Proteomics characterization of Chromosomal Common Fragile Site (CFS) - associated proteins uncovers ATRX as a regulator of CFS stability
Chromosomal Common Fragile Sites (CFSs) are conserved regions of our genome prone to break in conditions of replication stress (RS. Here, we have performed Chromatin Immunoprecipitation (ChIP) with FACND2 antibodies coupled to Mass Spectrometry to isolate CFSs from HeLa cells and identify the proteins enriched at these loci.
Sample Processing Protocol
ChIP eluates were separated by SDS-page and subjected to in-gel digestion with trypsin. Samples were stage-tipped and analysed analysed with an Easy-nLC 1000 system coupled to the Q Exactive HF-X instrument (Thermo Fisher Scientific) through a nanoelectrospray ion source.
Data Processing Protocol
All raw MS data was process using MaxQuant (v.18.104.22.168) and the integrated database search engine, Andromeda against the Uniprot database. Common contaminants were included in the database and excluded upon data analysis. False discovery rate was set to 1% for peptide spectral matches (PSMs) and protein level, calculated using the decoy database strategy.
Stephanie Munk, Copenhagen University
Andres Joaquin Lopez-Contreras, Department of Cellular and Molecular Medicine, Center for Chromosome Stability and Center for Healthy Aging, University of Copenhagen, Copenhagen 2200, Denmark ( lab head )
Pladevall-Morera D, Munk S, Ingham A, Garribba L, Albers E, Liu Y, Olsen JV, Lopez-Contreras AJ. Proteomic characterization of chromosomal common fragile site (CFS)-associated proteins uncovers ATRX as a regulator of CFS stability. Nucleic Acids Res. 2019 PubMed: 31180492