Proteomic Workflows for High-Quality Quantitative Proteome and Post-Translational Modification Analysis of Clinically Relevant Samples from Formalin-Fixed Paraffin-Embedded Archives

Well-characterized
archival formalin-fixed paraffin-embedded (FFPE)
tissues are of much value for prospective biomarker discovery studies,
and protocols that offer high throughput and good reproducibility
are essential in proteomics. Therefore, we implemented efficient paraffin
removal and protein extraction from FFPE tissues followed by an optimized
two-enzyme digestion using suspension trapping (S-Trap). The protocol
was then combined with TMTpro 16plex labeling and applied to lung
adenocarcinoma patient samples. In total, 9585 proteins were identified,
and proteins related to the clinical outcome were detected. Because
acetylation is known to play a major role in cancer development, a
fast on-trap acetylation protocol was developed for studying endogenous
lysine acetylation, which allows identification and localization of
the lysine acetylation together with quantitative comparison between
samples. We demonstrated that FFPE tissues are equivalent to frozen
tissues to study the degree of acetylation between patients. In summary,
we present a reproducible sample preparation workflow optimized for
FFPE tissues that resolves known proteomic-related challenges. We
demonstrate compatibility of the S-Trap with isobaric labeling and
for the first time, we prove that it is feasible to study endogenous
lysine acetylation stoichiometry in FFPE tissues, contributing to
better utility of the existing global tissue archives. The MS proteomic
data have been deposited to the ProteomeXchange Consortium via the
PRIDE partner repository with the data set identifiers PXD020157,
PXD021986, and PXD021964.