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

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.