Pathology Tissue-quantitative Mass Spectrometry Analysis to Profile Histone Post-translational Modification Patterns in Patient Samples

Abstract

Histone post-translational modifications (hPTMs) generate a complex combinatorial code that has been implicated with various pathologies, including cancer. Dissecting such a code in physiological and diseased states may be exploited for epigenetic biomarker discovery, but hPTM analysis in clinical samples has been hindered by technical limitations. Here, we developed a method (PAThology tissue analysis of Histones by Mass Spectrometry - PAT-H-MS) that allows to perform a comprehensive, unbiased and quantitative MS-analysis of hPTM patterns on formalin-fixed paraffin-embedded (FFPE) samples. In pairwise comparisons, histone extracted from formalin-fixed paraffin-embedded tissues showed patterns similar to fresh frozen samples for 24 differentially modified peptides from histone H3. In addition, when coupled with a histone-focused version of the super-SILAC approach, this method allows the accurate quantification of modification changes among breast cancer patient samples. As an initial application of the PAThology tissue analysis of Histones by Mass Spectrometry method, we analyzed breast cancer samples, revealing significant changes in histone H3 methylation patterns among Luminal A-like and Triple Negative disease subtypes. These results pave the way for retrospective epigenetic studies that combine the power of MS-based hPTM analysis with the extensive clinical information associated with formalin-fixed paraffin-embedded archives.

Alternative digestion approaches improve histone modification mapping by mass spectrometry in clinical samples

“…Recently, our group has developed a series of histone enrichment methods that allow the MS-based analysis of histones from patient-derived samples, including primary cells, frozen tissues, and formalin-fixed paraffin-embedded (FFPE) tissues. [9][10][11] Among them, the pathology tissue analysis of histones by mass spectrometry (PAT-H-MS) method allowed for the first time a comprehensive MS-based profiling of epigenetic marks in patient pathology tissues. We employed these different approaches to study histone H3 methylation and acetylation patterns in breast cancer subtypes, [12] and to investigate histone PTM changes in the transition from primary tumors to culture conditions and xenograft models.…”

“…We have previously shown that while FFPE storage is suitable for the MS analysis of at least 24 differentially modified histone H3 peptides, it causes the appearance/increase of artifactual methylations on specific residues, which therefore cannot be reliably quantified. [11] By exploiting the C18 StageTip column enrichment, which is compatible with an in-solution digestion that allows thoroughly dissecting histone H4 tail acetylations and K20 methylations, we aimed at verifying whether relative differences in histone H4 PTMs can be quantified in FFPE samples. We therefore applied an Arg-C digestion following C18 StageTip column enrichment to four ovarian cancer samples that were stored either as FFPE or fresh-frozen tissues, which were used as a reference (Table S1, Supporting Information).…”

“…However, MS has mostly been applied to study nonclinical samples. Recently, our group has developed a series ofhistone enrichment methods that allow the MS‐based analysis of histones from patient‐derived samples, including primary cells, frozen tissues, and formalin‐fixed paraffin‐embedded (FFPE) tissues . Among them, the pathology tissue analysis of histones by mass spectrometry (PAT‐H‐MS) method allowed for the first time a comprehensive MS‐based profiling of epigenetic marks in patient pathology tissues.…”

Recent advances in applying mass spectrometry and systems biology to determine brain dynamics

“…Despite immense efforts, there is still a need to further develop protocols for sample processing towards downstream proteomics analysis. In spite of these limitations, recent methodological developments have facilitated quantitative analyses by parallel reaction monitoring on FFPE breast cancer tissues [21] and PTM detection [22,23]. For a complete review of the proteomic developments in the analysis of FFPE tissues we refer to [24].…”

Quantitative Profiling of the Human Substantia Nigra Proteome from Laser-capture Microdissected FFPE Tissue

“…heating, there are still irreversible modifications with formaldehyde present. It has been shown recently that formylation and methylation on lysine are common modifications in FFPE tissue samples(46)(47)(48). However, these modifications are rarely included in the database search in studies using FFPE material.…”