Analysis of K-Ras interactions by biotin ligase tagging

Christopher Ritchie, Andrew Mack, Logan Harper, Ayna Alfadhli, Philip Stork, Xiaolin Nan, Eric Barklis

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

Background: Mutations of the human K-Ras 4B (K-Ras) G protein are associated with a significant proportion of all human cancers. Despite this fact, a comprehensive analysis of K-Ras interactions is lacking. Our investigations focus on characterization of the K-Ras interaction network. Materials and Methods: We employed a biotin ligase-tagging approach, in which tagged K-Ras proteins biotinylate neighbor proteins in a proximitydependent fashion, and proteins are identified via mass spectrometry (MS) sequencing. Results: In transfected cells, a total of 748 biotinylated proteins were identified from cells expressing biotin ligase-tagged K-Ras variants. Significant differences were observed between membrane-associated variants and a farnesylation-defective mutant. In pancreatic cancer cells, 56 K-Ras interaction partners were identified. Most of these were cytoskeletal or plasma membrane proteins, and many have been identified previously as potential cancer biomarkers. Conclusion: Biotin ligase tagging offers a rapid and convenient approach to the characterization of K-Ras interaction networks.

Original languageEnglish (US)
Pages (from-to)225-239
Number of pages15
JournalCancer Genomics and Proteomics
Volume14
Issue number4
DOIs
StatePublished - Jul 1 2017

Fingerprint

Ligases
Biotin
ras Proteins
Cells
Prenylation
Proteins
Cell membranes
Tumor Biomarkers
Pancreatic Neoplasms
GTP-Binding Proteins
Mass spectrometry
Blood Proteins
Mass Spectrometry
Membrane Proteins
Cell Membrane
Membranes
Mutation
Neoplasms

Keywords

  • Pancreatic cancer
  • Proximity mapping
  • Subcellular localization

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Genetics
  • Cancer Research

Cite this

Analysis of K-Ras interactions by biotin ligase tagging. / Ritchie, Christopher; Mack, Andrew; Harper, Logan; Alfadhli, Ayna; Stork, Philip; Nan, Xiaolin; Barklis, Eric.

In: Cancer Genomics and Proteomics, Vol. 14, No. 4, 01.07.2017, p. 225-239.

Research output: Contribution to journalArticle

@article{7cebd4144ccf4f96ade660284a460c7d,
title = "Analysis of K-Ras interactions by biotin ligase tagging",
abstract = "Background: Mutations of the human K-Ras 4B (K-Ras) G protein are associated with a significant proportion of all human cancers. Despite this fact, a comprehensive analysis of K-Ras interactions is lacking. Our investigations focus on characterization of the K-Ras interaction network. Materials and Methods: We employed a biotin ligase-tagging approach, in which tagged K-Ras proteins biotinylate neighbor proteins in a proximitydependent fashion, and proteins are identified via mass spectrometry (MS) sequencing. Results: In transfected cells, a total of 748 biotinylated proteins were identified from cells expressing biotin ligase-tagged K-Ras variants. Significant differences were observed between membrane-associated variants and a farnesylation-defective mutant. In pancreatic cancer cells, 56 K-Ras interaction partners were identified. Most of these were cytoskeletal or plasma membrane proteins, and many have been identified previously as potential cancer biomarkers. Conclusion: Biotin ligase tagging offers a rapid and convenient approach to the characterization of K-Ras interaction networks.",
keywords = "Pancreatic cancer, Proximity mapping, Subcellular localization",
author = "Christopher Ritchie and Andrew Mack and Logan Harper and Ayna Alfadhli and Philip Stork and Xiaolin Nan and Eric Barklis",
year = "2017",
month = "7",
day = "1",
doi = "10.21873/cgp.20034",
language = "English (US)",
volume = "14",
pages = "225--239",
journal = "Cancer Genomics and Proteomics",
issn = "1109-6535",
publisher = "International Institute of Anticancer Research",
number = "4",

}

TY - JOUR

T1 - Analysis of K-Ras interactions by biotin ligase tagging

AU - Ritchie, Christopher

AU - Mack, Andrew

AU - Harper, Logan

AU - Alfadhli, Ayna

AU - Stork, Philip

AU - Nan, Xiaolin

AU - Barklis, Eric

PY - 2017/7/1

Y1 - 2017/7/1

N2 - Background: Mutations of the human K-Ras 4B (K-Ras) G protein are associated with a significant proportion of all human cancers. Despite this fact, a comprehensive analysis of K-Ras interactions is lacking. Our investigations focus on characterization of the K-Ras interaction network. Materials and Methods: We employed a biotin ligase-tagging approach, in which tagged K-Ras proteins biotinylate neighbor proteins in a proximitydependent fashion, and proteins are identified via mass spectrometry (MS) sequencing. Results: In transfected cells, a total of 748 biotinylated proteins were identified from cells expressing biotin ligase-tagged K-Ras variants. Significant differences were observed between membrane-associated variants and a farnesylation-defective mutant. In pancreatic cancer cells, 56 K-Ras interaction partners were identified. Most of these were cytoskeletal or plasma membrane proteins, and many have been identified previously as potential cancer biomarkers. Conclusion: Biotin ligase tagging offers a rapid and convenient approach to the characterization of K-Ras interaction networks.

AB - Background: Mutations of the human K-Ras 4B (K-Ras) G protein are associated with a significant proportion of all human cancers. Despite this fact, a comprehensive analysis of K-Ras interactions is lacking. Our investigations focus on characterization of the K-Ras interaction network. Materials and Methods: We employed a biotin ligase-tagging approach, in which tagged K-Ras proteins biotinylate neighbor proteins in a proximitydependent fashion, and proteins are identified via mass spectrometry (MS) sequencing. Results: In transfected cells, a total of 748 biotinylated proteins were identified from cells expressing biotin ligase-tagged K-Ras variants. Significant differences were observed between membrane-associated variants and a farnesylation-defective mutant. In pancreatic cancer cells, 56 K-Ras interaction partners were identified. Most of these were cytoskeletal or plasma membrane proteins, and many have been identified previously as potential cancer biomarkers. Conclusion: Biotin ligase tagging offers a rapid and convenient approach to the characterization of K-Ras interaction networks.

KW - Pancreatic cancer

KW - Proximity mapping

KW - Subcellular localization

UR - http://www.scopus.com/inward/record.url?scp=85021369832&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85021369832&partnerID=8YFLogxK

U2 - 10.21873/cgp.20034

DO - 10.21873/cgp.20034

M3 - Article

C2 - 28647697

AN - SCOPUS:85021369832

VL - 14

SP - 225

EP - 239

JO - Cancer Genomics and Proteomics

JF - Cancer Genomics and Proteomics

SN - 1109-6535

IS - 4

ER -