Proteomic Workflows for High-Quality Quantitative Proteome and Post-Translational Modification Analysis of Clinically...

FFPE tissues are widely used in clinical studies due to their availability and long-term storage stability. However, these tissues pose challenges for proteomic analysis because of the cross-linking effects of formalin fixation. Prior research has shown that protein extraction from FFPE samples is less efficient than from fresh-frozen tissues. This gap motivated the need for improved protocols that maintain high throughput and reproducibility. Existing methods often fail to preserve post-translational modifications like acetylation, which are crucial for cancer research. No prior work had resolved the issue of reliably quantifying acetylated lysines in FFPE samples. This paper addresses these limitations by proposing a new workflow. It introduces a two-enzyme digestion strategy and an optimized labeling protocol. The study also explores the feasibility of using FFPE tissues for acetylation analysis.

The aim of this study was to develop and validate a reliable workflow for proteomic analysis of FFPE tissues. The specific problem addressed is the poor reproducibility and low yield of protein extraction from FFPE samples. The motivation stems from the need to utilize archived clinical samples for biomarker discovery. FFPE tissues are abundant in clinical archives but are rarely used in proteomics due to technical barriers. The study proposes a solution using a combination of efficient paraffin removal and a two-enzyme digestion approach. This workflow was tested using lung adenocarcinoma patient samples. The researchers also aimed to assess whether FFPE tissues could be used to study lysine acetylation, a modification linked to cancer progression. The goal was to demonstrate that FFPE tissues are as reliable as frozen tissues for proteomic and acetylation studies.

The study implemented a paraffin removal protocol followed by protein extraction from FFPE tissues. A two-enzyme digestion strategy was used with the S-Trap system to enhance reproducibility. The extracted proteins were labeled using TMTpro 16plex for quantitative analysis. The workflow was applied to lung adenocarcinoma patient samples to assess its performance. A fast on-trap acetylation protocol was developed to identify and quantify lysine acetylation. The method was validated by comparing acetylation levels between FFPE and frozen tissues. The S-Trap system was tested for compatibility with isobaric labeling techniques. The workflow was optimized for high-throughput processing and reproducibility.

The workflow identified 9585 proteins from FFPE tissues, with several proteins linked to clinical outcomes. The two-enzyme digestion using S-Trap improved reproducibility and yield. TMTpro 16plex labeling allowed for accurate quantification of proteins across samples. The acetylation protocol successfully identified and localized lysine acetylation sites. The study demonstrated that FFPE tissues are comparable to frozen tissues in terms of acetylation levels. The workflow proved to be reliable for high-throughput proteomic analysis. The S-Trap system was shown to be compatible with isobaric labeling. The method enabled the first successful quantification of endogenous lysine acetylation in FFPE tissues.

The study concludes that the proposed workflow is a reproducible and efficient method for proteomic analysis of FFPE tissues. The workflow addresses known challenges in FFPE sample preparation. The results suggest that FFPE tissues can be used effectively for biomarker discovery. The workflow is compatible with isobaric labeling techniques like TMTpro. The study proves that lysine acetylation can be quantified in FFPE tissues for the first time. The findings support the use of FFPE archives in proteomic research. The workflow enhances the utility of existing clinical tissue collections. The results suggest that FFPE tissues are as reliable as frozen tissues for proteomic and acetylation studies.