Two-color labeling of temporally defined protein populations in mammalian cells

Kimberly Beatty, David A. Tirrell

Research output: Contribution to journalArticle

45 Citations (Scopus)

Abstract

The proteome undergoes complex changes in response to disease, drug treatment, and normal cellular signaling processes. Characterization of such changes requires methods for time-resolved protein identification and imaging. Here, we describe the application of two reactive methionine (Met) analogues, azidohomoalanine (Aha) and homopropargylglycine (Hpg), to label two protein populations in fixed cells. Reactive lissamine rhodamine (LR), 7-dimethylaminocoumarin (DMAC), and bodipy-630 (BDPY) dyes were prepared and examined for use in selective dye-labeling of newly synthesized proteins in Rat-1 fibroblasts. The LR and DMAC, but not BDPY, fluorophores were found to enable selective, efficient labeling of subsets of the proteome; cells labeled with Aha and Hpg exhibited fluorescence emission three- to sevenfold more intense than that of control cells treated with Met. We also examined simultaneous and sequential pulse-labeling of cells with Aha and Hpg. After pulse-labeling, cells were treated with reactive LR and DMAC dyes, and labeled cells were imaged by fluorescence microscopy and analyzed by flow cytometry. The results of these studies demonstrate that amino acid labeling can be used to achieve selective two-color imaging of temporally defined protein populations in mammalian cells.

Original languageEnglish (US)
Pages (from-to)5995-5999
Number of pages5
JournalBioorganic and Medicinal Chemistry Letters
Volume18
Issue number22
DOIs
StatePublished - Nov 15 2008
Externally publishedYes

Fingerprint

Labeling
Color
Cells
Rhodamines
Coloring Agents
Population
Proteome
Proteins
Methionine
Cell signaling
Drug therapy
Imaging techniques
Flow cytometry
Fluorophores
Fluorescence microscopy
Fibroblasts
Rats
Labels
Fluorescence
Fluorescence Microscopy

Keywords

  • Alkyne
  • Azide
  • Click chemistry
  • Fluorescence
  • Microscopy
  • Proteomics

ASJC Scopus subject areas

  • Pharmaceutical Science
  • Drug Discovery
  • Organic Chemistry
  • Molecular Medicine
  • Molecular Biology
  • Clinical Biochemistry
  • Biochemistry

Cite this

Two-color labeling of temporally defined protein populations in mammalian cells. / Beatty, Kimberly; Tirrell, David A.

In: Bioorganic and Medicinal Chemistry Letters, Vol. 18, No. 22, 15.11.2008, p. 5995-5999.

Research output: Contribution to journalArticle

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