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

Beatty, Kimberly E. and Tirrell, David A. (2008) Two-color labeling of temporally defined protein populations in mammalian cells. Bioorganic and Medicinal Chemistry Letters, 18 (22). pp. 5995-5999. ISSN 0960-894X. PMCID PMC3182832. https://resolver.caltech.edu/CaltechAUTHORS:BEAbmcl08

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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.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1016/j.bmcl.2008.08.046DOIArticle
http://www.sciencedirect.com/science/article/pii/S0960894X08009657PublisherArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3182832PubMed CentralArticle
ORCID:
AuthorORCID
Tirrell, David A.0000-0003-3175-4596
Additional Information:© 2008 Elsevier. Received 12 July 2008; revised 11 August 2008; accepted 12 August 2008. Available online 19 August 2008. We thank S.E. Fraser and C. Waters (of the Biological Imaging Center of the Beckman Institute at Caltech) for advice on microscopy. We thank L. Brown and A. Spalla (City of Hope) and R. Diamond and D. Perez (Caltech) for assistance with flow cytometry, and M. Shahgholi for help with mass spectrometry. We appreciate the insightful comments on this work provided by M.L. Mock, Y.Y. Lu, and M.J. Hangauer. This work was supported by NIH Grant GM62523, by the ARO-sponsored Institute for Collaborative Biotechnologies, and by the Joseph J. Jacobs Institute for Molecular Engineering for Medicine. K.E.B. is grateful to the Hertz Foundation, PEO, and the AAAS (Alan E. Leviton Award) for supporting her research. Supplementary data: Experimental protocols and additional data. Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bmcl.2008.08.046.
Funders:
Funding AgencyGrant Number
NIHGM62523
Army Research Office (ARO)UNSPECIFIED
Joseph J. Jacobs Institute for Molecular Engineering for MedicineUNSPECIFIED
Fannie and John Hertz FoundationUNSPECIFIED
Philanthropic Educational Organization (PEO)UNSPECIFIED
American Association for the Advancement of Science (AAAS)UNSPECIFIED
Subject Keywords:Azide; Alkyne; Proteomics; Fluorescence; Click chemistry; Microscopy
Issue or Number:22
PubMed Central ID:PMC3182832
Record Number:CaltechAUTHORS:BEAbmcl08
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:BEAbmcl08
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:12672
Collection:CaltechAUTHORS
Deposited By: Archive Administrator
Deposited On:19 Dec 2008 03:58
Last Modified:03 Oct 2019 00:30

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