Unrestricted modification search reveals lysine methylation as major modification induced by tissue formalin fixation and paraffin embedding

Abstract

Formalin-fixed paraffin-embedded (FFPE) tissue is considered as an appropriate alternative to frozen/fresh tissue for proteomic analysis. Here we study formalin-induced alternations on a proteome-wide level. We compared LC-MS/MS data of FFPE and frozen human kidney tissues by two methods. First, clustering analysis revealed that the biological variation is higher than the variation introduced by the two sample processing techniques and clusters formed in accordance with the biological tissue origin and not with the sample preservation method. Second, we combined open modification search and spectral counting to find modifications that are more abundant in FFPE samples compared to frozen samples. This analysis revealed lysine methylation (+14 Da) as the most frequent modification induced by FFPE preservation. We also detected a slight increase in methylene (+12 Da) and methylol (+30 Da) adducts as well as a putative modification of +58 Da, but they contribute less to the overall modification count. Subsequent SEQUEST analysis and X!Tandem searches of different datasets confirmed these trends. However, the modifications due to FFPE sample processing are a minor disturbance affecting 2-6% of all peptide-spectrum matches and the peptides lists identified in FFPE and frozen tissues are still highly similar.

Isolation of Amyloid Plaques and Neurofibrillary Tangles from Archived Alzheimer’s Disease Tissue Using Laser-Capture Microdissection for Downstream Proteomics

“…Researchers have avoided using FFPE tissue for proteomics in the past because of concerns that the protein crosslinks generated during FFPE processing will prevent the detection of proteins using mass spectrometry or lead to inferior results compared to those using frozen, unfixed tissue. However, we (and an increasing number of other researchers, primarily in the cancer field) have shown that this is not the case [2–6]. The method described below has been specifically optimized to use FFPE tissue because the abundance of human FFPE tissue specimens is a potential gold mine for future studies of human disease.…”

Insights into autosomal dominant polycystic kidney disease by quantitative mass spectrometry-based proteomics

“…As PTM analyses are often based on single peptide identifications, they are more error-prone than protein studies. To identify potential sources of wrongly assigned PTMs in kidney disease proteomics, formalin-induced alterations in FFPE samples compared with freshly frozen samples of human kidney tissue have been characterized in a label-free proteomic approach (Zhang et al 2015). In an open modification search, the authors found +12, +14, +16, +30 and +58 Da mass shifts on various amino acid residues of peptides isolated from FFPE samples, shifts that were not present in freshly frozen samples.…”

“…Luckily, the overall number of peptides containing a formalin-induced methylation was in an acceptable low range (2-6%). However, since methylations on histone lysines often play a crucial role in the regulation of gene expression and chromatin remodeling, this formalininduced artifact should be taken into account when studying methylation events in renal FFPE specimens (Zhang et al 2015).…”

Proteomic analysis of FFPE tissue: barriers to clinical impact

“…This can be accomplished using two bioinformatics approaches. The more general approach uses an unrestricted modification search to extract the modifications in the sample directly from the MS/ MS data without the need for prior configuration [16]. Using this method, unexpected modifications are detected in an unbiased manner.…”

“…It is likely that formaldehyde-induced protein adducts undergo further reactions during histological processing. Using an unrestricted modification search method, Zhang et al [16] identified the presence of methylated lysine in FFPE proteomes: a reaction not seen in aqueous solutions of protein and formaldehyde. Ethoxymethylation of the primary amine is observed in 2′-deoxyadenosine 5′-monophosphate when the nucleotide is transferred to ethanol after reaction with formaldehyde in aqueous solution [18].…”