FFPE proteomics: getting usable data from archival blocks

What fixation does

Formalin cross-links proteins through methylene bridges, and paraffin embedding dehydrates the tissue. Proteins are still there, but they are insoluble in mild buffers and chemically modified. Decades-old blocks yield protein; what changes with age and fixation history is how much can be recovered and how clean it is.

What works

• Deparaffinise completely. Residual paraffin suppresses ionisation and clogs columns.
• Reverse the cross-links with heat and SDS. Heating in a strong SDS buffer at high temperature is the step that makes FFPE tractable; mild detergents do not do it. Suspension trapping was designed for exactly this kind of SDS lysate (Zougman et al., Proteomics 2014). Streamlined workflows for large FFPE cohorts are published (Coscia et al., J Pathol 2020).
• Use an SDS-tolerant digestion route. Because the lysis buffer is SDS-based, suspension trapping or SP3 is the natural digestion route; in-solution digestion forces a precipitation step that loses material.
• Expect modifications. Formaldehyde-derived modifications and methylation can be included as variable modifications in the search; they are informative about fixation quality.
• Assess before committing. Run a test section from a representative block before the cohort is cut. Yield and identifications from the test tell you what the cohort will give and whether the number of sections per sample needs to change.

Design points specific to FFPE

Block age, fixation time and storage are confounders; record them and include them in the model. Macrodissection or laser microdissection of the region of interest matters more than in fresh tissue, because surrounding stroma dominates a whole-section lysate. Pathologist review of an H&E section from the same block is a requirement, not a nicety.

Our S-Trap™ formats are the standard digestion route for FFPE in our laboratory, and tissue proteomics from archival blocks is a routine service.

References

1. Coscia F et al. A streamlined mass spectrometry-based proteomics workflow for large-scale FFPE tissue analysis. J Pathol 2020;251:100-112. Open access PubMed · DOI
2. Zougman A, Selby PJ, Banks RE. Suspension trapping (STrap) sample preparation method for bottom-up proteomics analysis. Proteomics 2014;14:1006. PubMed · DOI

Citations verified against PubMed records; “Open access” marks papers whose full text is freely available via PubMed Central or the journal.

Written by the omicslink proteomics team. Reviewed November 2026.