Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) Technology in Fission Yeast
| Autor: | Boris Macek, Kenneth E. Sawin, André Koch, Alejandro Carpy, Silke Hauf, Claudia C. Bicho, Weronika E. Borek |
|---|---|
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
chemistry.chemical_classification Proteomics Arginine biology Proteome Chemistry Lysine biology.organism_classification Phosphoproteins General Biochemistry Genetics and Molecular Biology Yeast Mass Spectrometry Amino acid Fungal Proteins 03 medical and health sciences 030104 developmental biology Biochemistry Stable isotope labeling by amino acids in cell culture Isotope Labeling Schizosaccharomyces pombe Schizosaccharomyces Amino Acids Shotgun proteomics |
| Zdroj: | Cold Spring Harbor protocols. 2017(6) |
| ISSN: | 1559-6095 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|