SWATH mass spectrometry: Quantitative mapping of soft tissue sarcomas by digital proteome profiling


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Lukas Krasny1,Martina Milighetti1,Alexander T.J. Lee1,Aik-Choon Tan2,Robin Jones3,Paul H. Huang1
1The Institute of Cancer Research, London, UK,2University of Colorado, Aurora, US,3Royal Marsden Hospital, London, UK

Abstract

Background

Soft tissue sarcomas (STS) are a rare and heterogeneous group of cancers. Our knowledge of STS biology is limited and although progress has been made in the genetic characterization of these diseases, comprehensive molecular profiling at the protein level has not been undertaken. One of the challenges associated with tumour proteomics is the presence of formalin-induced protein crosslinks in formalin-fixed paraffin-embedded (FFPE) tissue which complicates conventional proteomic workflows.

Method

In this study, we used digital proteome profiling by sequential window acquisition of all theoretical fragment ion spectra (SWATH) mass spectrometry (MS) to characterise a discovery cohort of STS cases (n=39) across 4 histological subtypes. Validation of findings was performed in an independent cohort of STS specimens (n=64) using a conventional proteomic approach that involves TMT labelling and off-line fractionation.

Results

Using SWATH-MS, we quantified 2865 proteins in the discovery cohort from starting material equivalent to a 5μm FFPE tissue section. Significantly altered expression levels of 884 proteins were found by ANOVA (FDR<0.05) in at least one of the four STS subtypes. Further analysis of these proteins by principal component analysis (PCA) and hierarchical clustering (HC) revealed distinct separation of samples into three subgroups corresponding to STS histological subtypes. Gene set enrichment analysis (GSEA) was employed to identify underlying biological processes and candidate therapeutics that are enriched in individual subgroups. In the validation cohort, 5642 proteins were quantified with 2114 proteins showing significantly altered expression levels in at least one of the STS subtypes (ANOVA, FDR<0.05) and analysis by PCA and HC showed similar histological subtype separation and biological pathways as the discovery cohort.

Conclusion

We demonstrate the potential of SWATH-MS for the molecular characterization of STS with minimal consumption of readily available FFPE tissue material. To our knowledge, this study is the most comprehensive analysis of the STS proteome to date.