Examining the complexity of human RNA polymerase complexes using HaloTag technology coupled to label free quantitative proteomics

Danette L. Daniels, Jacqui Méndez, Amber Mosley, Sreenivasa R. Ramisetty, Nancy Murphy, Hélène Benink, Keith V. Wood, Marjeta Urh, Michael P. Washburn

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

Efficient determination of protein interactions and cellular localization remains a challenge in higher order eukaryotes and creates a need for robust technologies for functional proteomics studies. To address this, the HaloTag technology was developed for highly efficient and rapid isolation of intracellular complexes and correlative in vivo cellular imaging. Here we demonstrate the strength of this technology by simultaneous capture of human eukaryotic RNA polymerases (RNAP) I, II, and III using a shared subunit, POLR2H, fused to the HaloTag. Affinity purifications showed successful isolation, as determined using quantitative proteomics, of all RNAP core subunits, even at expression levels near endogenous. Transient known RNAP II interacting partners were identified as well as three previously uncharacterized interactors. These interactions were validated and further functionally characterized using cellular imaging. The multiple capabilities of the HaloTag technology demonstrate the ability to efficiently isolate highly challenging multiprotein complexes, discover new interactions, and characterize cellular localization.

Original languageEnglish
Pages (from-to)564-575
Number of pages12
JournalJournal of Proteome Research
Volume11
Issue number2
DOIs
StatePublished - Feb 3 2012

Fingerprint

DNA-Directed RNA Polymerases
Proteomics
Labels
Technology
RNA Polymerase II
RNA Polymerase I
RNA Polymerase III
Imaging techniques
Multiprotein Complexes
Eukaryota
Purification
Proteins

Keywords

  • cellular imaging
  • mass spectrometry
  • MudPIT
  • normalized spectral abundance factor
  • protein interactions
  • quantitative proteomics
  • RNA polymerases

ASJC Scopus subject areas

  • Biochemistry
  • Chemistry(all)

Cite this

Examining the complexity of human RNA polymerase complexes using HaloTag technology coupled to label free quantitative proteomics. / Daniels, Danette L.; Méndez, Jacqui; Mosley, Amber; Ramisetty, Sreenivasa R.; Murphy, Nancy; Benink, Hélène; Wood, Keith V.; Urh, Marjeta; Washburn, Michael P.

In: Journal of Proteome Research, Vol. 11, No. 2, 03.02.2012, p. 564-575.

Research output: Contribution to journalArticle

Daniels, DL, Méndez, J, Mosley, A, Ramisetty, SR, Murphy, N, Benink, H, Wood, KV, Urh, M & Washburn, MP 2012, 'Examining the complexity of human RNA polymerase complexes using HaloTag technology coupled to label free quantitative proteomics', Journal of Proteome Research, vol. 11, no. 2, pp. 564-575. https://doi.org/10.1021/pr200459c
Daniels, Danette L. ; Méndez, Jacqui ; Mosley, Amber ; Ramisetty, Sreenivasa R. ; Murphy, Nancy ; Benink, Hélène ; Wood, Keith V. ; Urh, Marjeta ; Washburn, Michael P. / Examining the complexity of human RNA polymerase complexes using HaloTag technology coupled to label free quantitative proteomics. In: Journal of Proteome Research. 2012 ; Vol. 11, No. 2. pp. 564-575.
@article{1dcf7569c8914bc0b85efc2cc63fd4da,
title = "Examining the complexity of human RNA polymerase complexes using HaloTag technology coupled to label free quantitative proteomics",
abstract = "Efficient determination of protein interactions and cellular localization remains a challenge in higher order eukaryotes and creates a need for robust technologies for functional proteomics studies. To address this, the HaloTag technology was developed for highly efficient and rapid isolation of intracellular complexes and correlative in vivo cellular imaging. Here we demonstrate the strength of this technology by simultaneous capture of human eukaryotic RNA polymerases (RNAP) I, II, and III using a shared subunit, POLR2H, fused to the HaloTag. Affinity purifications showed successful isolation, as determined using quantitative proteomics, of all RNAP core subunits, even at expression levels near endogenous. Transient known RNAP II interacting partners were identified as well as three previously uncharacterized interactors. These interactions were validated and further functionally characterized using cellular imaging. The multiple capabilities of the HaloTag technology demonstrate the ability to efficiently isolate highly challenging multiprotein complexes, discover new interactions, and characterize cellular localization.",
keywords = "cellular imaging, mass spectrometry, MudPIT, normalized spectral abundance factor, protein interactions, quantitative proteomics, RNA polymerases",
author = "Daniels, {Danette L.} and Jacqui M{\'e}ndez and Amber Mosley and Ramisetty, {Sreenivasa R.} and Nancy Murphy and H{\'e}l{\`e}ne Benink and Wood, {Keith V.} and Marjeta Urh and Washburn, {Michael P.}",
year = "2012",
month = "2",
day = "3",
doi = "10.1021/pr200459c",
language = "English",
volume = "11",
pages = "564--575",
journal = "Journal of Proteome Research",
issn = "1535-3893",
publisher = "American Chemical Society",
number = "2",

