Miniaturized Digestion and Extraction of Surface Proteins from Candida albicans following Treatment with Histatin 5 for Mass Spectrometry Analysis.

Shirley Fan,Yizhi Xiao,Ken K.-C Yeung,Eduardo B Moffa,Walter L Siqueira

doi:10.1155/2016/9812829

Abstract

A common approach to isolate surface proteins from fungal and bacterial cells is to perform a proteolytic cleavage of proteins on the surface of intact cells suspended in solution. This paper describes miniaturization of this technique, in which cells are adhered on glass surfaces, and all sample treatments are conducted at μL volumes. Specifically, Candida albicans cells were attached onto HSA-coated glass slides. By depositing the appropriate reagent solutions on the adhered cells, we successfully performed cell washing, treatment with antifugal peptide, Histatin 5, and a proteolysis on intact cells with trypsin. The resulting peptides were subsequently analysed by mass spectrometry. In general, the data obtained was similar to that collected with suspended cells in much larger sample volumes. However, our miniaturized workflow offers the benefit of greatly reducing the consumption of cells and reagents.

Highlights

Candida albicans is a very common fungus found within the genitourinary and gastrointestinal tracts, as well as on the surface of skins [1]
The results indicated that preexposition of HTN5 to oral epithelial cells diminished the adhesion of C. albicans to the epithelium
Since the miniaturized assay in this work is performed on adhered cells, it is important to determine an optimal dosage of HTN5 which is sufficient to cause detected changes in protein abundance, but not too high to eradicate and desorb all cells from the glass surface

Summary

Introduction

Candida albicans is a very common fungus found within the genitourinary and gastrointestinal tracts, as well as on the surface of skins [1]. The most commonly used method to treat infections is to administer antifungal drugs orally or systematically into the circulatory system [2, 4] Despite these mechanisms, C. albicans has developed resistance towards them [4], and the discoveries of new therapeutic approaches are needed. Species identification through proteins and peptides from whole cell analyses has been reported [21,22,23]. When applying MS to study changes in protein levels of C. albicans upon treatment with HTN5, the global approach of studying the total proteome was certainly appropriate. In 2006, Rodrıguez-Ortega et al reported a protocol called “cell shaving,” which allowed them to focus on proteins partially exposed on the outside of cells [25]. The resulting MS data should be comparable to previous information obtained with the conventional, in solution, approach that required much larger quantities of reagent and cells

Materials and Methods

Results and Discussion

Conclusions