Targeted quantitative mass spectrometry analysis of protein biomarkers from previously stained single formalin-fixed paraffin-embedded tissue sections

ABSTRACT: Formalin-fixed, paraffin-embedded (FFPE) tissues represent a majority of all biopsy specimens commonly analyzed by histological or immunohistochemical (IHC) staining with adhesive coverslips attached. Mass spectrometry (MS) recently has been employed to precisely quantify proteins in samples consisting of multiple unstained FFPE sections. Here we report a MS method to analyze proteins from a single coverslipped 4-micron section previously stained with hematoxylin/eosin (H&E), Masson’s trichrome, or 3,3’-diaminobenzidine based IHC. We analyzed serial unstained and stained sections from non-small cell lung cancer (NSCLC) specimens for proteins of varying abundance (PD-L1, RB1, CD73, HLA-DRA). Coverslips were removed by soaking in xylene and, after tryptic digestion, peptides were analyzed by targeted high-resolution LC-MS/MS with stable isotope labeled peptide standards. The low abundance proteins RB1 and PD-L1 were quantified in 31 and 35 of 50 total sections analyzed, respectively, whereas higher abundance CD73 and HLA-DRA were quantified in 49 and 50 sections. Inclusion of targeted beta-actin (ACTB) measurement enabled normalization in samples where residual stain interfered with bulk protein quantitation by colorimetric assay. Measurement CVs for 5 replicate slides (H&E vs. unstained) from each block ranged from 3-18% for PD-L1, from 1-36% for RB1, from 3-21% for CD73 and from 4-29% for HLA-DRA. Collectively, these results demonstrate that targeted MS protein quantification can add a valuable data layer to clinical tissue specimens after assessment for standard pathology endpoints.

ORGANISM(S): Homo Sapiens

SUBMITTER: Ryan Morrison  

PROVIDER: PXD039290 | panorama | Fri Jun 12 00:00:00 BST 2026

REPOSITORIES: PanoramaPublic