Poster Presentation 28th Annual Lorne Proteomics Symposium 2023

Use of DIA-MS to determine the effects of storage temperature and time on FFPE tissue sections (#134)

Jennifer M. S. Koh 1 , Erin K. Sykes 1 , Jyoti Rukhaya 1 , Asim Anees 1 , Qing Zhong 1 , Rosemary Balleine 1 , Peter G. Hains 1 , Phillip J. Robinson 1
  1. ProCan, Children's Medical Research Institute, The University of Sydney, Westmead, NSW, Australia

Formalin-fixed paraffin-embedded (FFPE) tissues are invaluable resources for cancer proteomic studies and biomarker discovery. Very large numbers of them are stored in pathology laboratories and biobanks worldwide and often have associated clinical data available. FFPE tissue blocks are stable for decades when stored at room temperature (RT) and can be sectioned to produce samples for liquid chromatography-mass spectrometry (LC-MS) analysis. It is not known if the proteome of FFPE tissue after sectioning may be affected by storage temperature or time. To address this, we stored FFPE tissue sections at RT and -80°C for up to 336 days and analysed them at different timepoints to determine the proteome stability. A total of 297 FFPE 10 µm sections (triplicates of rat brain, kidney and liver) were cut from tissue blocks and stored at either RT or -80°C. Control samples were freshly cut sections from FFPE blocks. Samples were prepared for LC-MS analysis by digestion at 11 timepoints after storage (up to 336 days) and analysed by microflow HPLC on Triple TOF 6600 mass spectrometers (SCIEX) using data-dependent acquisition (DDA) mode. Kidney and liver digests were further analysed in data-independent acquisition (DIA) mode for quantitative analysis. Nine post-translational modifications (PTMs) that are specific for FFPE samples were identified from the DDA data analysis. These PTMs were monitored for quantitative changes in samples that were stored at different temperatures and for different time periods using DIA data. Overall, the storage temperature and time did not have significant effects on the proteomic analysis of FFPE sections. After FFPE blocks are cut, the tissue sections can be safely stored at either RT or -80°C for at least 1 year without significant impacts on the identified proteome. Such samples are suitable alternatives for fresh frozen tissues in proteomic studies.