application-masssquirm-for-quantitative-measurements-lysine

Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity

Recently, epigenetic regulators have been discovered as key players in many different diseases 1-3. As a result, these enzymes are prime targets for small ...

cutting-edge-technologies-driving-quantitative-mass-spectrometry

Cutting-Edge Technologies Driving Quantitative Mass Spectrometry

ARTICLES DISCUSSED:
Correa, C. N., Fiametti, L. O., Esquinca M. E. M., de Castro, L. M. Sample preparation and relative quantitation using reductive ...

In eukaryotes, DNA is wrapped around proteins called histones and is condensed into chromatin. Post-translational modification of histones can result in changes in gene expression. One of the most well-studied histone modifications is the methylation of lysine 4 on histone H3 (H3K4). This residue can be mono-, di- or tri-methylated and these varying methylation states have been associated with different levels of gene expression. Understanding exactly what the purpose of these methylation states is, in terms of gene expression, has been a topic of much research in recent years. Enzymes that can add (methyltransferases) and remove (demethylases) these modifications are of particular interest. The first demethylase discovered, LSD1, is the most well-classified and has been implicated in contributing to human cancers and to DNA damage response pathways. Currently, there are limited methods for accurately studying the activity of demethylases in vitro or in vivo. In this work, we present MassSQUIRM (mass spectrometric quantitation using isotopic reductive methylation), a quantitative method for studying the activity of demethylases capable of removing mono- and di-methyl marks from lysine residues. We focus specifically on LSD1 due to its potential as a prime therapeutic target for human disease. This quantitative approach will enable better characterization of the activity of LSD1 and other chromatin modifying enzymes in vitro, in vivo or in response to inhibitors.

MassSQUIRM: An assay for quantitative measurement of lysine demethylase activity.

In Eukaryoten DNA um Proteine genannt Histone gewickelt und wird in Chromatin kondensiert. Post-translationale Modifikation der Histone f&uuml;hrt zu Ver&auml;nderungen in der Genexpression. Eine der am meisten gut erforscht Histon &Auml;nderungen ist die Methylierung von Lysin 4 auf Histon H3 (H3K4). Diese R&uuml;ckst&auml;nde kann Mono-, di - oder Tri-methyliert und diese unterschiedlichen Methylierung-Staaten wurden mit verschiedenen Ebenen der Genexpression. Verstehen genau, was der Zweck dieser Methylierung-Staaten, in Bezug auf die Genexpression, ist ein Thema der viel Forschung in den letzten Jahren gewesen. Von besonderem Interesse sind Enzyme, die (Methyltransferasen) hinzuf&uuml;gen und entfernen (Demethylasen) k&ouml;nnen diese &Auml;nderungen. Die ersten Demethylase entdeckt, LSD1, ist die am meisten well-classified und hat verwickelt wurde, DNA Sch&auml;den Antwort Bildungswege zur menschlichen Krebserkrankungen beizutragen. Derzeit gibt es begrenzte Methoden f&uuml;r die Aktivit&auml;t der Demethylasen in vitro noch in vivo genau zu studieren. In dieser Arbeit pr&auml;sentieren wir Ihnen MassSQUIRM (massenspektrometrische Bestimmung mit Isotopen reduktive Methylierung), eine quantitative Methode zur Untersuchung der T&auml;tigkeit der Demethylasen f&auml;hig Mono - und di-Methyl zu entfernen markiert von Lysin-Resten. Wir setzen speziell auf LSD1 aufgrund seines Potentials als therapeutische Zielscheibe f&uuml;r menschliche Krankheit. Diese quantitative Ansatz erm&ouml;glicht bessere Charakterisierung der Aktivit&auml;t der LSD1 und andere Enzyme, die in-vitro, in vivo oder in Reaktion auf Inhibitoren &auml;ndern Chromatin.

MassSQUIRM: Ein Assay f&uuml;r die quantitative Messung von Lysin-Demethylase-Aktivit&auml;t.

DNA double-strand breaks are increased in human melanoma tissue as detected by histone H2AX phosphorylation.(1-3) We investigated two of the downstream effectors of DNA double-strand breaks, Rad50 and 53BP1 (tumor suppressor p53 binding protein 1), to determine if they are altered in human primary melanoma cells. Melanoma cases showed high Rad50 staining (81.8%; 9/11) significantly more frequently than conventional or atypical melanocytic nevi (0%; 0/18). In contrast, the staining pattern for 53BP1 appears similar between melanoma and nevi. This is the first study that shows activation and misregulation of the DNA repair pathway in human melanoma cells. The staining features of Rad50, a component of an essential DNA double-strand break repair complex, are clearly increased in melanoma cells with regards to both staining intensity and the number of positive melanoma cells. Interestingly, among the melanoma cases with increased Rad50 staining, most demonstrated cytoplasmic rather than nuclear staining (88.9%, 8/9). Further studies are needed to determine the cause of this mislocalization and its affects, if any, on DNA double-strand break repair in melanoma.

Misregulation of Rad50 expression in melanoma cells.

