PAQMAN: Protein-nucleic acid affinity quantification by MAss spectrometry in nuclear extracts.

Abstract

In recent years, various mass spectrometry-based approaches have been developed to determine global protein-DNA binding specificities using DNA affinity purifications from crude nuclear extracts. However, these assays are semi-quantitative and do not provide information about interaction affinities. We recently developed a technology that we call Protein-nucleic acid Affinity Quantification by MAss spectrometry in Nuclear extracts or PAQMAN, that can be used to determine apparent affinities between multiple nuclear proteins and a nucleic acid sequence of interest in one experiment. In PAQMAN, a series of affinity purifications with increasing bait concentrations and fixed amounts of crude nuclear extracts are combined with isobaric stable isotope labeling and quantitative mass spectrometry to generate Hill-like Kd curves for dozens of proteins in a single experiment. Here, we apply PAQMAN to determine apparent affinities for a genetic variant, rs36115365-C, which regulates TERT expression and is associated with an increased risk to develop various malignancies. Furthermore, we describe a detailed protocol for this method including important quality checks.