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

John Paul Savaryn, Owen S. Skinner, Luca Fornelli, Ryan T. Fellers, Philip D. Compton, Scott S. Terhune, Mike M. Abecassis, Neil L. Kelleher*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

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 293 T 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. This article is part of a Special Issue entitled: Protein Species. Biological significance: Characterizing transcription factor proteoforms in human cells is of high value to the field of molecular biology; many agree that post-translational modifications and combinations thereof play a critical role in modulating transcription factor activity. Thus, measuring these modifications promises increased understanding of molecular mechanisms governing the regulation of complex gene expression outcomes. To date, comprehensive characterization of transcription factor proteoforms within human cells has eluded measurement, owing primarily-with regard to top down mass spectrometry-to large protein size and low relative abundance. Here, we utilized HaloTag technology and recombinant protein expression to overcome these limitations and show top down mass spectrometry characterization of proteoforms of the 60 kDa NF-kB protein, p65. By optimizing the analytical procedure (i.e. purification, MS1, and MS2), our results make important progress toward the ultimate goal of targeted transcription factor characterization from endogenous loci.

Original languageEnglish (US)
Pages (from-to)76-84
Number of pages9
JournalJournal of Proteomics
Volume134
DOIs
StatePublished - Feb 16 2016

Keywords

  • Bottom up
  • HaloTag
  • Mass spectrometry
  • Native spray
  • Nuclear factor-kappa b
  • P65
  • Phosphorylation
  • Protein species
  • Proteoform
  • RelA
  • Top down

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry

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