Molecular pathways regulating melanoma initiation and progression are potential targets of therapeutic development for this aggressive cancer. Identification and molecular analysis of these pathways in patients has been primarily restricted to targeted studies on individual proteins. Here, we report the most comprehensive analysis of formalin-fixed paraffin-embedded human melanoma tissues using quantitative proteomics. From 61 patient samples, we identified 171 proteins varying in abundance among benign nevi, primary melanoma, and metastatic melanoma. Seventy-three percent of these proteins were validated by immunohistochemistry staining of malignant melanoma tissues from the Human Protein Atlas database. Our results reveal that molecular pathways involved with tumor cell proliferation, motility, and apoptosis are mis-regulated in melanoma. These data provide the most comprehensive proteome resource on patient melanoma and reveal insight into the molecular mechanisms driving melanoma progression.

Quantitative Proteomics Identifies Activation of Hallmark Pathways of Cancer in Patient Melanoma.

Arsenic is a widely distributed environmental toxicant that can cause multi-tissue pathologies. Proteomic assays allow for the identification of biological processes modulated by arsenic in diverse tissue types.

Proteomics-Based Identification of Differentially Abundant Proteins from Human Keratinocytes Exposed to Arsenic Trioxide.

Normal cell growth is characterized by a regulated epigenetic program that drives cellular activities such as gene transcription, DNA replication, and DNA damage repair. Perturbation of this epigenetic program can lead to events such as mis-regulation of gene transcription and diseases such as cancer. To begin to understand the epigenetic program correlated to the development of melanoma, we performed a novel quantitative mass spectrometric analysis of histone post-translational modifications mis-regulated in melanoma cell culture as well as patient tumors. Aggressive melanoma cell lines as well as metastatic melanoma were found to have elevated histone H3 Lys(27) trimethylation (H3K27me3) accompanied by overexpressed methyltransferase EZH2 that adds the specific modification. The altered epigenetic program that led to elevated H3K27me3 in melanoma cell culture was found to directly silence transcription of the tumor suppressor genes RUNX3 and E-cadherin. The EZH2-mediated silencing of RUNX3 and E-cadherin transcription was also validated in advanced stage human melanoma tissues. This is the first study focusing on the detailed epigenetic mechanisms leading to EZH2-mediated silencing of RUNX3 and E-cadherin tumor suppressors in melanoma. This study underscores the utility of using high resolution mass spectrometry to identify mis-regulated epigenetic programs in diseases such as cancer, which could ultimately lead to the identification of biological markers for diagnostic and prognostic applications.

Quantitative Histone Mass Spectrometry Identifies Elevated Histone H3 Lysine 27 (Lys27) Trimethylation in Melanoma.

Cancer immunotherapy provides durable clinical benefit in only a small fraction of patients, and identifying these patients is difficult due to a lack of reliable biomarkers for prediction and evaluation of treatment response. Here, we demonstrate the first application of label-free Raman spectroscopy for elucidating biomolecular changes induced by anti-CTLA4 and anti-PD-L1 immune checkpoint inhibitors (ICI) in the tumor microenvironment (TME) of colorectal tumor xenografts. Multivariate curve resolution-alternating least squares (MCR-ALS) decomposition of Raman spectral datasets revealed early changes in lipid, nucleic acid, and collagen content following therapy. Support vector machine classifiers and random forests analysis provided excellent prediction accuracies for response to both ICIs and delineated spectral markers specific to each therapy, consistent with their differential mechanisms of action. Corroborated by proteomics analysis, our observation of biomolecular changes in the TME should catalyze detailed investigations for translating such markers and label-free Raman spectroscopy for clinical monitoring of immunotherapy response in cancer patients. SIGNIFICANCE: This study provides first-in-class evidence that optical spectroscopy allows sensitive detection of early changes in the biomolecular composition of tumors that predict response to immunotherapy with immune checkpoint inhibitors.

ArticleTotal : 2	Year
Publication title Cited by 1	2012
Publication title Cited by 1	2023

Article	Year
MassSQUIRM: An assay for quantitative measurement of lysine demethylase activity. Epigenetics : official journal of the DNA Methylation Society\| PubMed ID: 21273814	2011
Journal of cutaneous pathology\| PubMed ID: 21883366	2011
Misregulation of Rad50 expression in melanoma cells. Journal of cutaneous pathology\| PubMed ID: 22725639	2012
Quantitative Proteomics Identifies Activation of Hallmark Pathways of Cancer in Patient Melanoma. Journal of proteomics & bioinformatics\| PubMed ID: 23976835	2013
Proteomics-Based Identification of Differentially Abundant Proteins from Human Keratinocytes Exposed to Arsenic Trioxide. Journal of proteomics & bioinformatics\| PubMed ID: 25419056	2014
Quantitative Histone Mass Spectrometry Identifies Elevated Histone H3 Lysine 27 (Lys27) Trimethylation in Melanoma. Molecular & cellular proteomics : MCP\| PubMed ID: 26621846	2016
Raman Spectroscopy and Machine Learning Reveals Early Tumor Microenvironmental Changes Induced by Immunotherapy. Cancer research\| PubMed ID: 34645610	2021

Nathan L. Avaritt

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