Targeted analysis of recombinant NF kappa B (RelA/p65) by denaturing and native top down mass spectrometry.

ABSTRACT: Measuring post-translational modifications on transcription factors by targeted mass spectrometry is hampered by low protein abundance and inefficient isolation. Here, we utilized HaloTag technology to overcome these limitations and evaluate various top down mass spectrometry approaches for measuring NF-κB p65 proteoforms isolated from human cells. We show isotopic resolution of N-terminally acetylated p65 and determined it is the most abundant proteoform expressed following transfection in 293T cells. We also show MS(1) evidence for monophosphorylation of p65 under similar culture conditions and describe a high propensity for p65 proteoforms to fragment internally during beam-style MS(2) fragmentation; up to 71% of the fragment ions could be matched as internals in some fragmentation spec

SUBMITTER: Savaryn JP 

PROVIDER: S-EPMC4633404 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature