Spatial epi-proteomics enabled by histone post-translational modification analysis from low-abundance clinical samples

Abstract

Background Increasing evidence linking epigenetic mechanisms and different diseases, including cancer, has prompted in the last 15 years the investigation of histone post-translational modifications (PTMs) in clinical samples. Methods allowing the isolation of histones from patient samples followed by the accurate and comprehensive quantification of their PTMs by mass spectrometry (MS) have been developed. However, the applicability of these methods is limited by the requirement for substantial amounts of material. Results To address this issue, in this study we streamlined the protein extraction procedure from low-amount clinical samples and tested and implemented different in-gel digestion strategies, obtaining a protocol that allows the MS-based analysis of the most common histone PTMs from laser microdissected tissue areas containing as low as 1000 cells, an amount approximately 500 times lower than what is required by available methods. We then applied this protocol to breast cancer patient laser microdissected tissues in two proof-of-concept experiments, identifying differences in histone marks in heterogeneous regions selected by either morphological evaluation or MALDI MS imaging. Conclusions These results demonstrate that analyzing histone PTMs from very small tissue areas and detecting differences from adjacent tumor regions is technically feasible. Our method opens the way for spatial epi-proteomics, namely the investigation of epigenetic features in the context of tissue and tumor heterogeneity, which will be instrumental for the identification of novel epigenetic biomarkers and aberrant epigenetic mechanisms.

The histone code of the fungal genus Aspergillus uncovered by evolutionary and proteomic analyses

“…The samples were then desalted on handmade StageTips columns (46). Peptide mixtures were separated by reversed-phase chromatography on an EASY-Spray column (Thermo Fisher Scientic), 25-cm long (inner diameter 75 µm, PepMap C18, 2 µm particles), which was connected online to a Q Exactive Plus instrument (Thermo Fisher Scientific) through an EASY-Spray™ Ion Source (Thermo Fisher Scientific), as described (45).…”

“…Variable modifications included lysine propionylation, monomethylation + propionylation, dimethylation, trimethylation and acetylation. N-terminal PIC labeling was set as a fixed modification (45). To reduce the search time and the rate of false positives, with increasing the number of variable modifications included in the database search, the raw data were analyzed through multiple parallel MaxQuant jobs (47), setting different combinations of variable modifications.…”

Integrating computational pathology and proteomics to address tumor heterogeneity

“…This technique was demonstrated in a recent study by Noberini et al , where they applied MALDI MSI methods to a cohort of breast cancer tissue specimens to identify intratumoral regions with distinct histone posttranslational modifications. Following their identification, ROIs were then extracted using laser capture microdissection and profiled using a histone‐optimized mass‐spectrometry based‐analysis [41]. As demonstrated by their ability to analyze histone posttranslational modifications from a small tissue sample and their identification of spatially defined epigenetic changes within individual tumors, the combination of MSI and sensitive mass‐spectrometry profiling approaches may circumvent the traditional MSI limitations in depth of profiling.…”

“…These distinctions have further been demonstrated by some groups to be feasible at the level of individual cells, by identifying and classifying morphologically distinct cells with machine learning-based semiautomated annotation and subsequently correlating the individual cells with MSI data [38]. Although MSI methods are traditionally limited in resolution and may only identify biomolecules vulnerable to fragmentation when ionized, various studies have highlighted the utility of MSI in selecting tumor regions of interest (ROIs) for more in-depth profiling [21,40,41]. With material isolation techniques such as laser capture microdissection, tumor regions identified as molecularly distinct by MSI may be selected and profiled with increased sensitivity.…”

High Risk-Human Papillomavirus in HNSCC: Present and Future Challenges for Epigenetic Therapies

“…Dissecting hPTMs profiles in cancer is an emerging strategy with important implications in different directions such as discovery of new specific biomarkers for patients’ stratification, and for identification of new therapeutic targets. In this context, the technological advances in novel and more accurate quantitative mass-spectrometry based approaches represent a powerful tool for a fine profiling of the epigenetic landscapes in cancers [ 49 , 50 , 51 ].…”