Kaneva, I.N., Longworth, J., Sudbery, P.E. et al. (1 more author) (2018) Quantitative Proteomic Analysis in Candida albicans using SILAC-based Mass Spectrometry. Proteomics, 18 (5-6). 1700278. ISSN 1615-9853
Abstract
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 .
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2017 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim. This is an author-produced version of a paper subsequently published in Proteomics. Uploaded in accordance with the publisher's self-archiving policy. |
Keywords: | Candida albicans; SILAC; native SILAC; quantitative proteomics |
Dates: |
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Institution: | The University of Sheffield |
Academic Units: | The University of Sheffield > Faculty of Engineering (Sheffield) > Department of Chemical and Biological Engineering (Sheffield) The University of Sheffield > Faculty of Science (Sheffield) > School of Biosciences (Sheffield) > Department of Molecular Biology and Biotechnology (Sheffield) |
Funding Information: | Funder Grant number BIOTECHNOLOGY AND BIOLOGICAL SCIENCES RESEARCH COUNCIL (BBSRC) BB/E003273/1 BIOTECHNOLOGY AND BIOLOGICAL SCIENCES RESEARCH COUNCIL (BBSRC) BB/M012166/1 |
Depositing User: | Symplectic Sheffield |
Date Deposited: | 07 Feb 2018 12:48 |
Last Modified: | 17 Dec 2020 18:00 |
Status: | Published |
Publisher: | Wiley |
Refereed: | Yes |
Identification Number: | 10.1002/pmic.201700278 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:127153 |