Nic1 inactivation enables stable isotope labeling with 13C615N4-arginine in Schizosaccharomyces pombe

Carpy, Alejandro, Patel, Avinash, Tay, Ye Dee, Hagan, Iain M. and Macek, Boris 2015. Nic1 inactivation enables stable isotope labeling with 13C615N4-arginine in Schizosaccharomyces pombe. Molecular and Cellular Proteomics 14 (1) , pp. 243-250. 10.1074/mcp.O114.045302

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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, 13C615N4-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 13C615N4-arginine is catabolized by arginase and urease activity to 15N1-labeled ammonia that is used as a precursor for general amino acid biosynthesis. We show that disruption of Ni2+-dependent urease activity, through deletion of the sole Ni2+ transporter Nic1, blocks this recycling in ammonium-supplemented EMMG medium to enable 13C615N4-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.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Biosciences
Publisher:	American Society for Biochemistry and Molecular Biology
ISSN:	1535-9476
Funders:	Cancer Research UK
Last Modified:	23 Jul 2024 13:30
URI:	https://orca.cardiff.ac.uk/id/eprint/170128

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