Critical comparison of sample preparation strategies for shotgun proteomic analysis of formalin-fixed, paraffin-embedded samples: insights from liver tissue

Clinical Proteomics

Salvatore Pisanu¹  Maria Filippa Addis¹  Sergio Uzzau²  Alessandro Tanca¹  Marcello Abbondio¹  Daniela Pagnozzi¹ 

[1] Porto Conte Ricerche, Tramariglio, Alghero, ItalyPorto Conte Ricerche, Tramariglio, Alghero, ItalyPorto Conte Ricerche, Tramariglio, Alghero, Italy;Porto Conte Ricerche, Tramariglio, Alghero, ItalyDipartimento di Scienze Biomediche, Università di Sassari, Sassari, ItalyPorto Conte Ricerche, Tramariglio, Alghero, ItalyPorto Conte Ricerche, Tramariglio, Alghero, ItalyDipartimento di Scienze Biomediche, Università di Sassari, Sassari, ItalyDipartimento di Scienze Biomediche, Università di Sassari, Sassari, ItalyPorto Conte Ricerche, Tramariglio, Alghero, ItalyDipartimento di Scienze Biomediche, Università di Sassari, Sassari, Italy

关键词: Archival tissues;    FASP;    FFPE;    LC-MS/MS;    Protein extraction;   

DOI  :  10.1186/1559-0275-11-28

来源: Humana Press Inc

【 摘 要 】

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.

【 授权许可】

Unknown