Quantitative Proteomic Analysis in Candida albicans using SILAC-based Mass Spectrometry

Stable isotope labelling by amino acids in cell culture (SILAC) in conjunction with mass spectrometry analysis is a sensitive and reliable technique for quantifying relative differences in protein abundance and post-translational modifications between cell populations. We have developed and utilised 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 were auxotrophic for lysine were successfully labelled with both lysine and arginine stable isotopes. We found that prototrophic C. albicans preferentially uses exogenous arginine and downregulates internal production, which allowed it to achieve high incorporation rates. However, similar to other yeast, C. albicans was able to metabolise heavy arginine to heavy proline, which compromised the accuracy of protein quantification. A computational method was developed to correct for the incorporation of heavy proline. In addition, we utilised the developed SILAC labelling in Candida albicans for the global quantitative proteomic analysis of a strain expressing a phosphatase-dead mutant Cdc14PD