}

TY - JOUR

T1 - Examining the complexity of human RNA polymerase complexes using HaloTag technology coupled to label free quantitative proteomics

AU - Daniels, Danette L.

AU - Méndez, Jacqui

AU - Mosley, Amber

AU - Ramisetty, Sreenivasa R.

AU - Murphy, Nancy

AU - Benink, Hélène

AU - Wood, Keith V.

AU - Urh, Marjeta

AU - Washburn, Michael P.

PY - 2012/2/3

Y1 - 2012/2/3

N2 - Efficient determination of protein interactions and cellular localization remains a challenge in higher order eukaryotes and creates a need for robust technologies for functional proteomics studies. To address this, the HaloTag technology was developed for highly efficient and rapid isolation of intracellular complexes and correlative in vivo cellular imaging. Here we demonstrate the strength of this technology by simultaneous capture of human eukaryotic RNA polymerases (RNAP) I, II, and III using a shared subunit, POLR2H, fused to the HaloTag. Affinity purifications showed successful isolation, as determined using quantitative proteomics, of all RNAP core subunits, even at expression levels near endogenous. Transient known RNAP II interacting partners were identified as well as three previously uncharacterized interactors. These interactions were validated and further functionally characterized using cellular imaging. The multiple capabilities of the HaloTag technology demonstrate the ability to efficiently isolate highly challenging multiprotein complexes, discover new interactions, and characterize cellular localization.

AB - Efficient determination of protein interactions and cellular localization remains a challenge in higher order eukaryotes and creates a need for robust technologies for functional proteomics studies. To address this, the HaloTag technology was developed for highly efficient and rapid isolation of intracellular complexes and correlative in vivo cellular imaging. Here we demonstrate the strength of this technology by simultaneous capture of human eukaryotic RNA polymerases (RNAP) I, II, and III using a shared subunit, POLR2H, fused to the HaloTag. Affinity purifications showed successful isolation, as determined using quantitative proteomics, of all RNAP core subunits, even at expression levels near endogenous. Transient known RNAP II interacting partners were identified as well as three previously uncharacterized interactors. These interactions were validated and further functionally characterized using cellular imaging. The multiple capabilities of the HaloTag technology demonstrate the ability to efficiently isolate highly challenging multiprotein complexes, discover new interactions, and characterize cellular localization.

KW - cellular imaging

KW - mass spectrometry

KW - MudPIT

KW - normalized spectral abundance factor

KW - protein interactions

KW - quantitative proteomics

KW - RNA polymerases

UR - http://www.scopus.com/inward/record.url?scp=84856654953&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84856654953&partnerID=8YFLogxK

U2 - 10.1021/pr200459c

DO - 10.1021/pr200459c

M3 - Article

VL - 11

SP - 564

EP - 575

JO - Journal of Proteome Research

JF - Journal of Proteome Research

SN - 1535-3893

IS - 2

ER -