critical comparison of sample preparation strategies for shotgun proteomic analysis of formalin-fixed, paraffin-embedded samples: insights from liver tissue
| dc.contributor.author | Tanca, Alessandro | |
| dc.contributor.author | Abbondio, Marcello | |
| dc.contributor.author | Pisanu, Salvatore | |
| dc.contributor.author | Pagnozzi, Daniela | |
| dc.contributor.author | Uzzau, Sergio | |
| dc.contributor.author | Addis, Maria Filippa | |
| dc.date.accessioned | 2014-12-09T12:37:56Z | |
| dc.date.available | 2014-12-09T12:37:56Z | |
| dc.date.issued | 2014-07-08 | |
| dc.description.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. Experimental design: 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. | IT |
| dc.description.status | Pubblicato | IT |
| dc.identifier.doi | 10.1186/1559-0275-11-28 | IT |
| dc.identifier.eissn | 1559-0275 | |
| dc.identifier.issn | 1542-6416 | |
| dc.identifier.uri | http://hdl.handle.net/11050/1133 | |
| dc.language.iso | en | IT |
| dc.publisher | BioMed Central | IT |
| dc.relation.ispartof | Clinical Proteomics | IT |
| dc.relation.ispartofseries | 11;28 | |
| dc.rights | Attribuzione - Non commerciale - Non opere derivate 3.0 Italia | * |
| dc.rights.uri | http://creativecommons.org/licenses/by-nc-nd/3.0/it/ | * |
| dc.subject | archival tissues | IT |
| dc.subject | FASP | IT |
| dc.subject | FFPE | IT |
| dc.subject | LC-MS/MS | IT |
| dc.subject | protein extraction | IT |
| dc.subject.een-cordis | EEN CORDIS::SCIENZE BIOLOGICHE ::Ricerca sul genoma ::Espressione genica, ricerca proteomica | IT |
| dc.title | Critical comparison of sample preparation strategies for shotgun proteomic analysis of formalin-fixed, paraffin-embedded samples: insights from liver tissue | IT |
| dc.type | Articolo | IT |