Critical comparison of sample preparation strategies for shotgun proteomic analysis of formalin-fixed, paraffin-embedded samples

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dc.contributor.authorAbbondio, Marcello
dc.contributor.authorTanca, Alessandro
dc.contributor.authorPisanu, Salvatore
dc.contributor.authorUzzau, Sergio
dc.contributor.authorPagnozzi, Daniela
dc.contributor.authorAddis, Maria Filippa
dc.date.accessioned2014-12-17T09:04:33Z
dc.date.available2014-12-17T09:04:33Z
dc.date.issued2014-10-06
dc.description.abstractIntroduction and objectives. The growing field of formalin-fixed paraffin-embedded (FFPE) tissue proteomics holds promise for improving translational research. Worldwide archival tissue banks hold a significant number and variety of tissue samples, as well as a wealth of retrospective information regarding diagnosis, prognosis, and response to therapy. This makes them an important resource for protein biomarker discovery and validation. 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 LC-MS/MS analysis of FFPE samples, but a critical comparison of the different methods is currently lacking. Methods DT was preceded by homogenization in ammonium bicarbonate, while ISD and FASP comprised protein extraction in SDS based-buffer, followed by SDS depletion with Detergent Removal Spin Columns and Microcon Ultracel YM-30 filtration devices, respectively. The three workflows were applied to consecutive tissue sections cut from an FFPE liver tissue block, and peptide mixtures were finally analyzed according to a label-free quantitative MS approach. Data were evaluated in terms of method reproducibility and protein/peptide distribution according to localization, MW, pI and hydrophobicity. Results and Discussion. 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. Conclusion. 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.conferencedate2014-10-06
dc.description.conferencelocationMadrid, SpainIT
dc.description.conferencetitle13th Human Proteome Organization World CongressIT
dc.identifier.urihttp://hdl.handle.net/11050/1139
dc.language.isoen_USIT
dc.rightsCC0 1.0 Universal*
dc.rights.urihttp://creativecommons.org/publicdomain/zero/1.0/*
dc.subjectFASPIT
dc.subjectFFPEIT
dc.subjectLC-MS/MSIT
dc.subjectprotein extractionIT
dc.subjectarchival tissuesIT
dc.subject.een-cordisEEN CORDIS::SCIENZE BIOLOGICHE ::Ricerca sul genoma ::Espressione genica, ricerca proteomicaIT
dc.titleCritical comparison of sample preparation strategies for shotgun proteomic analysis of formalin-fixed, paraffin-embedded samplesIT
dc.typeContributo a convegnoIT
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