Related Experiment Video
Updated: Aug 16, 2025
09:20
Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue
Published on: September 2, 2013
27.8K
Highly Accurate and Robust Absolute Quantification of Target Proteins in Formalin-Fixed Paraffin-Embedded (FFPE)
Jie Pu1, Chao Xue2, Shihan Huo1
1Department of Pharmaceutical Sciences, University at Buffalo, State University of New York, Buffalo, New York 14214, United States.
Analytical Chemistry
|December 19, 2022
Summary
Accurate protein quantification in formalin-fixed paraffin-embedded (FFPE) tissues is now possible using liquid chromatography-mass spectrometry (LC-MS). New methods ensure reliable peptide recovery and accurate calibration, overcoming storage and quantification challenges for FFPE samples.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Biotechnology
Background:
- Formalin-fixed paraffin-embedded (FFPE) tissues offer vast potential for pharmaceutical and clinical research but accurate protein quantification remains a challenge.
- Existing methods face hurdles in efficient peptide recovery, understanding long-term storage effects, and preparing reliable calibration standards for FFPE samples.
Purpose of the Study:
- To develop and validate an LC-MS-based strategy for accurate and precise targeted protein quantification in FFPE tissues.
- To address limitations in peptide recovery, storage effects, and calibration standard preparation for FFPE analysis.
Main Methods:
- A surfactant cocktail-based procedure (f-SEPOD) was developed for efficient and reproducible peptide recovery from FFPE tissues.
- Heat-accelerated degradation studies assessed the impact of long-term storage on protein integrity for LC-MS analysis.
- A novel calibration approach using FFPE-treated standards was implemented to ensure accurate quantification.
Main Results:
- The f-SEPOD procedure demonstrated high and reproducible recovery of target peptides from FFPE tissues.
- Long-term FFPE storage primarily affected immunohistochemistry (IHC) signals, with LC-MS signals remaining stable, indicating minimal quantitative bias.
- The FFPE-treated calibration standard approach enabled accurate and precise protein quantification, outperforming conventional methods.
Conclusions:
- The developed LC-MS strategy enables reliable, absolute quantification of proteins in FFPE tissues, overcoming previous limitations.
- FFPE tissue storage does not significantly impact quantitative accuracy for LC-MS-based protein analysis.
- This approach facilitates novel applications, such as the first pharmacokinetics measurement of a monoclonal antibody and its target in FFPE tissues.
