Proteomic workflows for high quality quantitative proteome and PTM analysis of clinically relevant samples from FFPE archives

ABSTRACT: Well-characterized archival FFPE tissues are of much value for prospective biomarker discovery studies. Protocols that offer high-throughput and good reproducibility are essential in proteomics. Therefore, we developed a workflow implementing efficient paraffin removal and protein extraction from FFPE tissues, followed by digestion using suspension Trapping (S-Trap). The protocol was then combined with isobaric labeling/TMTpro 16plex and applied to lung adenocarcinoma patient samples. In total, 9,585 proteins were quantified, and proteins related to clinical outcome and histopathological subtype were detected. Since acetylation is known to play a major role in cancer development, an on-filter acetylation protocol was developed for studying endogenous lysine acetylation. Our method allows identification and localization of lysine acetylation and quantitative comparison between samples. We identified 1,839 acetylated peptides belonging to 1,339 protein groups and showed that the data obtained from FFPE tissues is in concordance with acetylation data from frozen tissues. 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 TMTpro 16plex 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.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Skin

DISEASE(S): Melanoma

SUBMITTER: Magdalena Kuras  

LAB HEAD: Gyorgy Marko-Varga

PROVIDER: PXD021986 | Pride | 2020-12-18

REPOSITORIES: Pride