108 research outputs found
Mass-spectrometry analysis of histone post-translational modifications in pathology tissue using the PAT-H-MS approach
No full textAbstractAberrant histone post-translational modifications (hPTMs) have been implicated with various pathologies, including cancer, and may represent useful epigenetic biomarkers. The data described here provide a mass spectrometry-based quantitative analysis of hPTMs from formalin-fixed paraffin-embedded (FFPE) tissues, from which histones were extracted through the recently developed PAT-H-MS method. First, we analyzed FFPE samples from mouse spleen and liver or human breast cancer up to six years old, together with their corresponding fresh frozen tissue. We then combined the PAT-H-MS approach with a histone-focused version of the super-SILAC strategy-using a mix of histones from four breast cancer cell lines as a spike-in standard- to accurately quantify hPTMs from breast cancer specimens belonging to different subtypes. The data, which are associated with a recent publication (Pathology tissue-quantitative mass spectrometry analysis to profile histone post-translational modification patterns in patient samples (Noberini, 2015) [1]), are deposited at the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier http://www.ebi.ac.uk/pride/archive/projects/PXD002669
Advances in enrichment methods for mass spectrometry-based proteomics analysis of post-translational modifications
No full textPost-translational modifications (PTMs) occur during or after protein biosynthesis and increase the functional diversity of proteome. They comprise phosphorylation, acetylation, methylation, glycosylation, ubiquitination, sumoylation (among many other modifications), and influence all aspects of cell biology. Mass-spectrometry (MS)-based proteomics is the most powerful approach for PTM analysis. Despite this, it is challenging due to low abundance and labile nature of many PTMs. Hence, enrichment of modified peptides is required for MS analysis. This review provides an overview of most common PTMs and a discussion of current enrichment methods for MS-based proteomics analysis. The traditional affinity strategies, including immunoenrichment, chromatography and protein pull-down, are outlined together with their strengths and shortcomings. Moreover, a special attention is paid to chemical enrichment strategies, such as capture by chemoselective probes, metabolic and chemoenzymatic labelling, which are discussed with an emphasis on their recent progress. Finally, the challenges and future trends in the field are discussed
Emocha. Enhanced Museum of Cultural Heritage & Arts
No full textDigital archives, repository, mostre virtual
A Super-SILAC Approach for Profiling Histone Posttranslational Modifications
No full textHistone posttranslational modifications (PTMs) play an important role in the regulation of gene expression and have been implicated in a multitude of physiological and pathological processes. During the last decade, mass spectrometry (MS) has emerged as the most accurate and versatile tool to quantitate histone PTMs. Stable-isotope labeling by amino acids in cell culture (SILAC) is an MS-based quantitation strategy involving metabolic labeling of cells, which has been applied to global protein profiling as well as histone PTM analysis. The classical SILAC approach is associated with reduced experimental variability and high quantitation accuracy, but provides limited multiplexing capabilities and can be applied only to actively dividing cells, thus excluding clinical samples. Both limitations are overcome by an evolution of classical SILAC involving the use of a mix of heavy-labeled cell lines as a spike-in standard, known as "super-SILAC". In this chapter, we will provide a detailed description of the optimized protocol used in our laboratory to generate a histone-focused super-SILAC mix and employ it as an internal standard for histone PTM quantitation
PAT-H-MS coupled with laser microdissection to study histone post-translational modifications in selected cell populations from pathology samples
