Digestion-Free Analysis of Peptides from 30-year-old Formalin-Fixed, Paraffin-Embedded Tissue by Mass Spectrometry Imaging

Abstract

Formalin-fixed neuroendocrine tissues from American cockroaches (Periplaneta americana) embedded in paraffin more than 30 years ago were recently analyzed by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), to reveal the histological localization of more than 20 peptide ions. These represented protonated, and other cationic species of, at least, 14 known neuropeptides. The characterization of peptides in such historical samples was made possible by a novel sample preparation protocol rendering the endogenous peptides readily amenable to MSI analysis. The protocol comprises brief deparaffinization steps involving xylene and ethanol, and is further devoid of conventional aqueous washing, buffer incubations, or antigen retrieval steps. Endogenous secretory peptides that are typically highly soluble are therefore retained in-tissue with this protocol. The method is fully “top-down”, that is, without laborious in situ enzymatic digestion that typically disturbs the detection of low-abundance endogenous peptides by MSI. Peptide identifications were supported by accurate mass, on-tissue tandem MS analyses, and by earlier MALDI-MSI results reported for freshly prepared P. americana samples. In contrast to earlier literature accounts stating that MALDI-MSI detection of endogenous peptides is possible only in fresh or freshly frozen tissues, or exceptionally, in formalin-fixed, paraffin-embedded (FFPE) material of less than 1 year old, we demonstrate that MALDI-MSI works for endogenous peptides in FFPE tissue of up to 30 years old. Our findings put forward a useful method for digestion-free, high-throughput analysis of endogenous peptides from FFPE samples and offer the potential for reinvestigating archived and historically interesting FFPE material, such as those stored in hospital biobanks.

Complementary neuropeptide detection in crustacean brain by mass spectrometry imaging using formalin and alternative aqueous tissue washes

“…The choice of 10% NBF was motivated by (1) the fact that it is the most commonly used tissue fixative and (2) previous studies showing neuropeptides can escape formalin-mediated crosslinking during formalin fixation and therefore are able to be detected using MS imaging from formalin-fixed paraffin-embedded (FFPE) tissue without applying antigen retrieval. In a study performed by Paine et al, their modified deparaffinization method allowed for detection of neuropeptides from FFPE cockroach tissue without antigen retrieval [32], likely from crosslinking interfering species and allowing neuropeptides to be available for MS detection.…”

“…These discoveries launched studies to utilize MS to analyze proteins from FFPE tissue, such as characterization of potential protein modifications due to formalin fixation [28] and optimization of protocols involving deparaffinization, antigen retrieval, and proteolytic digestion [29][30][31]. Recently, a study emerged demonstrating that a protocol only including deparaffinization and omitting antigen retrieval allowed detection of endogenous peptide species from FFPE tissues in MS imaging studies [32]. At first, this discovery is counterintuitive to previous studies which utilize antigen retrieval and proteolytic digestion for peptide detection in MS measurements.…”

“…At first, this discovery is counterintuitive to previous studies which utilize antigen retrieval and proteolytic digestion for peptide detection in MS measurements. However, these studies reveal that some neuropeptides have characteristics that allow them to escape from crosslinking during formalin fixation, such as lack of free amine groups as indicated by the lack of lysine residues and the presence of a blocked N-terminus (pyroglutamic acid) [32]. One potential reason for formalin fixation preserving neuropeptides is that catabolic enzymes are cross-linked and inactivated.…”

Visualizing the dynamics of tuberculosis pathology using molecular imaging

“…MALDI-MSI is label-free and can localize ionized molecules (e.g., drugs, metabolites, lipids, proteins) and overlay them onto histologically stained sections to enable spatial distribution of each ion of interest with cellular and subcellular resolution ( 31 , 32 ). MALDI-MSI can also be applied to archived tissue blocks dating back decades ( 33 ).…”

A recommended and verified procedure for in situ tryptic digestion of formalin‐fixed paraffin‐embedded tissues for analysis by matrix‐assisted laser desorption/ionization imaging mass spectrometry

“…First, removal of paraffin is required for analysis, and this typically uses organic solvents that remove endogenous lipids and some proteins. Endogenous peptides and metabolites can be imaged without further sample preparation prior to matrix application. However, the intramolecular crosslinks formed during formalin fixation make it difficult to analyse proteins directly.…”