Format

Send to

Choose Destination
Mol Cell Proteomics. 2014 Jul;13(7):1866-76. doi: 10.1074/mcp.O113.037119. Epub 2014 May 12.

Cell-specific labeling enzymes for analysis of cell-cell communication in continuous co-culture.

Author information

1
From the ‡Division of Cancer Biology, The Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB, UK; §Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139.
2
From the ‡Division of Cancer Biology, The Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB, UK;
3
§Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139.
4
From the ‡Division of Cancer Biology, The Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB, UK; claus.jorgensen@cruk.manchester.ac.uk.

Abstract

We report the orthologous screening, engineering, and optimization of amino acid conversion enzymes for cell-specific proteomic labeling. Intracellular endoplasmic-reticulum-anchored Mycobacterium tuberculosis diaminopimelate decarboxylase (DDC(M.tub-KDEL)) confers cell-specific meso-2,6-diaminopimelate-dependent proliferation to multiple eukaryotic cell types. Optimized lysine racemase (Lyr(M37-KDEL)) supports D-lysine specific proliferation and efficient cell-specific isotopic labeling. When ectopically expressed in discrete cell types, these enzymes confer 90% cell-specific isotopic labeling efficiency after 10 days of co-culture. Moreover, DDC(M.tub-KDEL) and Lyr(M37-KDEL) facilitate equally high cell-specific labeling fidelity without daily media exchange. Consequently, the reported novel enzyme pairing can be used to study cell-specific signaling in uninterrupted, continuous co-cultures. Demonstrating the importance of increased labeling stability for addressing novel biological questions, we compare the cell-specific phosphoproteome of fibroblasts in direct co-culture with epithelial tumor cells in both interrupted (daily media exchange) and continuous (no media exchange) co-cultures. This analysis identified multiple cell-specific phosphorylation sites specifically regulated in the continuous co-culture. Given their applicability to multiple cell types, continuous co-culture labeling fidelity, and suitability for long-term cell-cell phospho-signaling experiments, we propose DDC(M.tub-KDEL) and Lyr(M37-KDEL) as excellent enzymes for cell-specific labeling with amino acid precursors.

PMID:
24820872
PMCID:
PMC4083121
DOI:
10.1074/mcp.O113.037119
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for HighWire Icon for PubMed Central
Loading ...
Support Center