RRC ID 76733
Author Ray J, Kruse A, Ozer A, Kajitani T, Johnson R, MacCoss M, Heck M, Lis JT.
Title RNA aptamer capture of macromolecular complexes for mass spectrometry analysis.
Journal Nucleic Acids Res
Abstract Specific genomic functions are dictated by macromolecular complexes (MCs) containing multiple proteins. Affinity purification of these complexes, often using antibodies, followed by mass spectrometry (MS) has revolutionized our ability to identify the composition of MCs. However, conventional immunoprecipitations suffer from contaminating antibody/serum-derived peptides that limit the sensitivity of detection for low-abundant interacting partners using MS. Here, we present AptA-MS (aptamer affinity-mass spectrometry), a robust strategy primarily using a specific, high-affinity RNA aptamer against Green Fluorescent Protein (GFP) to identify interactors of a GFP-tagged protein of interest by high-resolution MS. Utilizing this approach, we have identified the known molecular chaperones that interact with human Heat Shock Factor 1 (HSF1), and observed an increased association with several proteins upon heat shock, including translation elongation factors and histones. HSF1 is known to be regulated by multiple post-translational modifications (PTMs), and we observe both known and new sites of modifications on HSF1. We show that AptA-MS provides a dramatic target enrichment and detection sensitivity in evolutionarily diverse organisms and allows identification of PTMs without the need for modification-specific enrichments. In combination with the expanding libraries of GFP-tagged cell lines, this strategy offers a general, inexpensive, and high-resolution alternative to conventional approaches for studying MCs.
Volume 48(15)
Pages e90
Published 2020-9-4
DOI 10.1093/nar/gkaa542
PII 5866105
PMID 32609809
PMC PMC7470977
MeSH Aptamers, Nucleotide / chemistry* Aptamers, Nucleotide / genetics Green Fluorescent Proteins / genetics Heat Shock Transcription Factors / chemistry* Heat Shock Transcription Factors / genetics Histones / chemistry Humans Immunoprecipitation Macromolecular Substances / chemistry Macromolecular Substances / isolation & purification* Mass Spectrometry* Molecular Chaperones / chemistry Molecular Chaperones / genetics Peptides / chemistry Protein Binding Protein Processing, Post-Translational
IF 11.502