ChAP-MS: A Method for Identification of Proteins and Histone Posttranslational Modifications at a Single Genomic Locus

Abstract

Summary The field of epigenomics has been transformed by chromatin immunoprecipitation approaches that provide for the localization of a defined protein or posttranslationally-modified protein to specific chromosomal sites. While these approaches have helped us conceptualize epigenetic mechanisms, the field has been limited by the inability to define features like the proteome and histone modifications at a specific genomic locus in an unbiased manner. We developed an unbiased approach whereby a unique native genomic locus was isolated, which was followed by high resolution proteomic identification of specifically associated proteins and histone posttranslational modifications. This Chromatin Affinity Purification with Mass Spectrometry (ChAP-MS) technique was used to specifically enrich a ~1,000 base-pair section of GAL1 chromatin under transcriptionally active and repressive conditions, and to identify the specifically bound proteins and histone posttranslational modifications. ChAP-MS should yield unprecedented insight into the regulatory mechanisms of transcription and help identify factors that epigenetically control chromatin function.

Evidence That G-quadruplex DNA Accumulates in the Cytoplasm and Participates in Stress Granule Assembly in Response to Oxidative Stress

“…LC-MS Analysis-Protein gel bands were excised, then digested with trypsin (107). Tryptic peptides were separated using a nanoAcquity UPLC system (Waters) coupled to a LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific) (107,108).…”

“…Tryptic peptides were separated using a nanoAcquity UPLC system (Waters) coupled to a LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific) (107,108). Proteins were identified by a database search using PEAKS Studio v7 (Bioinformatics Solutions).…”

Reading the second genomic code

“…In response to this shortfall, Tackett and his Arkansas colleagues, along with scientists at the Johns Hopkins School of Medicine, have developed chromatin affinity purification with mass spectrometry (ChAP-MS) 3 . The approach involves cutting out a 1,000-base-pair region of a chromosome, purifying it and determining all the epigenetic changes that are present.…”

A CRISPR-based approach for proteomic analysis of a single genomic locus

“…[2][3][4][5][6][7][8][9] Purification of a small region of chromatin from the cellular milieu is one of the most challenging aspects of these approaches as the proteins and histone PTMs specifically isolated with the targeted chromatin typically constitute a small fraction of the identified proteins -most of which are non-specific associations. 2,3,10 We developed two approaches using quantitative high resolution mass spectrometry that distinguish whether proteins and histone PTMs identified during epiproteome measurements are "specific" to the target chromatin or are "non-specific" contaminants. 2,3 These quantitative approaches are critical components of our ChAP-MS (Chromatin Affinity Purification with Mass Spectrometry) platform of technologies that enable local epiproteome analysis.…”

“…2,3,10 We developed two approaches using quantitative high resolution mass spectrometry that distinguish whether proteins and histone PTMs identified during epiproteome measurements are "specific" to the target chromatin or are "non-specific" contaminants. 2,3 These quantitative approaches are critical components of our ChAP-MS (Chromatin Affinity Purification with Mass Spectrometry) platform of technologies that enable local epiproteome analysis. Included in this platform are the first generation ChAP-MS and second generation TAL-ChAP-MS approaches.…”