Mass spectrometric quantitation of covalently bound cell wall proteins in Saccharomyces cerevisiae Qing Yuan Yin1, Piet W. J. de Groot1, Luitzen de Jong1, Frans M. Klis2 & Chris G. De Koster1 1Laboratory for Biomacromolecular Mass Spectrometry; and 2Molecular Biology and Microbial Food Safety, Swammerdam Institute for Life Sciences, University of Amsterdam, WV Amsterdam, The Netherlands OnlineOpen: This article is available free online at www.blackwell-synergy.com Correspondence: Frans M. Klis, Swammerdam Institute for Life Sciences, University of Amsterdam, Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands. Tel.: 131 20 5257834; fax: 131 20 5257924; e-mail: f.m.klis@uva.nl Received 9 March 2007; revised 7 May 2007; accepted 7 May 2007. First published online 6 July 2007. DOI:10.1111/j.1567-1364.2007.00272.x Editor: Richard Calderone Keywords cell wall protein; proteomics; MS; glycosylphosphatidylinositol-anchored protein; pir protein; transglycosylase. Abstract The cell wall of yeast consists of an internal skeletal layer and an external layer of glycoproteins covalently linked to the stress-bearing polysaccharides. The cell wall protein (CWP) population consists of over 20 different proteins, and may vary in composition. We present two complementary methods for quantifying CWPs, based on isobaric tagging and tandem MS: (1) absolute quantitation of individual CWPs, allowing estimation of surface densities; and (2) relative quantitation of CWPs, allowing monitoring of the dynamics of the CWP population. For absolute quantitation, we selected a representative group of ﬁve proteins (Cwp1p, Crh1p, Scw4p, Gas1p, and Ecm33p), which had 67 103, 44 103, 38 103, 11 103 and 6.5 103 of wall-bound copies per cell, respectively. As Cwp1p is predominantly incorporated in the birth scar, this corresponds to a protein density of c. 22 103 copies mm 2. For relative quantitation, we compared wild-type cells to gas1D cells, in which the cell wall integrity pathway is constitutively activated. The levels of Crh1p, Crh2p, Ecm33p, Gas5p, Pst1p and Pir3p increased about three- to ﬁvefold, whereas the level of Scw4p was signiﬁcantly decreased. We propose that our methods are widely applicable to other fungi. Introduction Fungal cells are surrounded by a sturdy wall, enabling them to withstand the internal osmotic pressure, and providing protection against mechanical injury. The fungal wall is also instrumental in adhesion to abiotic and biotic surfaces and in pathogenesis. The cell wall of Saccharomyces cerevisiae has two layers: an internal layer, which consists of a network of branched b-1,3-glucan molecules extended with covalently attached b-1,6-glucan and chitin molecules, and an outer layer, which is mainly composed of mannoproteins covalently linked to the underlying glycans (Klis et al., 2006). A similar cell wall structure is also found in other ascomycetous yeasts, such as the human pathogens Candida albicans, C. glabrata, Exophiala dermatitidis and Sporothrix schenckii, in the ﬁssion yeast Schizosaccharomyces pombe, and to a lesser extent in mycelial ascomycetous fungi (Travassos, 1985; Brul et al., 1997; Montijn et al., 1997; De Groot et al., 2004, 2007; Weig et al., 2004; Damveld et al., 2005; Klis et al., 2006). In the external protein layer of baker’s yeast, at least 20 different types of cell wall proteins (CWPs) have been identiﬁed (Protchenko et al., 2001; Yin et al., 2005; Verstre- FEMS Yeast Res 7 (2007) 887–896 pen & Klis, 2006). Most of them are glycosylphosphatidyli- nositol (GPI)-modiﬁed and are therefore called GPI-CWPs. GPI-CWPs include ﬂocculins, adhesins, transglycosidases, proteins involved in iron utilization, and proteins of un- known function (Klis et al., 2006). A smaller group of CWPs are directly linked to the b-1,3-glucan network through an alkali-sensitive linkage (ASL), including the family of Pir- CWPs (Pir, proteins with internal repeats) (Mrsa et al., 1997). Pir-CWPs may be involved in crosslinking the b-1,3- glucan network through their repeats, presumably by the formation of a carboxyl ester bond between a speciﬁc glutamine residue and a glucosyl hydroxyl group (Ecker et al., 2006). The composition of the cell wall proteome is highly dynamic, and depends on growth conditions such as nutrient availability, temperature, external pH, and oxygen level (Klis et al., 2006). Furthermore, the expression and incorporation into the cell wall of CWPs is temporally and spatially controlled. When the cell wall is stressed, either constitutively due to a genetic defect, or temporarily and dose-dependently by growing the cells in the presence of cell wall-perturbing compounds, the cells tend to become swol- len, as their walls are less capable of withstanding the turgor c 2007 The Authors Journal compilation c 2007 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd.