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In-depth mass spectrometry-based proteomics of formalin-fixed, paraffin embedded tissues with a spatial resolution of 50–200 µm

ABSTRACT: Formalin-fixed, paraffin-embedded (FFPE) tissues are banked in large repositories as a cost-effective means of preserving invaluable specimens for subsequent study, including for clinical proteomics in translational medicine. With the rapid growth of spatial proteomics, FFPE tissue samples can serve as a more accessible alternative to commonly used fresh frozen tissues. However, extracting proteins from FFPE tissue for analysis by mass spectrometry has been challenging due to crosslinks formed between proteins and formalin, particularly when studying limited samples. We have previously demonstrated that nanoPOTS (Nanodroplet Processing in One Pot for Trace Samples) is an enabling technology for high-resolution and in-depth spatial and single-cell proteomics measurements, but only fresh frozen tissues had been previously analyzed. Here we have adapted the nanoPOTS sample processing workflows for proteome profiling of 10-µm-thick FFPE tissues with lateral dimensions as small as 50 µm. Following a comparison of extraction solvents, times, and temperatures, and under the most favorable conditions, we respectively identified an average of 1180 and 2990 proteins from FFPE preserved mouse liver tissues having dimensions of 50 µm and 200 µm. This was on average 87% of the coverage achieved for fresh frozen mouse liver samples analyzed with the same general procedure. We also characterized the performance of our fully automated sample preparation and analysis workflow, termed autoPOTS, for FFPE spatial proteomics. These workflows provide the greatest depth of coverage reported to date for high-resolution spatial proteomics applied to FFPE tissues.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Liver

SUBMITTER: Ryan Kelly

LAB HEAD: Ryan T Kelly

PROVIDER: PXD029729 | Pride | 2022-08-02

REPOSITORIES: Pride

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			Action	DRS
	100um_ffpe_nanopots_rep1.raw	Raw
	100um_ffpe_nanopots_rep2.raw	Raw
	100um_ffpe_nanopots_rep3.raw	Raw
	100um_ffpe_nanopots_rep4.raw	Raw
	100um_frfr_nanopots_rep1.raw	Raw

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Project description:For the effectiveness of targeted immunotherapy, which is currently one of the most promising treatments for brain tumors, it is necessary to know specific tumor-associated antigens (TAA). This project aimed to validate the suitability of formalin-fixed and paraffin-embedded (FFPE) material instead of fresh-frozen material for RNA sequencing and downstream TAA identification. Purpose: Detect potential oligodendroglioma-associated antigens using both fresh-frozen and FFPE brain tissues; assess the suitability of the FFPE material for TAA identification. Results: A comparative analysis based on the RNA-seq of canine oligodendroglioma showed that formalin fixation of the samples had a significant effect on the RNA-seq library in terms of quality. Read distribution analysis showed that the FFPE samples contained fewer reads mapping to exonic regions and were enriched with reads mapped to introns and intergenic regions, while the fresh-frozen samples were mostly enriched in reads mapping to exons. Mismatch profile and SNVs calling analyses revealed some substitution artefacts present in the FFPE samples and absent in the corresponding fresh-frozen samples. However, according to the Principal Component Analysis, 36% of the variance between the samples could be explained by the types of conservation, while 43% of the variance could still be attributed to different expression profiles between oligodendroglioma and control conditions. A differential expression analysis of fresh-frozen oligodendroglioma versus fresh-frozen control samples identified 62 potential TAA strongly up-regulated in tumor tissue. The same analysis using the FFPE samples showed 80% of these genes (49/62) also to be differentially expressed. Comparative analysis showed good agreement between FFPE and fresh-frozen RNA-seq libraries in terms of the accuracy of gene expression measurements indicating that archived FFPE tissue can be used for identification of TAA candidates by RNA-seq providing a wealthy source of clinical samples for research. The following 10 genes encoding cell surface proteins have been identified by both approaches as potential TAA candidates based on their strong overexpression in oligodendrogliomas: PDGFRA, NOTCH1, DLL1, GPER1, TNR, IQGAP3, CD44, ERBB3, BCAS1 and ROR2. Future projects still need to investigate their suitability as TAA for targeted immunotherapy. Conclusion: Formalin fixation of the samples had a significant impact on the RNA-seq quality in terms of the accuracy of gene expression measurements, as well as the quality of the nucleotide sequences. Nevertheless, the comparative analysis showed good agreement between FFPE and fresh-frozen RNA-seq libraries.

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In-depth mass spectrometry-based proteomics of formalin-fixed, paraffin embedded tissues with a spatial resolution of 50–200 µm

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