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Title: Targeted analysis of recombinant NF kappa B (RelA/p65) by denaturing and native top down mass spectrometry Open Access Deposited

https://creativecommons.org/licenses/by/3.0/us/
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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-?B 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 MS1 evidence for monophosphorylation of p65 under similar culture conditions and describe a high propensity for p65 proteoforms to fragment internally during beam-style MS2 fragmentation; up to 71% of the fragment ions could be matched as internals in some fragmentation spectra. Finally, we used native spray mass spectrometry to measure proteins copurifying with p65 and present evidence for the native detection of p65, 71 kDa heat shock protein, and p65 homodimer. Collectively, our work demonstrates the efficient isolation and top down mass spectrometry analysis of p65 from human cells, and we discuss the perturbations of overexpressing tagged proteins to study their biochemistry.
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  • 06/20/2024
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Date issued
  • 2015-05-04
Description sponsorship
  • The following grants are acknowledged: NIH RO1 GM067193, NIH P30 DA018310, NSF ABI-1062432, NIH HHSN261200800001E, NIH T32 5T32DK077662-07 to M.M.A., and NSF GRFP fellowship 2014171659 to O.S.S.
To Cite this Work:
Targeted analysis of recombinant NF kappa B (RelA/p65) by denaturing and native top down mass spectrometry [Data set]. Indiana University - DataCORE.

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Files (Count: 3; Size: 1.49 KB)

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TargetedAnalysisOfRecombinantNFK...B.zip 2024-08-16 2024-08-16 Open Access
README.txt 2024-08-16 2024-08-16 1.3 KB Open Access
Targeted_analysis_of_recombinant...t.txt 2024-08-16 2024-08-16 202 Bytes Open Access

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