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HERO ID
2966876
Reference Type
Journal Article
Title
Nic1 inactivation enables stable isotope labeling with 13C615N4-arginine in Schizosaccharomyces pombe
Author(s)
Carpy, A; Patel, A; Tay, YD; Hagan, IM; Macek, B
Year
2015
Is Peer Reviewed?
1
Journal
Molecular and Cellular Proteomics
ISSN:
1535-9476
EISSN:
1535-9484
Volume
14
Issue
1
Page Numbers
243-250
Language
English
PMID
25368411
DOI
10.1074/mcp.O114.045302
Abstract
Stable Isotope Labeling by Amino Acids (SILAC) is a commonly used method in quantitative proteomics. Because of compatibility with trypsin digestion, arginine and lysine are the most widely used amino acids for SILAC labeling. We observed that Schizosaccharomyces pombe (fission yeast) cannot be labeled with a specific form of arginine, (13)C(6) (15)N(4)-arginine (Arg-10), which limits the exploitation of SILAC technology in this model organism. We hypothesized that in the fission yeast the guanidinium group of (13)C(6) (15)N(4)-arginine is catabolized by arginase and urease activity to (15)N1-labeled ammonia that is used as a precursor for general amino acid biosynthesis. We show that disruption of Ni(2+)-dependent urease activity, through deletion of the sole Ni(2+) transporter Nic1, blocks this recycling in ammonium-supplemented EMMG medium to enable (13)C(6) (15)N(4)-arginine labeling for SILAC strategies in S. pombe. Finally, we employed Arg-10 in a triple-SILAC experiment to perform quantitative comparison of G1 + S, M, and G2 cell cycle phases in S. pombe.
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