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Targeted analysis of recombinant NF kappa B (RelA/p65) by denaturing and native top down mass spectrometry. J Proteomics 2016 Feb 16;134:76-84

Date

05/09/2015

Pubmed ID

25952688

Pubmed Central ID

PMC4633404

DOI

10.1016/j.jprot.2015.04.025

Scopus ID

2-s2.0-84958173605   11 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-I?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 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 spectra. Finally, we used native spray mass spectrometry to measure proteins copurifying with p65 and present evidence for the native detection of p65, 71kDa 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 60kDa NF-kB protein, p65. By optimizing the analytical procedure (i.e. purification, MS(1), and MS(2)), our results make important progress toward the ultimate goal of targeted transcription factor characterization from endogenous loci.

Author List

Savaryn JP, Skinner OS, Fornelli L, Fellers RT, Compton PD, Terhune SS, Abecassis MM, Kelleher NL

Author

Scott Terhune PhD Professor in the Microbiology and Immunology department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

Humans
Mass Spectrometry
Protein Isoforms
Protein Multimerization
Recombinant Proteins
Transcription Factor RelA