Formalin-Fixed, Paraffin-Embedded Tissues (FFPE) as a Robust Source for the Profiling of Native and Protease-Generated Protein Amino Termini

Abstract

Dysregulated proteolysis represents a hallmark of numerous diseases. In recent years, increasing number of studies has begun looking at the protein termini in hope to unveil the physiological and pathological functions of proteases in clinical research. However, the availability of cryopreserved tissue specimens is often limited. Alternatively, formalin-fixed, paraffin-embedded (FFPE) tissues offer an invaluable resource for clinical research. Pathologically relevant tissues are often stored as FFPE, which represent the most abundant resource of archived human specimens. In this study, we established a robust workflow to investigate native and protease-generated protein N termini from FFPE specimens. We demonstrate comparable N-terminomes of cryopreserved and formalin-fixed tissue, thereby showing that formalin fixation/paraffin embedment does not proteolytically damage proteins. Accordingly, FFPE specimens are fully amenable to N-terminal analysis. Moreover, we demonstrate feasibility of FFPE-degradomics in a quantitative N-terminomic study of FFPE liver specimens from cathepsin L deficient or wild-type mice. Using a machine learning approach in combination with the previously determined cathepsin L specificity, we successfully identify a number of potential cathepsin L cleavage sites. Our study establishes FFPE specimens as a valuable alternative to cryopreserved tissues for degradomic studies. Molecular & Cellular Proteomics 15:

Reproducible proteomics sample preparation for single FFPE tissue slices using acid-labile surfactant and direct trypsinization

“…Kidneys of a male, 6-months old C57 black 6 mouse lacking cathepsin L (Ctsl −/− ) were removed immediately after sacrificing it. Formalin fixation and paraffin embedding were performed as described previously [ 24 ]. 10 µm thick sections were sliced from one kidney FFPE tissue block using a microtome and mounted onto glass slides.…”

OpenMS: a flexible open-source software platform for mass spectrometry data analysis

“…In a recent study, Lai et al [42] used quantitative proteomics and stable isotope labeling to identify proteolytically generated protein neo-termini from formalin-fixed, paraffinembedded (FFPE) specimens (which represent the vast majority of samples stored in clinical archives) using biochemical enrichment methods. OpenMS enabled the parallel and unbiased analysis of multiple, prevalent N-terminal modifications in different, isotopically labeled states.…”

“…Typically, this situation is prone to distorted quantification due to chromatographic background signals [43] ; a problem that can be resolved by accurate peptide feature detection in OpenMS. The workflow characterized thousands of protein N-termini in FFPE specimens and identified 23 novel potential substrates for the lysosomal protease cathepsin L [42] .…”

LysargiNase enhances protein identification on the basis of trypsin on formalin‐fixed paraffin‐embedded samples

“…Large numbers of Formalin‐Fixed Paraffin‐Embedded (FFPE) samples are well preserved for routine clinical diagnostics and archival purposes. The specimens are clinically annotated and stored for long periods at room temperature, which is convenient and cost‐effective . Although cryopreserved samples are considered to be the gold standard for many molecular assays for its minimal introduction of chemical modification, it is challenging to collect a sufficient number of cryopreserved tissue samples for robust study.…”

“…The specimens are clinically annotated and stored for long periods at room temperature, which is convenient and cost-effective. 1 Although cryopreserved samples are considered to be the gold standard for many molecular assays for its minimal introduction of chemical modification, it is challenging to collect a sufficient number of cryopreserved tissue samples for robust study. It is easier and more cost-effective to collect FFPE tissues than cryopreserved tissues, so FFPE tissue collection is gaining more attention.…”