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Mass Spectrometry: Complex Analysis01:21

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Quantitative Proteomic Analysis in Candida albicans Using SILAC-Based Mass Spectrometry.

Iliyana N Kaneva1,2, Joseph Longworth1, Peter E Sudbery2

  • 1ChELSI Institute, Department of Chemical and Biological Engineering, University of Sheffield, Sheffield, UK.

Proteomics
|December 28, 2017
PubMed
Summary

We developed a new method for quantitative proteomics in Candida albicans using stable isotope labeling by amino acids in cell culture (SILAC). This approach overcomes limitations in arginine metabolism, enabling accurate protein quantification and analysis of fungal pathogens.

Keywords:
Candida albicansSILACnative SILACquantitative proteomics

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Area of Science:

  • Proteomics
  • Molecular Biology
  • Mycology

Background:

  • Stable isotope labeling by amino acids in cell culture (SILAC) coupled with mass spectrometry (MS) is a powerful quantitative proteomics technique.
  • Arginine metabolism is crucial for fungal pathogenesis, but its manipulation in Candida albicans complicates SILAC experiments.
  • Existing methods require engineering arginine auxotrophs, limiting research applications.

Purpose of the Study:

  • To develop and implement robust SILAC-MS workflows for quantitative proteomics in the fungal pathogen Candida albicans.
  • To overcome challenges associated with arginine metabolism in C. albicans for SILAC analysis.
  • To enable accurate proteomic quantification in C. albicans, including the analysis of specific mutant strains.

Main Methods:

  • Utilized SILAC labeling with stable isotopes of lysine and arginine in lysine-auxotrophic C. albicans.
  • Developed a computational method to correct for the conversion of heavy arginine to heavy proline.
  • Applied the optimized SILAC workflow to analyze a strain expressing a phosphatase-dead mutant Cdc14 (Cdc14PD).

Main Results:

  • Achieved high stable isotope incorporation rates in prototrophic C. albicans by leveraging exogenous arginine.
  • Identified and computationally corrected for the metabolic conversion of heavy arginine to heavy proline, a common issue in yeast SILAC.
  • Successfully performed global quantitative proteomic analysis of a Cdc14PD mutant strain using the developed SILAC method.

Conclusions:

  • The developed SILAC-MS strategy effectively enables quantitative proteomics in C. albicans without requiring arginine auxotrophs.
  • The computational correction for heavy proline formation significantly improves the accuracy of protein quantification.
  • This methodology provides a valuable tool for studying C. albicans biology, pathogenesis, and drug target identification.