FFPE tissues hold unmatched potential for molecular discovery, but unlocking their proteomes has long required complex, expert-driven workflows. Here, we present a practical, scalable workflow that enables deep, reproducible proteome profiling from FFPE tissue sections using short-gradient DIA mass spectrometry on the Orbitrap Astral platform.
This approach integrates a streamlined FFPE sample prep protocol, including paraffin removal with xylene and graded ethanol washes, mechanical homogenization, and probe sonication to assist lysis. Proteins are prepared into peptides using a 96-well compatible, easy-to-use kit. Peptides are analyzed using three LC-MS configurations designed for flexibility: 3-, 7-, and 25-minute gradients. The Orbitrap Astral’s fast, sensitive DIA acquisition ensures robust quantitation, even at inputs as low as 20 ng.
Early results using breast and lung FFPE tissues (tumor and normal) demonstrate consistent protein recovery and preliminary experiments identify >3,500 protein identifications in some gradients, with higher depth expected in final datasets. Biological patterns, including PCA-based separation of tumor and normal samples, are readily observed. This workflow is fully compatible with spatially annotated slides and preliminary data quantifies key proteins such as CRABP2, previously observed to increase in lung tumors. Biological results indicate the utility of studying FFPE tissue and highlight the contribution of proteomics to the pathological interrogation of FFPE samples.
By removing longstanding barriers- no fractionation, no high input, no long runs- this workflow makes deep FFPE proteomics both practical and approachable. Its compatibility with 96-well formats and easy-to-use prep enables adoption beyond expert labs, inviting spatial biologists, pathologists, and transcriptomics researchers to access thousands of protein measurements from tissue they already work with. Whether for biomarker discovery, retrospective cohorts, or spatial biology, this end-to-end method unlocks the proteomic potential of the world’s FFPE archives.