Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) Technology in Fission Yeast
- Boris Maček1,7,
- Alejandro Carpy1,
- André Koch2,4,
- Claudia C. Bicho3,5,
- Weronika E. Borek3,
- Silke Hauf2,6 and
- Kenneth E. Sawin3,7
- 1Proteome Center Tuebingen, Interfaculty Institute for Cell Biology, University of Tuebingen, 72076 Tuebingen, Germany
- 2Friedrich Miescher Laboratory of the Max Planck Society, Tuebingen, 72076, Germany
- 3Wellcome Trust Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3BF, United Kingdom
- ↵7Correspondence: boris.macek{at}uni-tuebingen.de; ken.sawin{at}ed.ac.uk
Abstract
Shotgun proteomics combined with stable isotope labeling by amino acids in cell culture (SILAC) is a powerful approach to quantify proteins and posttranslational modifications across the entire proteome. SILAC technology in Schizosaccharomyces pombe must cope with the “arginine conversion problem,” in which isotope-labeled arginine is converted to other amino acids. This can be circumvented by either using stable isotope-marked lysine only (as opposed to the more standard lysine/arginine double labeling) or using yeast genetics to create strains that only very inefficiently convert arginine. Both strategies have been used successfully in large-scale (phospho)proteomics projects in S. pombe. Here we introduce methods for performing a typical SILAC-based experiment in fission yeast, including generation of SILAC-compatible strains, sample preparation, and measurement by mass spectrometry.
Footnotes
-
From the Fission Yeast collection, edited by Iain M. Hagan, Antony M. Carr, Agnes Grallert, and Paul Nurse.
