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

Roberta Noberini,Andrea Uggetti,Giancarlo Pruneri,Saverio Minucci,Tiziana Bonaldi

doi:10.1074/mcp.m115.054510

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.

Highlights

From the ‡Center for Genomic Science of IIT@SEMM, Istituto Italiano di Tecnologia, Via Adamello 16, 20139 Milan, Italy; §Biobank for Translational Medicine Unit, Department of Pathology, European Institute of Oncology, Via Ripamonti 435, 20141 Milano; ¶School of Medicine, University of Milan, 20122 Milan, Italy; ʈDepartment of Experimental Oncology, European Institute of Oncology, Via Adamello 16, 20139 Milan, Italy; **Drug Development Program, European Institute of Oncology, Via Adamello 16, 20139 Milan, Italy; ‡‡Department of Bioscience, University of Milan, 20133 Milan, Italy
We report for the first time the successful application of MS-based analysis of Histone post-translational modifications (hPTMs) to human clinical samples, focusing in particular on the development and validation of a method (PAT-H-MS) to extract histones from formalin-fixed paraffin embedded (FFPE) tissues in yield and purity sufficient to enable the subsequent use of a proteomic workflow optimized for hPTM analysis [13]
Set-up of the PAT-H-MS Protocol in Mouse Tissues—To set-up the technology to profile by mass spectrometry hPTMs from FFPE tissue we used different murine tissues, which can be obtained in large amounts and offer the possibility to compare FFPE and frozen tissue from the same samples stored for up to several years

Summary

EXPERIMENTAL PROCEDURES

Preparation of Frozen and FFPE Tissues—Leukemic blasts were isolated from acute promyelocytic leukemia transgenic mice and 1 ϫ 106 cells were i.v. injected in a syngenic recipient mouse to induce secondary leukemia [14]. To identify a pool of cell lines that would appropriately represent the heterogeneous range of modifications possibly found in breast tumor samples, we analyzed the hPTM patterns of five breast cancer cell lines belonging to different subtypes and with different immunoprofiles (supplemental Fig. S4A) For this purpose we used a previously described spike-in approach [15], where unlabeled normal breast MCF10A cells are spiked into each of the five heavy-labeled breast cancer cell line samples and used as an internal standard for quantitation. We evaluated histone H3 hPTM patterns of acetylations and methylations of lysine residues based on H/L ratios of relative abundances (supplemental Fig. S4C–S4D) Because they showed the most divergent patterns, we chose MDA-MB-231, MDAMB-468, MDA-MB-453 and MDA-MB-361 to generate a super-SILAC mix that can be spiked into breast cancer samples prior to GeLC-MS analysis (supplemental Fig. S4E). Images were acquired on a ChemiDoc XRS instrument (Bio-Rad) and quantified using the ImageJ software (rsb.info.nih.gov)

RESULTS

C LuA 1 LuA 2 LuA 3 LuA 4 LuA 5

DISCUSSION