Making FFPE sample preparation easier with BeatBox^® and iST

A xylene-free, high-throughput workflow for in-depth tissue proteome analysis

The challenge

Formalin-Fixed, Paraffin-Embedded (FFPE) tissue samples are a highly valuable source for proteomic analysis. They are routinely collected and stored by hospitals and readily available in biobanks worldwide for retrospective studies, such as discovering new biomarkers. However, preparing FFPE samples for proteomic analysis is very challenging as formalin fixation hinders protein extraction.

Besides, current protocols require an initial xylene-based deparaffinization step as paraffin interferes in the downstream LC-MS analysis. This process is time-consuming, low reproducible, and hazardous due to high toxicity.

The solution

An efficient and straightforward solution for processing FFPE tissues for proteomic analysis is combining PreOmics®’ BeatBox® tissue homogenizer and the iST technology. In the BeatBox® FFPE workflow, the high-toxic xylene-based deparaffinization step is eliminated, avoiding sample loss. Fast and reproducible processing of up to 96 samples can be performed in parallel from starting material to clean peptides within one working day.

Results

Compatible sample types

BeatBox® FFPE workflow has been successfully used to extract proteins from different FFPE sample formats, i.e., “full” curls, sections, and macrodissections.

Comparison of protein identifications after BeatBox^®- and sonication-based FFPE workflows

10 µm FFPE tissue “full” curls of mouse cardiac muscle, kidney, and liver were processed with the BeatBox® FFPE workflow and benchmarked against sonication methods. The BeatBox® FFPE workflow increased the proteomic depth on average by 19–43%, depending on the tissue type.

Technical variability of the BeatBox FFPE workflow

Coefficients of variation (CV) within quadruplicates from “full” curl FFPE samples were <10% for all assessed tissue types.

Comparison of identified proteins and dynamic range for FFPE and fresh-frozen tissue

FFPE “full” curls (10 µm) and fresh frozen tissue (1-2 mg) samples of mouse cardiac muscle, kidney, and liver were prepared using the BeatBox® and iST and analyzed by LC-MS. The number of proteins identified and protein depth using “full” curls and fresh frozen samples were comparable.

"We routinely analyze a large number of tissue samples at once, and to improve our efficiency, we have successfully adapted the BeatBox technology for tissue homogenization. Our latest discovery is that the BeatBox technology can efficiently homogenize paraffin-embedded tissue scrolls. This breakthrough now allows us to process large cohorts of FFPE tissue quickly, reproducibly, and with greater precision, opening up the possibility of large-scale biobank projects. By leveraging this technology, we are excited to uncover novel deep proteomics insights in the field of pathology research."
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Dr. Michael Wierer

Director of the Proteomics Research Infrastructure, University of Copenhagen, Denmark

Key benefits of the BeatBox^® FFPE workflow

Reliable results
Easy-to-use and standardized protocol for efficient protein extraction and high reproducibility
Simplified FFPE sample preparation
Straightforward xylene-free workflow without a separate deparaffinization step for LC-MS-based proteomics
Comparable to fresh frozen tissues
The protein depth of FFPE samples is equivalent to that for fresh frozen tissue
Suitable for large-scale studies
Get clean peptides from up to 96 samples/day