Evaluating Spatiotemporal Dynamics of Phosphorylation of RNA Polymerase II Carboxy-Terminal Domain by Ultraviolet Photodissociation Mass Spectrometry

This is the authors' abstract. We don't add key points for this paper.

The critical role of site-specific phosphorylation in eukaryotic transcription has motivated efforts to decipher the complex phosphorylation patterns exhibited by the carboxyl-terminal domain (CTD) of RNA polymerase II. Phosphorylation remains a challenging post-translational modification to characterize by mass spectrometry owing to the labile phosphate ester linkage and low stoichiometric prevalence, two features that complicate analysis by high-throughput MS/MS methods. Identifying phosphorylation sites represents one significant hurdle in decrypting the CTD phosphorylation, a problem exaggerated by a large number of potential phosphorylation sites. An even greater obstacle is decoding the dynamic phosphorylation pattern along the length of the periodic CTD sequence. Ultraviolet photodissociation (UVPD) is a high-energy ion activation method that provides ample backbone cleavages of peptides while preserving labile post-translational modifications that facilitate their confident localization. Herein, we report a quantitative parallel reaction monitoring (PRM) method developed to monitor spatiotemporal changes in site-specific Ser5 phosphorylation of the CTD by cyclin-dependent kinase 7 (CDK7) using UVPD for sequence identification, phosphosite localization, and differentiation of phosphopeptide isomers. We capitalize on the series of phospho-retaining fragment ions produced by UVPD to create unique transition lists that are pivotal for distinguishing the array of phosphopeptides generated from the CTD.