Proteomic Workflows for High-Quality Quantitative Proteome and Post-Translational Modification Analysis of Clinically Relevant Samples from Formalin-Fixed Paraffin-Embedded Archives

Abstract

Well-characterized archival formalin-fixed paraffin-embedded (FFPE) tissues are of much value for prospective biomarker discovery studies, and protocols that offer high throughput and good reproducibility are essential in proteomics. Therefore, we implemented efficient paraffin removal and protein extraction from FFPE tissues followed by an optimized two-enzyme digestion using suspension trapping (S-Trap). The protocol was then combined with TMTpro 16plex labeling and applied to lung adenocarcinoma patient samples. In total, 9585 proteins were identified, and proteins related to the clinical outcome were detected. Because acetylation is known to play a major role in cancer development, a fast on-trap acetylation protocol was developed for studying endogenous lysine acetylation, which allows identification and localization of the lysine acetylation together with quantitative comparison between samples. We demonstrated that FFPE tissues are equivalent to frozen tissues to study the degree of acetylation between patients. In summary, we present a reproducible sample preparation workflow optimized for FFPE tissues that resolves known proteomic-related challenges. We demonstrate compatibility of the S-Trap with isobaric labeling and for the first time, we prove that it is feasible to study endogenous lysine acetylation stoichiometry in FFPE tissues, contributing to better utility of the existing global tissue archives. The MS proteomic data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifiers PXD020157, PXD021986, and PXD021964.

The Human Melanoma Proteome Atlas—Complementing the melanoma transcriptome

“…Incubation was followed by a brief centrifugation at 14 000 g at 4˚C for 3 minutes, removal of the EnVision solution and the paraffin. These steps were repeated until complete paraffin removal as previously described 81 …”

“…These findings are in agreement with previous results reported by our group and others. [79][80][81][82] Metabolic pathways such as glycolysis, the TCA cycle, and amino acid and fatty acid metabolism, were significantly enriched in the melanoma acetylome (Figure 6B). Coincidently, these pathways have been found dysregulated in melanoma with important implications , left axis) of peptides in the 60 samples submitted to acetylome analysis.…”

Protein Extraction From FFPE Kidney Tissue Samples: A Review of the Literature and Characterization of Techniques

“…Continuous studies have provided solutions related to the FFPE protein extraction methods to expand the proteomic search. In this way, several papers have been recently published where the authors made a comparison between procedures and buffers to improve the protein extraction of FFPE samples (9,16,26,28,31,46). This work aims to develop a protein extraction protocol from healthy renal FFPE tissue, unlike other studies (47), testing several tissue disaggregation methods, deparaffinization procedures, and protein extraction buffers for proteomics analysis.…”

“…In recent years, considerable advances have been made regarding the proteomic analysis of FFPE samples ( 9 ), improving the coverage and the number of proteins identified, studying post-translational modifications ( 9 , 14 ), using iTRAQ-label-based proteomics or “label-free” assays for the quantitative analysis ( 8 , 15 ). For example, recently, using a xylene-free method for the deparaffinization step and a combination of SDS or urea as buffers for the protein extraction step, were possible the identification and quantification of a set of proteins from FFPE tissues using a tandem mass tag (TMT) labeling approach ( 16 ).…”

The human melanoma proteome atlas—Defining the molecular pathology

“…61 (See Material and Methods of Supporting Information) FFPE derived protein extracts were digested using the Strap method following the manufactures' instructions with a few modifications as reported. 59 4.5.5 TMT 11 plex labeling TMT11 plex labeling was performed according to manufacturer's instructions.…”