Clinical proteomics workflow for high quality quantitative proteome and PTM analysis of biologically relevant FFPE tissue samples

ABSTRACT: In recent years, the use of formalin-fixed paraffin-embedded (FFPE) tissues has increased within cancer research and clinical proteomics. However, the use of FFPE tissues can be demanding due to paraffin embedding and cross-links introduced by the formaldehyde. Here, we developed a workflow implementing efficient paraffin removal and protein extraction from FFPE tissues, followed by optimized digestion using suspension trapping (S-trap), with different clinical proteomics strategies. To gain proteomic depth, the optimized sample preparation protocol was combined with isobaric labeling and applied to three lung adenocarcinoma patient samples. The peptides eluted from the S-trap were labeled with TMT without any desalting step in between. In total, 8,163 proteins were commonly quantified across all channels, and specific proteins related to clinical outcome and histopathological subtype were detected. In addition, we developed a protocol in the S-trap for studying endogenous lysine acetylation stoichiometry using FFPE tonsil tissue. Our method allows identification and localization of lysine acetylation and relative quantification comparison between samples. We could identify 1,839 acetylated peptides belonging to 1,339 protein groups. The results were compared to frozen tissue samples and no notable differences in acetylation pattern were observed. In summary, we present a reproducible sample preparation workflow optimized for FFPE tissues that is easy to scale up using the S-trap 96-well plate. Our results demonstrate compatibility of the S-trap with TMT labeling. And, for the first time, we showed that it is biologically relevant to study endogenous lysine acetylation in FFPE tissues, contributing to a better use of the existing FFPE collections around the world.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Tonsil, Lung

DISEASE(S): Non-small Cell Lung Carcinoma

SUBMITTER: Magdalena Kuras  

LAB HEAD: Gyorgy Marko-Varga

PROVIDER: PXD020157 | Pride | 2020-12-18

REPOSITORIES: Pride