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Nic1 Inactivation Enables Stable Isotope Labeling with (13)C(6)(15)N(4)-Arginine in Schizosaccharomyces pombe
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 labe...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
The American Society for Biochemistry and Molecular Biology
2015
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4288259/ https://www.ncbi.nlm.nih.gov/pubmed/25368411 http://dx.doi.org/10.1074/mcp.O114.045302 |
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author | Carpy, Alejandro Patel, Avinash Tay, Ye Dee Hagan, Iain M. Macek, Boris |
author_facet | Carpy, Alejandro Patel, Avinash Tay, Ye Dee Hagan, Iain M. Macek, Boris |
author_sort | Carpy, Alejandro |
collection | PubMed |
description | 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)N(1)-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. |
format | Online Article Text |
id | pubmed-4288259 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | The American Society for Biochemistry and Molecular Biology |
record_format | MEDLINE/PubMed |
spelling | pubmed-42882592015-01-12 Nic1 Inactivation Enables Stable Isotope Labeling with (13)C(6)(15)N(4)-Arginine in Schizosaccharomyces pombe Carpy, Alejandro Patel, Avinash Tay, Ye Dee Hagan, Iain M. Macek, Boris Mol Cell Proteomics Technological Innovation and Resources 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)N(1)-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. The American Society for Biochemistry and Molecular Biology 2015-01 2014-11-03 /pmc/articles/PMC4288259/ /pubmed/25368411 http://dx.doi.org/10.1074/mcp.O114.045302 Text en © 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Author's Choice—Final version full access. |
spellingShingle | Technological Innovation and Resources Carpy, Alejandro Patel, Avinash Tay, Ye Dee Hagan, Iain M. Macek, Boris Nic1 Inactivation Enables Stable Isotope Labeling with (13)C(6)(15)N(4)-Arginine in Schizosaccharomyces pombe |
title | Nic1 Inactivation Enables Stable Isotope Labeling with (13)C(6)(15)N(4)-Arginine in Schizosaccharomyces pombe |
title_full | Nic1 Inactivation Enables Stable Isotope Labeling with (13)C(6)(15)N(4)-Arginine in Schizosaccharomyces pombe |
title_fullStr | Nic1 Inactivation Enables Stable Isotope Labeling with (13)C(6)(15)N(4)-Arginine in Schizosaccharomyces pombe |
title_full_unstemmed | Nic1 Inactivation Enables Stable Isotope Labeling with (13)C(6)(15)N(4)-Arginine in Schizosaccharomyces pombe |
title_short | Nic1 Inactivation Enables Stable Isotope Labeling with (13)C(6)(15)N(4)-Arginine in Schizosaccharomyces pombe |
title_sort | nic1 inactivation enables stable isotope labeling with (13)c(6)(15)n(4)-arginine in schizosaccharomyces pombe |
topic | Technological Innovation and Resources |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4288259/ https://www.ncbi.nlm.nih.gov/pubmed/25368411 http://dx.doi.org/10.1074/mcp.O114.045302 |
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