Stable isotope labelling by amino acids in cell culture (SILAC) in conjunction with MS analysis is a sensitive and reliable technique for quantifying relative differences in protein abundance and posttranslational modifications between cell populations. We develop and utilise SILAC-MS workflows for quantitative proteomics in the fungal pathogen Candida albicans. Arginine metabolism provides important cues for escaping host defences during pathogenesis, which limits the use of auxotrophs in Candida research. Our strategy eliminates the need for engineering arginine auxotrophs for SILAC experiments and allows the use of ARG4 as selectable marker during strain construction. Cells that are auxotrophic for lysine are successfully labelled with both lysine and arginine stable isotopes. We find that prototrophic C. albicans preferentially uses exogenous arginine and down-regulates internal production, which allow it to achieve high incorporation rates. However, similar to other yeast, C. albicans is able to metabolise heavy arginine to heavy proline, which compromised the accuracy of protein quantification. A computational method is developed to correct for the incorporation of heavy proline. In addition, we utilise the developed SILAC labelling in C. albicans for the global quantitative proteomic analysis of a strain expressing a phosphatase-dead mutant Cdc14 PD .
|Publication status||Published - Mar 2018|
- Candida albicans
- native SILAC
- quantitative proteomics