Setting proteins free: Progresses and achievements in proteomics of formalin‐fixed, paraffin‐embedded tissues

Abstract

Formalin fixation, followed by paraffin embedding, is long established as the standard procedure for the stabilization and preservation of tissue architecture, essential for enabling microscopic examination and long-term storage of samples. During the years, this has led to the generation of a worldwide repository of patient tissues with associated complete clinical records. As such, this represents a golden mine for all those attempting to identify proteomic signatures of disease, aimed to the understanding of pathological processes and to the identification of new biomarkers. However, access to this resource has been hampered by the stable cross-linked network generated on tissue molecules during formalin fixation. Recently, researchers have been actively working to overcome this limitation, reaching unexpected achievements. This review aims to discuss and compare the various strategies devised for extracting full-length proteins or peptides from fixed tissues, and to provide a general perspective on studies comparing matched fixed and fresh-frozen tissue proteomes, applying proteomic techniques for biomarker discovery from archival tissues, and attempting to exploit gel-based approaches. In addition, the concomitant progresses in understanding the impact of tissue processing variables and the extent and nature of formaldehyde-induced modifications are presented. In conclusion, the future perspectives and open challenges in this field are discussed.

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

“…Archival tissue repositories are indeed a valuable resource for protein biomarker discovery and validation, since they hold a considerable number and variety of tissue specimens, including those from patients with rare malignancies, and provide retrospective information concerning diagnosis, survival, and response to therapy [2,3]. …”

“…In order to exploit this enormous potential, in the last years researchers have been aiming at evaluating the suitability of a wide array of techniques for FFPE tissue proteome analysis, including gel-based approaches (2DE-MS, DIGE-MS, GeLC-MS/MS [3-7]), shotgun LC-MS/MS (both label-based and label-free [8-13]), as well as targeted MS [14] and imaging MS [15]. With respect to shotgun LC-MS/MS, direct tissue trypsinization (DT) and protein extraction followed by in solution digestion (ISD) are the most commonly used strategies; more recently, an efficient alternative for processing FFPE protein extracts through the filter-aided sample preparation (FASP) workflow has been introduced [3,8,11].…”

“…With respect to shotgun LC-MS/MS, direct tissue trypsinization (DT) and protein extraction followed by in solution digestion (ISD) are the most commonly used strategies; more recently, an efficient alternative for processing FFPE protein extracts through the filter-aided sample preparation (FASP) workflow has been introduced [3,8,11]. These sample preparation workflows are all preceded by tissue deparaffinization and rehydration and usually comprise a high temperature treatment, but exhibit several differences in the other steps.…”

Quantification of HER2 by Targeted Mass Spectrometry in Formalin-Fixed Paraffin-Embedded (FFPE) Breast Cancer Tissues

“…eral studies have shown that although the individual peptides retrieved and identified from fresh-frozen and FFPE tissues may differ, the biological information obtained from both types of material in terms of number of proteins identified, cellular location and molecular function is very similar (5)(6)(7)(8)(9)(10). A number of proteomics studies were reported, which used untargeted MS on FFPE tissues to compare diseased and healthy samples in the search for potential novel biomarkers (10).…”

Label-free mass spectrometry-based proteomics for biomarker discovery and validation

“…Proteome-level mining of this treasure trove of biomarkers was long hampered by the inaccessibility of the proteins due to extensive formalininduced covalent cross-linking. In a recent review, Tanca et al described various strategies devised for the extraction of fulllength proteins or peptides from fixed tissues, and the impact of tissue processing variables [91]. In addition, the feasibility of the analysis of the phosphoproteome and N-glycoproteome from FFPE material has also been recently shown [92].…”