Parallel Proteomic Workflow for Mass Spectrometric Analysis of Tissue Samples Preserved by Different Methods

Formalin-fixed
and paraffin-embedded (FFPE) and optimal cutting
temperature (OCT)-embedded and frozen tissue specimens in biobanks
are highly valuable in clinical studies but proteomic and post-translational
modification (PTM) studies using mass spectrometry (MS) have been
limited due to structural arrangement of proteins and contaminations
from embedding material. This study aims to develop a parallel proteomic
workflow for FFPE and OCT/frozen samples that allows for large-scale,
quick, reproducible, qualitative, and quantitative high-resolution
MS analysis. The optimized protocol gives details on removal of embedding
material, protein extraction, and multienzyme digestion using filter-aided
sample preparation method. The method was evaluated by investigating
the protein expression levels in nonmuscle-invasive and muscle-invasive
bladder cancer samples in two cohorts and MS spectra were carefully
reviewed for contaminations. More than 2000 and 3000 proteins in FFPE
and OCT/frozen samples, respectively, were identified, and samples
could be clustered in different tumor stages based on their protein
expression. Furthermore, more than 250 and 400 phosphopeptides could
be identified from specific patient samples of FFPE and OCT/frozen,
respectively, using titanium dioxide enrichment. The paper presents
unique data describing the similarities and differences observed in
FFPE and OCT/frozen samples and shows the feasibility to detect proteins
and site-specific phosphorylation even after long-term storage of
clinical samples.