Characterization of histone-related chemical modifications in formalin-fixed paraffin-embedded and fresh-frozen human pancreatic cancer xenografts using LC-MS/MS

Abstract Post-translational modifications (PTMs) of histones including acetylation, methylation, and ubiquitination are known to be involved in the epigenetic regulation of gene expression and thus can have an important role in tumorigenesis. A number of PTMs have been linked to pancreatic cancer and are frequently studied as potential targets for cancer therapy or diagnosis. The availability of biobank-stored, formalin-fixed, paraffin-embedded (FFPE) materials and advanced proteomic analytical tools make it possible to detect histone-related PTMs using predicted mass shifts caused by specific modification. It is, however, important to take into account the fact that formaldehyde (FA) present in the FFPE material is chemically reactive and may undergo condensation reactions, for example, with terminal amino groups and active CH functionalities of the studied proteins. As supported by the results of this study, the possibility to misinterpret such protein condensation product as endogenous PTMs should be taken into consideration in all proteomic analytical work involving FFPE materials. In this study, we used liquid chromatography-tandem mass spectrometry to assess preassumed modification of the lysine residues of histone proteins in FFPE or fresh-frozen (FF) tumor xenografts, derived from the human pancreatic cancer cell line, Capan-1. Here we report modifications with a defined mass shift of +14.016, +28.031, +42.011, or +114.043 Da, corresponding to apparent methylation, dimethylation, acetylation, or ubiquitination that were differentially distributed between the groups. The identified modifications were significantly more frequent in FFPE samples as compared with FF samples. Our results indicate that FFPE tissue processing may result in persistent chemical modifications of histones, which correspond in mass shift of important PTMs. Herein, we highlight the importance to investigate and report FA-formed modifications in FFPE-treated tissues, as well as the necessity of careful manual examination of observed modifications to eliminate false-positive PTMs. Main In eukaryotic nuclei, chromosomal DNA is tightly packed in a compact chromatin, a macromolecular structure composed of repetitive fundamental units, nucleosomes. Nucleosomes are in turn formed of 166 bp DNA wrapped around a histone octamer core consisting of histone H3–H4 tetramer and two copies of histone H2A–H2B dimers. Adjacent linker histones such as histone H1 can further package the nucleosome into the higher-order chromatin. Basic histones are often subjected to post-translational modifications (PTMs) located on both the tail and the globular domain of the protein.1, 2, 3, 4 PTMs of histones, including acetylation, methylation, and/or ubiquitination typically occurring on lysine, arginine, or serine residues, are known to be involved in epigenetic regulation of gene expression and thus have an important role in tumorigenesis.5 A number of PTMs of histones, linked to diverse malignancies including pancreatic cancer, are frequently studied as potential targets for cancer therapy. Novel cancer-specific histone PTMs could also provide information for the discovery of biomarkers that would be suitable for diagnostic, prognostic, or predictive purposes.6, 7, 8 Owing to the insufficient availability of required amounts of fresh tissue biopsies, studies are often based on archived formaldehyde-fixed, paraffin-embedded (FFPE) tissue blocks, serving as an important clinical resource. In this respect, the fixing with paraformaldehyde (PFA) followed by embedding in paraffin is a classical standard method in pathology for preservation and morphology analysis of clinical tissue material. Several techniques have been developed to extract effectively proteins from FFPE tissue, allowing subsequent proteomic analyses of the digested proteins.9, 10, 11, 12 Liquid chromatography-tandem mass spectrometry (LC-MS/MS) approaches are commonly applied to detect PTM sites on histones, by tracing predicted mass shifts caused by a particular modification.13 FFPE-preserved tissues offer many advantages as compared with fresh-frozen (FF) biopsies, in particular regarding the high stability at room temperature and the extensive availability from global biobanks. Formaldehyde (FA) fixation of the tissue also provides an outstanding structural preservation of the tissue morphology, which is not possible when the tissue is stored at ultralow temperatures.14 It is however important to consider that FA present in FFPE materials may undergo condensation reaction with, for example, free N-terminal amino groups, other amino- and thiol-residues, as well as with active CH functionalities of the proteins, forming attached methyl and methylol groups, via intermediate Schiff bases or imines and crosslinking methylene bridges between amino groups.15, 16 In addition, as reported previously, N-terminal amino groups and particularly lysine side chains account for a great majority of FA fixation-induced modifications. Methylation corresponding to a mass shift of +14.016 Da, methylene adducts (+12 Da), methylol adducts (+30.0106 Da), and formylation (+27.9949 Da) were identified as the most significant.17, 18, 19 It is clear that tissue specimens are subjected to a wide range of chemical processing before the final proteomic analysis, starting with formalin fixation and paraffin impregnation for tissue preservation, followed by heat-induced antigen retrieval to reverse the effect of the previous treatment as well as to improve the protein yield.20, 21 The pattern of chemical modifications in tissue proteins after formalin treatment has been studied previously.18, 19 It has, however, not yet been fully elucidated whether chemical reactions occurring during FFPE preservation of tissue could result in irreversible alterations of histone proteins to induce shifts in a multifold of masses that could be incorrectly interpreted as endogenously formed PTMs. In this study, we evaluated and compared chemical modifications on the basic residues of the histone proteins originating from pancreatic tumor xenografts tissue, subjected to FFPE or FF processing. The focus was directed towards modifications resulting in a defined mass shift of +14.016, +28.031, +42.047, +42.011, and +114.043 Da, assigned as methylation, dimethylation, trimethylation, acetylation, or ubiquitination, respectively, considered as PTMs potentially causing epigenetic changes frequently occurring in diverse malignancies. Materials and methods Materials The following chemicals and solvents were purchased from Sigma-Aldrich (St Louis, MO, USA); Tris-HCl, guanidine-HCl, ammonium bicarbonate (AMBIC), dithiothreitol (DTT), iodoacetamide (IAA), formic acid, and acetonitrile (ACN). PFA, xylene and paraffin were obtained from Histolab Products AB (Gotemburg, Sweden) and ethanol (EtOH) from Solveco (Rosenberg, Sweden). BCA assay, Pierce quantitative colorimetric peptide assay, and Pier (...truncated)