Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)-Based Quantitative Proteomics and Phosphoproteomics in Fission Yeast
- Alejandro Carpy1,
- André Koch2,4,
- Claudia C. Bicho3,5,
- Weronika E. Borek3,
- Silke Hauf2,6,
- Kenneth E. Sawin3,7 and
- Boris Maček1,7
- 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: ken.sawin{at}ed.ac.uk; boris.macek{at}uni-tuebingen.de
Abstract
Modern mass spectrometry (MS)-based approaches are capable of identifying and quantifying thousands of proteins and phosphorylation events in a single biological experiment. Here we present a (phospho)proteomic workflow based on in-solution proteome digestion of samples labeled by stable isotope labeling by amino acids in cell culture (SILAC) and phosphopeptide enrichment using strong cation exchange (SCX) and TiO2 chromatographies. These procedures are followed by high-accuracy MS measurement on an Orbitrap mass spectrometer and subsequent bioinformatic processing using MaxQuant software.
Footnotes
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From the Fission Yeast collection, edited by Iain M. Hagan, Antony M. Carr, Agnes Grallert, and Paul Nurse.