No full textBackground: Aberrations in histone post-translational modifications (hPTMs) have been linked with various pathologies, including cancer, and could not only represent useful biomarkers but also suggest possible targetable epigenetic mechanisms. We have recently developed an approach, termed pathology tissue analysis of histones by mass spectrometry (PAT-H-MS), that allows performing a comprehensive and quantitative analysis of histone PTMs from formalin-fixed paraffin-embedded pathology samples. Despite its great potential, the application of this technique is limited by tissue heterogeneity. Methods: In this study, we further implemented the PAT-H-MS approach by coupling it with techniques aimed at reducing sample heterogeneity and selecting specific portions or cell populations within the samples, such as manual macrodissection and laser microdissection (LMD). Results: When applied to the analysis of a small set of breast cancer samples, LMD-PAT-H-MS allowed detecting more marked changes between luminal A-like and triple negative patients as compared with the classical approach. These changes included not only the already known H3 K27me3 and K9me3 marks, but also H3 K36me1, which was found increased in triple negative samples and validated on a larger cohort of patients, and could represent a potential novel marker distinguishing breast cancer subtypes. Conclusions: These results show the feasibility of applying techniques to reduce sample heterogeneity, including laser microdissection, to the PAT-H-MS protocol, providing new tools in clinical epigenetics and opening new avenues for the comprehensive analysis of histone post-translational modifications in selected cell populations
Spatial epi-proteomics enabled by histone post-translational modification analysis from low-abundance clinical samples
No full textBackground: 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 quan-tification 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
A truncated and catalytically inactive isoform of KDM5B histone demethylase accumulates in breast cancer cells and regulates H3K4 tri-methylation and gene expression
No full textKDM5B histone demethylase is overexpressed in many cancers and plays an ambivalent role in oncogenesis, depending on the specific context. This ambivalence could be explained by the expression of KDM5B protein isoforms with diverse functional roles, which could be present at different levels in various cancer cell lines. We show here that one of these isoforms, namely KDM5B-NTT, accumulates in breast cancer cell lines due to remarkable protein stability relative to the canonical PLU-1 isoform, which shows a much faster turnover. This isoform is the truncated and catalytically inactive product of an mRNA with a transcription start site downstream of the PLU-1 isoform, and the consequent usage of an alternative ATG for translation initiation. It also differs from the PLU-1 transcript in the inclusion of an additional exon (exon-6), previously attributed to other putative isoforms. Overexpression of this isoform in MCF7 cells leads to an increase in bulk H3K4 methylation and induces derepression of a gene cluster, including the tumor suppressor Cav1 and several genes involved in the interferon-alpha and -gamma response. We discuss the relevance of this finding considering the hypothesis that KDM5B may possess regulatory roles independent of its catalytic activity.</p
Immunofluorescence imaging-guided laser microdissection for ultralow input spatial tissue proteomics
No full textLaser microdissection (LMD) enables the precise isolation of specific cells or regions of interest from tissue sections, guiding downstream molecular analyses with high spatial resolution. In this protocol, we describe an optimized protocol combining whole-slide immunofluorescence imaging, LMD, and low-input liquid chromatography (LC) mass spectrometry (MS)-based proteomics of FFPE tissue sections
Targeting Eph receptors with chemical compounds and peptides
No full textThe Eph receptors are a large family of tyrosine kinases involved in many biological processes and have recently emerged as promising drug target candidates. Indeed, they have been implicated in numerous pathological processes, including cancer progression and metastasis, pathological forms of angiogenesis and inhibition of spinal cord regeneration after injury. Therefore, inhibitors of Eph receptors function could be useful for diverse medical applications, in addition to helping elucidate the mechanism of receptor-ligand interaction.
In this study we applied two different screening approaches, high throughput screening and combinatorial library screening, to the identification of chemical compounds that target EphA4 and EphA2. Two 2,5-dimethylpyrrolyl benzoic acid derivatives were identified in the high throughput screen for EphA4 antagonists. These compounds selectively inhibit ephrin binding to EphA4 and EphA2 and the characteristic biological activities of the two receptors, showing for the first time that the Eph receptors can be successfully targeted by small molecules. By combinatorial library screening, we also identified platinum(II) tetraamines as potential selective inhibitors of EphA2 activity. Finally, we modified TNYL-RAW, a potent peptide antagonist for EphB4, in order to improve its stability and delay its in vivo clearance from the blood circulation by coupling it with poly ethylene glycol (PEG) or the Fc portion of human IgG
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